Amelioration of cobalt oxide nanoparticles induced genomic and mitochondrial DNA damage and oxidative stress by omega-3 co-administration in mice

ABSTRACT

Increased human exposure to cobalt oxide nanoparticles (CoO-NPs) because of their widespread use in many industrial and medical applications makes the study of genotoxicity and mutagenicity necessary. Therefore, this study was conducted to assess the genomic and mitochondrial DNA damage induction by CoO-NPs and the possible protective role of omega-3 in mice. The simultaneous administration of omega-3 significantly reduced DNA damage caused by CoO-NPs, which was evaluated by restoring the normal intact appearance of genomic DNA running on the agarose gel and normalizing fractionated DNA. The elevated malondialdehye (MDA) level and the reduced catalase (CAT) and glutathione peroxidase (Gpx) activities by CoO-NPs oral administration were normalized by omega-3 co-administration. Moreover, the omega-3 co-administration reduced the incidence of mutations induced by CoO-NPs in mitochondrial D-loop from 67% to 33%. We concluded that genomic and mitochondrial DNA damage and oxidative stress caused by CoO-NPs was mitigated by omega-3 co-administration.

KEYWORDS:

• Cobalt oxide nanoparticles
• genotoxicity
• mutagenicity
• omega-3
• mice

Introduction

Because of the unique interesting properties of cobalt oxide nanoparticles (CoO-NPs), they are increasingly used in a wide range of applications, in particular in biomedical applications as a primary material for building dextran coating and magnetic polymer microspheres, as a magnetic resonance imaging contrast agent in conjunction with iron, gold, graphite and platinum, as well as their uses in anaerobic waste water treatment, cancer therapy and as a non-viral DNA carrier in gene therapy (Liu et al. 2005, 2010; Papis et al. 2009; Rebello et al. 2010; Magaye et al. 2012; Yin et al. 2012; Jiang et al. 2013).

In addition, uses of cobalt oxides as pigments in the glass and ceramics industries, as catalysts in the oil and chemical industries, as paint and printing ink driers and as trace metal additives for agricultural and medical uses, as well as the presence of cobalt as active center in cobalamin coenzymes, indicate the broad use of and exposure to cobalt and cobalt compounds (Kirkland et al. 2015).

However, CoO-NPs have been shown to be genotoxic and carcinogenic both in vitro and in vivo (Bucher et al. 1999; Magaye et al. 2012; Uboldi et al. 2016). In vitro studies of CoO-NPs have demonstrated them to stimulate the formation of micronuclei, oxidative DNA damage and double strand DNA breaks in various experimental cell lines (Alarifi et al. 2013; Cavallo et al. 2015; Uboldi et al. 2016); an in vivo study has shown that CoO-NPs given to rats over two years caused benign lung tumor, bronchio-alveolar carcinoma, adenocarcinomas and bronchio-alveolar adenomas (Bucher et al. 1999).

Oxidative stress is now considered a acceptable mechanism for CoO-NPs induced genotoxicity. Many studies have demonstrated the generation of reactive oxygen species (ROS) by CoO-NPs using different experimental systems (Monteiller et al. 2007; Papis et al. 2009; Chattopadhyay et al. 2015). Thus, the use of natural antioxidants to diminish the genotoxicity of these nanoparticles increases.

Omega-3 fatty acids are essential long chain, polyunsaturated fatty acids (PUFA) derived from various dietary sources including hemp, flax seed, canola and walnuts, which are rich sources of omega-3 PUFA alpha-linolenic acid (ALA); in addition, fish oil is rich in omega-3 fatty acids including eicosapentanoic acid (EPA), docosahexanoic acid (DHA) and alpha linoleic acid (Alan et al., 2003; Kumar et al. 2011).

Omega-3 is a powerful antioxidant that recovers the antioxidant status by reducing the level of malondialdehyde and increasing the level of reduced glutathione and the activities of superoxide dismutase and glutathione peroxidase enzymes that have been adversely affected by methotrexate administration (Kumar et al. 2011). Omega-3 fatty acid supplements have become very important because the majority of diets contain a large amount of omega-6 and insufficient omega-3 fatty acids.

With increasing interest in the use of CoO-NPs in medical and industrial applications because of their remarkable properties, the human exposure to these nanoparticles and their risks increases, which necessitates the assessment of CoO-NPs genotoxicity and reduction of their possible genotoxicity using natural products. Therefore, the present study was designed to investigate the effect of CoO-NPs on the genomic and mitochondrial DNA and the modulatory effect of omega-3 dietary supplement on CoO-NPs induced genotoxicity in mice.

Materials and methods

Animals

Male Swiss albino mice aged 10–12 weeks and weighing about 25–30 g were purchased from National Research Center (NRC) animal house (Dokki, Giza, Egypt). All experimental procedures and animal maintenance were conducted in accordance with the accepted standards of animal care per cage (Council of Europe, European Convention for the Protection of Vertebrate Animals 2006). All experimentations and animals handling were performed in accordance to both the European Community Directive (86/609/EEC) and National Rules on Animal Care and the guidelines of Institutional Animal Care and Use Committee (IACUC) of Faculty of Science Cairo University with approval number CUIF/16/18 from IACUC.

Chemicals

The CoO-NPs used in this study were purchased in from Sigma Chemical Company (St. Louis, MO, USA) as black powder with particles size ≤ 50 nm and suspended in deionized distilled water to prepare the genotoxic dose 20 mg kg^–1 bw on the basis of Shaikh et al. (2015) study. Dietary supplement omega-3 was purchased from a pharmacy; it was produced by Medizan Pharmaceutical Industries (Borg El Arab, Alexandria, Egypt) and packed by the Arab Company for Gelatin & Pharmaceutical products of Montana Pharmaceutical Company, Alexandria, Egypt.

Characterization of CoO-NPs

In order to ensure the size of CoO-NPs, X-ray diffraction (XRD) pattern was detected using a charge coupled device diffractometer (XPERT-PRO, PANalytical, Almelo,  Netherlands). The particles size was also calculated using Scherrer’s relationship (D = 0.9 k/Bcosh) where k is the wavelength of X-ray, B is the expansion of the diffraction line measured as the half of its maximum intensity in radians, and h is the Bragg’s diffraction angle.

The size of agglomeration and zeta potential were measured using the Malvern Instrument Zetasizer Nano Series (Malvern Instruments, Westborough, MA, USA) equipped with a He-Ne laser (λ = 633 nm, max 5 mW). Finally, the particles size and CoO-NPs morphology in the aqueous media were assessed by transmission electron microscope (TEM) imaging of ultra-sonicated CoO-NPs in Milli-Q water for about 25 min at 40 W and placed on carbon-coated copper TEM grids using a Tecnai G20, Super twin, double tilt TEM at an accelerating voltage of 200 kV.

Treatment schedule

Twelve mice were randomly divided into four groups; three mice per group, and orally administrated for three consecutive days with deionized distilled water representing negative control group (Group 1), omega-3 oil 250 mg kg^–1 bw (Group 2) based on the study of Kumar et al. (2011), CoO-NPs suspension 20 mg kg^–1 (Group 3) or omega-3 simultaneously with CoO-NPs suspension (Group 4). All animals were then sacrificed after 24 h of the last administration and dissected to expose internal organs. Liver, kidney and testis were washed with cold PBS and stored at −20°C until used for experimentations.

Qualitative laddered DNA fragmentation assay

To study the influence of CoO-NPs and/or omega-3 on the apoptotic DNA damage in liver, kidney and testis tissues, laddered DNA fragmentation assay was carried out based on the protocol described by Sriram et al. (2010). Briefly, small pieces of tissues were gently homogenized and lysed in Tris EDTA (TE) lysis buffer, then added 0.5 mg ml^–1 RNase A and incubated at 37°C for 1 h. Proteinase K (0.2 mg ml^–1) was added and the sample was incubated at 50°C overnight. After that, DNA was extracted by the phenol extraction method and precipitated by 7.5 M ammonium acetate and isopropanol. Extracted genomic DNA was electrophoresed in 1% agarose gel at 70 V and visualized using a UV trans-illuminator and finally photographed.

Quantitative measurement of DNA fragmentation using diphenylamine assay

Colorimetric diphenylamine assay was performed to further assess DNA fragmentation quantitatively in liver, kidney and testis tissues according to Gercel-Taylor (2005) protocol. In brief: a small tissue piece was homogenized in ice-cold hypotonic lysis Tris EDTA (TE) buffer (10 mM Tris HCl, 1 mM EDTA, 0.2% Triton X-100, pH 8.0) and incubated in TE lysis buffer for 45 min for lysis. It was centrifuged to separate the fragmented and damaged DNA in the supernatant fraction and the pellet re-suspended in TE buffer. Trichloroacetic acid (TCA) (10% [v/v]) was added to both the supernatant and the pellet, then it was incubated at room temperature for 20 min. It was then centrifuged, and the precipitates re-suspended in 5% TCA, boiled for 15 min at 100°C and centrifuged to remove proteins at 4°C. Diphenylamine (0.088 M) was added to the supernatants then left to react at room temperature for about 16-20 h and the absorbance was measured spectrophotometrically at 600 nm. The DNA fragmentation was expressed as a percentage of total DNA.

Screening mutations in mitochondrial D-loop using SSCP-PCR

Single strand conformational polymorphism polymerase chain reaction (SSCP-PCR) was carried out to screen mutations in the mitochondrial D-loop. First mitochondrial DNA was extracted from the liver and kidney tissues based on the protocol described by Noshy et al. (2013), by gently homogenizing a small amount of tissue in cold homogenization buffer (100 mM Tris–HC1, pH 7.4, 250 mM sucrose, 10 mM EDTA), and centrifuged to remove nuclei and cellular debris. The supernatant was further centrifuged for 10 min at 4°C at 10,500 rpm (10,000 × g) then the mitochondrial pellet was re-suspended in high-salt buffer (10 mM Tris–HCl pH 7.6, 10 mM KCl, 10 mM MgCl2, 0.4 M NaCl and 2 mM EDTA). For complete denaturation and solubilization of protein, 10% SDS was added and incubated at 55°C. After that 6 M NaCl was added for complete salting out of protein, and centrifuged. Finally cold absolute ethanol was added to precipitate mitochondrial DNA, dried and re-suspended in deionized distilled water.

Second amplification of the two fragments of the mitochondrial DNA D-loop was carried out in the Thermal Cycler (Programmable Thermal Cycler, PTC-100TM thermal cycler, Model 96; MJ Research, Inc., Watertown, MA, USA) using the primers sequences previously designed by Dai et al. (2005) and listed in Table 1 for both the fragment 1 (P1) and fragment 2 (P2) of the mitochondrial D-loop. The PCR reaction was initiated at 94°C for 5 min, then cycle of denaturation at 94°C for 30 s, primer annealing at 55°C for 1 min, and primer extension at 72°C for 1 min was repeated 30 times and followed by final extension for 10 min at 72°C for complete amplification. PCR products were electrophoresed on a 1.5% ethidium bromide-treated agarose gel to confirm the amplification of the desired sequences (Sigma, Gillingham, UK).

Table 1. Sequences of primers used for amplification of mitochondrial D-loop fragment 1(P1) and fragment 2 (P2).

Primer	Sequences
P1	Forward 5-CCACCACCAGCACCCAAAG-3
	Reverse 5-CGGGTTGTTGGTTTCACGG-3
P2	Forward 5-AGGCATGAAAGGACAGCAC-3
	Reverse 5-ATAAGGCCAGGACCAAACC-3

Finally, the mix of PCR products, loading dye and TE buffer was electrophoresed in 9% polyacrylamide gel (acrylamide/bisacrylamide = 49:1, v/v) at 90 V until the bands reached the bottom of the plate. Then the gel was stained with ethidium bromide to visualize the DNA bands and finally photographed using gel documentation system.

Estimation of hepatic oxidative stress markers

The level of hepatic malondialdehye (MDA) was measured as an indicator of lipid peroxidation using the protocol of Ohkawa et al. (1979), based on the reaction between thiobarbituric acid (TBA) and MDA in acidic medium at temperature of 95°C for about 45 min resulting in TBA reactive products; the absorbance of its pink color was measured spectrophotometrically at 534 nm. Results were expressed as nmol g^–1 tissue used. The antioxidant activity of both hepatic catalase (CAT) and glutathione peroxidase (Gpx) enzymes was measured based on the protocol described by Aebi (1984) and Paglia and Valentine (1967) respectively. Results were expressed as U g^–1 tissue used.

Statistical analysis

All data in this study were expressed as mean ± SD and analyzed using the Statistical Package for the Social Sciences (version 20) (copyright by IBM SPSS software, US). One-way analysis of variance (ANOVA) was performed to test the effect of CoO-NPs and/or omega-3 on the tested parameters.

Results

Characterization of CoO-NPs

The appearance of peaks at the 18.99°, 31.31°, 36.86°, 38.66°, 44.75°, 59.35° and 65.21° diffraction angles in the XRD curve confirmed the purchased form of CoO-NPs, and their particles sizes were about 31.60 ± 16.19 nm using Scherrer’s equation (Figure 1(A)). The value of polydispersity index (PdI) of 0.200 (d.nm) and the zeta potential value of −2.11 mV confirmed the stability of CoO-NPs in the aqueous media (Figure 1(B, C)). Imaging of CoO-NPs using TEM revealed their good dispersion in deionized distilled water with average size of about 20.56 ± 5.14 nm and a spherical-cubic shape of CoO-NPs (Figure 1(D)).

Figure 1. Characterization of CoO-NPs. (A) XRD pattern of CoO-NPs. (B) Zeta potential distribution. (C) TEM imaging. (D) Size distribution by number.

Laddered DNA fragmentation

As shown in Figure 2 oral administration of CoO-NPs at the dose level 20 m kg^–1 bw resulted in dramatic damage in the genomic DNA as shown by the fragmentized pattern of genomic DNA of liver, kidney and testis tissues compared to the intact pattern of negative control DNA. Conversely, the pattern of genomic DNA running on agarose gel revealed that simultaneous co-administration of omega-3 with CoO-NPs reduced the incidence of CoO-NPs induced apoptotic DNA fragmentation as shown by appearance of less DNA fragmentation bands. On the other hand, no DNA damage was caused by oral administration of omega-3 alone, as shown by the appearance of intact genomic DNA pattern similar to that of negative control pattern (Figure 2).

Figure 2. Pattern of mice liver, kidney and genomic DNA running on ethidium bromide stained agarose gel. Abbreviations: M: DNA marker (100 base pairs); N: negative control; O: omega-3; C: cobalt oxide nanoparticles.

Quantitative DNA fragmentation

The oral administration of omega-3 (250 mg kg^–1) alone did not result in any significant changes (p > 0.05) in the % fragmented DNA as it is still at the control level in contrast to oral administration of CoO-NPs (20 mg kg^–1 bw) for three consecutive days caused significant elevations in the % fragmented DNA in the liver (p < 0.001), kidney (p < 0.001) and testicular (p < 0.05) tissues compared to the negative control group level, as shown in Figure 3.

Figure 3. Level of genomic DNA fragmentation in liver, kidney and testis tissues of mice orally administered CoO-XPs and/or omega-3 for three consecutive days. One-way ANOVA followed by Duncan’s test was done and statistical significance is indicated by different letters. Results are expressed as mean ± SD. Abbreviations: a: non-significant difference from the negative control group; b: significant difference from the negative control group.

However, simultaneous co-administration of omega-3 with CoO-NPs attenuated CoO-NPs induced DNA fragmentation as revealed by the observed significant reductions in the % fragmented DNA in the liver (p < 0.001), kidney (p < 0.001) and testicular (p < 0.05) tissues compared with those in the CoO-NPs administered group (Figure 3).

Mutation screening in mitochondrial d-loop

In the liver tissues there were no mutations induced by oral administration of omega-3 or/and CoO-NPs as the PCR-SSCP pattern of both fragments P1 and P2 of the mitochondrial DNA loop of the three groups administered orally omega-3 or/and CoO-NPS (Groups 3–4) were identical to that of the negative control group as shown in Figure 4.

Figure 4. PCR-SSCP pattern of mitochondrial D-loop fragment 1 (Pl) and 2 (P2) in negative control group (lanes 1–2), omega-3 group (lanes 3–4), CoO-NPs group (lanes 5–6) and omega-3 plus CoO-NPs group (lanes 7–8). Mutation is indicated by arrow.

On the other hand, mutations in kidney tissues induced by oral administration of CoO-NPs in the fragment P1 of mitochondrial DNA loop were reduced from 67% (two mice out of three) to 33% (one mouse out of three) by simultaneous co-administration of omega-3 while no changes were observed in the SSCP pattern of fragment P2 of the mitochondrial DNA loop in kidney tissues of all treated groups compared with the negative control pattern (Figure 4).

Hepatic oxidative stress markers

Oral administration of omega-3 at the dose level 250 mg kg^–1 resulted in non-significant changes in the level of MDA and activities of CAT and Gpx enzymes compared with the negative control levels. Conversely, oral administration of CoO-NPs at the dose level 20 mg kg^–1 caused statistically significant increases in the MDA level (p < 0.001) and statistical significant declines in the activities of both CAT (p < 0.001) and Gpx (p < 0.01) enzymes compared with the negative levels as shown in Table 2.

Table 2. Malondialdehyde (MDA) level and activities of catalase (CAT) and glutathione peroxidase (Gpx) enzymes in mice orally administered CoO-NPs and/or omega-3.

Group	Treatment	Dose (mg kg^–1)	MDA level (nmol g^–1 tissue)	CAT activity (U g^–1 tissue)	Gpx activity (U g^–1 tissue)
1	Negative control	0	180.76 ± 13.06^a	7.65 ± 0.77^a	8.31 ± 0.60^a
2	Omega-3	250	169.81 ± 6.73^a	6.72 ± 0.90^a	8.60 ± 0.78^a
3	CoO-NPs	20	245.38 ± 5.57^b	3.21 ± 0.92^b	4.91 ± 0.97^b
4	Omega-3+CoO-NPs	250 + 20	183.43 ± 14.69^a	7.33 ± 1.04^a	8.27 ± 1.17^a
One-way ANOVA			F = 30.35 p < 0.001	F = 15.50 p < 0.01	F = 11.18 p < 0.01

Results are expressed as mean ± SD and analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s test. Similar letters indicate non-significance between the different groups in the same column.

Simultaneous co-administration of omega-3 resulted in attenuation of CoO-NPs induced oxidative stress as revealed by significant reduction in the MDA level and significant increases in the CAT and Gpx activities compared to those in the CoO-NPs, even reached the negative control levels (Table 2).

Discussion

With the increasing uses of CoO-NPs in a wide range of industrial and medical applications, human exposure to these nanoparticles is constantly increasing and requires study of their effects on living cells. Therefore, the current study was designed to assess the genotoxicity and mutagenicity of CoO-NPs and the possible modulatory effect of omega-3 in mice.

The DNA damage induction by CoO-NPs demonstrated in this study by the fragmentized appearance of genomic DNA and significant elevations in the % fragmented DNA could be attributed either to the reported direct uptake of these nanoparticles by cells or/and to their partial dissolution, leading to increased levels of Co++ ions in the tissues, which subsequently affects the cells and causes the production of reactive oxygen species (ROS) (Papis et al. 2009; Chattopadhyay et al. 2015). Moreover, our finding of low zeta potential (−2.11 mV) of CoO-NPs has revealed a low surface charge that is also a factor favoring cell internalization and, consequently, DNA damage.

The imbalance in the production and manifestation of ROS increases the cellular oxidative stress and leads to cells losing their ability to detoxify reactive intermediates or to repair the resulting damage. Our finding of a significant increase in the level of MDA, an indicator of lipid peroxidation, by oral administration of CoO-NPs administration revealed high ROS generations that exhausted the antioxidant defense system, resulting in a significant reduction in the activities of CAT and Gpx causing oxidative stress induction, in agreement with previous studies (Monteiller et al. 2007; Papis et al. 2009; Chattopadhyay et al. 2015).

The critical cellular objective for ROS is mitochondrial DNA and chronic exposure to ROS leads to increased mitochondrial-generated ROS, reduced mitochondrial function, and continuous mitochondrial DNA damage (Higuchi 2012). As a result, mutations that have been demonstrated in the renal fragment P1 of mitochondrial D-loop in this study using PCR-SSCP could be attributed to oxidative stress induction by CoO-NPs administration that weakens the cells’ ability to repair oxidative DNA damage and cause mutations. Meanwhile, the absence of mutations in both fragments P1 and P2 of the mitochondrial D-loop in liver tissues can be explained by high dividing and regeneration abilities of liver cells, that leads to rapid elimination of mutated cells (Michalopoulos and Defrances 1997; Fausto et al. 2006).

Although the mitochondria D-loop is a non-coding region; it is responsible for regulating mitochondrial DNA replication and transcription due to the presence of the primary site for heavy chain replication and promotors for heavy and light chain transcription. Thus, any mutation in mitochondrial DNA causes mutations in the coding region, altering protein synthesis, and affecting the function of the respiratory chain that hampers the energy supply of the cells and produces an excessive volume of ROS that ultimately harms the genomic DNA (Bianchi et al., 2001; Zhao et al., 2005).

Therefore, there is increasing interest in using natural antioxidants to diminish the genotoxicity of nanoparticles. Omega-3 is safe and non-genotoxic as the oral administration of omega-3 for three consecutive days did not cause any change in the integrity of the genomic DNA, PCR-SSCP pattern of the mitochondrial D-loop fragments P1 and P2 and oxidative stress markers in a line with previous studies (El-Ansary et al. 2011; Kumar et al. 2011; Elelaimy et al. 2012; Arunagiri et al. 2014).

Omega-3 is a strong free radicals scavenger due to its fatty acids rich in unsaturated bonds, e.g. docosahexaeonoic acid (DHA) and eicosapentaennoic acid (EPA). Thus, restoring the structural integrity of the genomic DNA and the normal oxidative status by simultaneous co-administration of omega-3 with CoO-NPs could be attributed to the omega-3 fatty acids DHA and EPA acting as antioxidants which scavenge ROS and also provide the building blocks for healthy cell membranes (Le-Niculescu et al. 2011; Arunagiri et al. 2014).

Furthermore, our finding of reduction in the incidence of CoO-NPs induced mutation from 67% to 33% by simultaneous co-administration of omega-3 could be explained by the free radical scavenger ability of omega-3, as revealed by observed declines in the level of MDA and increasing the antioxidant CAT and Gpx enzymatic activities even to the normal control level.

Conclusion

Oral administration of CoO-NPs for three consecutive days damaged the genomic DNA and caused mutations in the mitochondrial D-loop by stimulating oxidative stress. However, synchronized administration of omega-3 has declined the CoO-NPs induced genotoxicity and mutagenicity and restored the normal antioxidant status disturbed by CoO-NPs administration.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Faculty of Science, Cairo University.