Estimation of genomic and mitochondrial DNA integrity in the renal tissue of mice administered with acrylamide and titanium dioxide nanoparticles

The Kidneys remove toxins from the blood and move waste products into the urine. However, the accumulation of toxins and fluids in the body leads to kidney failure. For example, the overuse of acrylamide and titanium dioxide nanoparticles (TiO2NPs) in many food and consumer products increases human exposure and risks; however, there are almost no studies available on the effect of TiO2NPs coadministration with acrylamide on the integrity of genomic and mitochondrial DNA. Accordingly, this study was conducted to estimate the integrity of genomic and mitochondrial DNA in the renal tissue of mice given acrylamide and TiO2NPs. To achieve this goal, mice were administrated orally TiO2NPs or/and acrylamide at the exposure dose levels (5 mg/kg b.w) and (3 mg/kg b.w), respectively, five times per week for two consecutive weeks. Concurrent oral administration of TiO2NPs with acrylamide caused remarkable elevations in the tail length, %DNA in tail and tail moment with higher fragmentation incidence of genomic DNA compared to those detected in the renal tissue of mice given TiO2NPs alone. Simultaneous coadministration of TiO2NPs with acrylamide also caused markedly high elevations in the reactive oxygen species (ROS) production and p53 expression level along with a loss of mitochondrial membrane potential and high decreases in the number of mitochondrial DNA copies and expression level of β catenin gene. Therefore, from these findings, we concluded that concurrent coadministration of acrylamide with TiO2NPs augmented TiO2NPs induced genomic DNA damage and mitochondrial dysfunction through increasing intracellular ROS generation, decreasing mitochondrial DNA Copy, loss of mitochondrial membrane potential and altered p53 and β catenin genes expression. Therefore, further studies are recommended to understand the biological and toxic effects resulting from TiO2NPs with acrylamide coadministration.

observed after acrylamide treatment 5,[10][11][12][13] .The study of Alzahrani 14 , also found that the oral acrylamide single administration (10, 20, or 30 mg/kg or repeated administration (10 mg/kg) for one and two weeks significantly increased the frequency of micronuclei and chromosomal abnormalities induction compared to the negative control values.
Unfortunately, humans are exposed to many other chemicals such as nanoparticles together with acrylamide.Titanium dioxide nanoparticles (TiO 2 NPs) are one of the widest used nanoparticles in many food, medical and consumer products such as pharmaceuticals, toothpastes, sweets, chewing jam and cosmetics 15 , as well as, nanoparticles are extensively used in wastewater treatment to remove harmful and xenobiotic chemicals due to their corrosion resistance and high stability 16,17 .All these applications along with the high uses of TiO 2 NPs in increasing crop yield by accelerating photosynthesis of and increasing agricultural productivity specific crops 15 increase human exposure to TiO 2 NPs and require studying the effect of exposure of nanoparticles on human health.
Upon ingestion of pharmaceuticals, food, water and other consumer products containing TiO 2 NPs nanoparticles such as chewing gums, candies, sweets and artificial flavors, the TiO 2 NPs can diffuse throughout the circulation and enter cells of various organs by endocytosis or/and free diffusion causing gastric toxicity, hepatotoxicity, neurotoxicity and nephrotoxicity [18][19][20][21][22] .Nano-TiO 2 particles can also directly or indirectly interact with genomic DNA disrupting the integrity of genomic DNA through induction of chromosomal aberrations, DNA breakages, mutations and alterations of apoptotic genes expressions in various experimental models 19,20,[22][23][24][25] .
As seen, all of the above studies demonstrated toxicity after exposure to acrylamide or TiO 2 NPs separately and almost no studies have investigated the genotoxicity of both acrylamide and TiO 2 NPs although human exposure to acrylamide and TiO 2 NPs together can be done simultaneously.Therefore, the current focused on studying the effect of concurrent simultaneous administration of TiO 2 NPs and acrylamide on genomic and mitochondrial DNA integrity in the kidney tissues of mice.Genomic DNA integrity was assessed using alkaline comet and laddered DNA fragmentation assays, while, mitochondrial membrane potential and level of intracellular reactive oxygen species (ROS) were studied using Rhodamine and 2,7 dichlorofluorescin diacetate dyes, respectively.Moreover, quantitative real time polymerase chain reaction (qRT-PCR) was done to measure the number of mitochondrial DNA copy and expression level of apoptotic genes.

Animals.
Swiss Webster male mice weighting 20-25 gm and aging 10-12 weeks were purchased from Animal House of the National Organization for Drug Control and Research (NODCAR).For acclimatization mice were kept in the animal house conditions at the Department of Zoology, Faculty of Science Cairo University under standard dark/light cycle and supplied with standard diet pellets and water that were given ad libitum.

Chemicals.
Acrylamide and TiO 2 NPs were purchased as white powder from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).Powder of TiO 2 NPs < 100 nm was immediately suspended in deionized distilled water prior to use for preparing the given dose of 5 mg/kg b.w equivalent to the human exposure dose 26,27 , while, acrylamide was dissolved in deionized distilled water to prepare the tested dose of 3 mg/kg b.w equivalent to the human exposure dose 3 .Other used chemicals and reagents used in the experiments of this study were of analytical and molecular biology grade.
Characterization of TiO 2 NPs.Powder of TiO 2 NPs purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA) with CAS number 13463-67-7 and a purity 99.5% has been characterized in recent study 13 using X-ray diffraction (XRD) to ensure the purity of the TiO 2 NPs crystallites and transmission electron microscopy (TEM) to detect the shape and average particle size of suspended TiO 2 NPs in aqueous media.

Ethical consideration and treatment schedule.
The experimentations and study plan were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Cairo University with accreditation number (CU/I/F/15/18).This study was reported according to ARRIVE guidelines and also Animal handling and experimentations were conducted in accordance with the Guidelines of the National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures.
Twenty-four male mice were acclimated for one week and then randomly divided into four groups, six animals per group.The negative control group (group I) was orally given distilled deionized water and the three treated groups (Groups II to IV) were orally administered acrylamide at a dose level of 3 mg/kg body weight 3 or/and TiO 2 NPs at a dose level of 5 mg/kg body weight 26,27 separately or simultaneously five times per week for two successive weeks.Mice from all groups were sacrificed 24 h after the last administration and dissected.The obtained kidney tissues were stored at − 80 °C for further molecular studies.

Estimation of genomic DNA integrity.
To assess the effect of acrylamide or/ and TiO 2 NPs administration on the integrity of genomic DNA laddered DNA fragmentation and alkaline comet assays were conducted in the renal tissues of all groups.
Ladder DNA fragmentation assay.Laddered DNA fragmentation assay was done by gently homogenizing small pieces of renal tissues in cold Tris EDTA (TE) and sodium dodecyl sulfate lysis buffer, RNase A then added and incubating samples for 1 h in lysis buffer at 37 °C.After lysis, proteinase K was added to samples, incubated at 50 °C, and precipitated genomic DNA using cold absolute ethanol.The precipitated genomic DNA was dissolved in deionized dist.water and 15 µl of the dissolved genomic DNA (3 µg) was electrophoresed in 1% agarose gel at 70 V, visualized and photographed using a UV trans-illuminator 28 .

Mitochondrial membrane potential.
The mitochondrial membrane permeability potential was studied in the renal tissues of control and treated mice with the fluorescent Rhodamine-123 dye 30 .Small pieces of renal tissues were gently homogenized in phosphate buffered saline (PBS), the clear cell suspension was mixed with Rhodamine-123 fluorescent dye and left for 1 h at 37 °C in the dark.Renal cells were then washed twice with PBS and the intensity of fluorescent light emitted from cells stained with Rhodamine-123 imaged and analyzed using an epi-fluorescence microscope at 20 × magnification.
Mitochondrial DNA copy.To quantify the number of mitochondrial DNA copy in renal tissues of negative control and treated mice, a quantitative real time polymerase chain reaction (RT-PCR) was done: total RNA and DNA was first extracted from renal tissues using GeneJET Purification Kit (Thermo Scientific, USA) and then the RNA was reversely converted into complementary DNA (cDNA) using Oligo(dT) 18 and Random Hexamer primers according to the manufacturer's instructions for the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA).A separate RT-PCR was then conducted in the 7500 Fast system thermal cycler (Applied Biosystem 7500, Clinilab, Egypt) to amplify the mitochondrial 12S ribosomal RNA (12S rRNA) gene using the pre-deigned primers shown in Table 1 31 .The obtained data were standardized against the nuclear 18S rRNA gene as a housekeeping gene and the number of mitochondrial DNA copies was then determined using a comparative Ct (DDCt) method.Results were expressed as mean ± S.D.

mRNA expression of p53 and β-catenin genes.
To measure the mRNA expression level of p53 and β-Catenin genes in the renal tissues of negative control and treated mice; whole RNA was extracted and reversely transcribed into complementary DNA (cDNA) using the GeneJET RNA Purification and Revert Aid First Strand cDNA Synthesis Kits (Thermo Scientific, USA), respectively.For cDNA synthesis each a µg of template RNA and 1 µl of primer were added to a sterile nuclease free water to complete the final volume to 12 µl in nuclease free tube on ice.Then added reaction buffer, RNase Inhibitor, nucleotides and Revert Aid M-MuLV RT completed the final volume to 20 µl.Samples were mixed gently, incubated 42 °C for 60 min and terminated the reaction by heating at 70 °C for 5 min.Amplification of p53 and β-Catenin genes was then conducted using the primers' sequences listed in Table 1 and previously designed by 32,33 .The results obtained from RT-PCR were then normalized to the housekeeping β-actin gene and the comparative Ct (DDCt) method was used to calculate the fold changes in the expression level of p53 β-Catenin genes.

Generation of intracellular ROS.
The generation level of ROS within renal cells was evaluated by mixing renal cells suspension with 2,7 dichlorofluorescin diacetate dye that enters passively.Mixed cells with dye were left in dark for thirty minutes to allow interaction between dye and generated ROS within cells.After incubation cells were spread on a clean slide, visualized and photographed the forming the highly fluorescent dichlorofluorescein compound under epi-fluorescence microscope at 20 × magnification 34 .
Table 1.The sequences of used primers.

Results
Characterization of TiO 2 NPs.Characterization of TiO 2 NPs in our recent study 13 confirmed the purity of purchased TiO 2 NPs powders through the appearance of unique bands for TiO 2 NPs crystals and also demonstrated the polyhedral shape the of well dispersed TiO 2 NPs with about 60 nm median particles size shown in TEM image.
Integrity of genomic DNA.Laddered DNA fragmentation.As displayed in Fig. 1 oral administration of TiO 2 NPs (5 mg/kg) and acrylamide (3 mg/kg) separately or simultaneously together caused dramatic damage to renal genomic DNA as displayed by the seen smeared fragmentized pattern of DNA running on agarose gel compared to the intact DNA pattern of the negative control mice.However, the degree of DNA damage observed after administration of TiO 2 NPs alone was lower than that observed after administration of acrylamide alone or in combination with TiO 2 NPs as displayed in Fig. 1).

Alkaline comet assay.
Results of Comet assay demonstrated the induction of DNA breakages by TiO 2 NPs (5 mg/kg) and acrylamide (3 mg/kg) administered separately or simultaneously together by the statistically significant (p < 0.001) elevations in tail length, %DNA in tail and tail moment noticed in renal tissues of mice given TiO 2 NPs or/and acrylamide compared to the negative control values (Table 2).Otherwise, the DNA damage induced by administration of acrylamide alone or simultaneously with TiO 2 NPs was significantly (p < 0.001) higher than that induced by administration of TiO 2 NPs alone.This was manifested by the statistical significant (p < 0.001) elevations in the three Comet parameters: tail length, %DNA in tail and tail moment observed after administration of acrylamide alone or in combination with TiO 2 NPs compared to their values in renal tissues of mice given TiO 2 NPs alone (Table 2).Examples for the scored Comet nuclei in the renal tissues of the negative control group and mice given acrylamide or/and TiO 2 NPs are displayed in Fig. 2.

Integrity of mitochondrial DNA. Mitochondrial membrane potential. Staining the renal cells with
Rhodamine-123 dye showed that oral administration of acrylamide and TiO 2 NPs separately or together simultaneously caused a large reduction in the integrity of the mitochondrial membrane permeability potential as manifested by the greater decreases in the intensity of the fluorescence light emitted from the treated renal cells compared with the light emitted from the untreated control renal cells (Fig. 3).Moreover, oral administration of TiO 2 NPs alone caused less reduction in the integrity of mitochondrial membrane potential compared to reductions resulting from administration of acrylamide alone or in combination with TiO 2 NPs (Fig. 3).
Mitochondrial DNA copy.The copy number of mitochondrial DNA was statistically significantly (p < 0.001) decreased after multiple oral administration of acrylamide or/and TiO 2 NPs compared to the copy of www.nature.com/scientificreports/ the negative control mitochondrial DNA as seen in Fig. 4. Otherwise, oral administration of acrylamide alone resulted in less reduction in mitochondrial DNA copy than those resulting from administration of TiO 2 NPs alone or in combination with acrylamide (Fig. 4).

Expression of p53 and β-Catenin genes.
As noted in Table 3, multiple oral administration of acrylamide and TiO 2 NPs caused statistical significant (p < 0.001) elevations in the expression level of p53 gene compared with that of the negative control renal tissue.Meanwhile, the expression level of the p53 gene in renal cells Table 2. Induction of DNA damage in the renal tissue of negative control mice and mice given orally acrylamide or/and TiO 2 NPs.Six mice were used for each group.Results are expressed as mean ± SD. Results were analyzed using one-way analysis of variance followed by Duncan's test to test the similarity between the control and three treated groups.According to Duncan's test means with different letters indicates statistical significant difference between the compared groups in the same column at a significant level of p < 0.001.www.nature.com/scientificreports/ of mice orally given TiO 2 NPs alone was significantly (p < 0.001) lower than that expressed in the renal tissues of mice given acrylamide alone or simultaneously with TiO 2 NPs (Table 3).On the contrary, several oral administration of acrylamide and TiO 2 NPs caused statistically significant (p < 0.001) decreases in the expression level of the β-Catenin gene compared to that of the negative control (Table 3).On the other hand, the expression level of β-Catenin gene in renal tissue of mice administered TiO 2 NPs alone was statistically (p < 0.001) higher than that of mice given acrylamide alone or simultaneously with TiO 2 NPs (Table 3).

Generation of intracellular ROS.
Examination of renal cells stained with the 2,7 dichlorofluorescin diacetate (DCFH-DA) dye using epi-fluorescent microscope showed that ROS are highly generated after oral administration of TiO 2 NPs or/and acrylamide as the intensity of the fluorescent light emitted from the stained renal cells was remarkably higher compared to those emitted from the negative control renal cells (Fig. 5).

Discussion
Intensive uses of various consumer and food products containing acrylamide or TiO 2 NPs increase the risk of human exposure to acrylamide TiO 2 NPs together.However, almost no studies have investigated the effect of acrylamide and TiO 2 NPs coadministration on the integrity of genomic and mitochondrial DNA in kidney tissues.Therefore, this study was done to estimate the effect of simultaneous co-administration acrylamide and TiO 2 NPs on the integrity of genomic and mitochondrial DNA in renal tissues of mice.
In this study, acrylamide and TiO 2 NPs in this study were orally administered to mice at the dose level 3 mg/ kg and 5 mg/kg, respectively, equivalent to the human exposure dose, because human excessive consumption of high temperature cooked starchy foods and other acrylamide containing foodstuffs reach human daily intake of acrylamide to 3 mg/kg by 3,35,36 .Similarly, the massive use of TiO 2 NPs in plastics, cosmetics toothpaste, Candy, sweets, gums, artificial flavors and other foods due to their high stability and anti-corrosion properties increases human ingestion of a large amount of TiO 2 NPs exceeding 5 mg/kg 26,27,37 .
Our results demonstrated the genotoxicity of acrylamide (3 mg/kg) or/and TiO 2 NPs (5 mg/kg) given orally daily over two weeks through the noticed fragmented smeared pattern of genomic DNA electrophoresed on ethidium bromide stained agarose gel compared to the intact appearance of the negative control genomic DNA.Similarly, the noticed remarkable elevations in measured Comet parameters: tail length, %DNA in tail and tail moment confirmed the induction of DNA breakage after multiple administration of the tested low doses of acrylamide or/and TiO 2 NPs in kidney tissues of mice because alkaline Comet assay sensitively detects single and double stranded DNA breakages 29 .These findings thus supported the demonstrated genotoxicity of acrylamide and TiO 2 NPs administered separately in previous studies 19,20,23,24,[38][39][40] .
The generation of ROS normally occurs within cells during numerous metabolic processes.However, exposure to various toxic agents leads the production of intracellular ROS over the normal level leading to a disruption of the balance between ROS generation and antioxidant defense system inducing oxidative stress 13,[41][42][43] .Excessive generation of ROS by acrylamide or/and TiO 2 NPs was manifested in this study by marked increases in the intensity of fluorescent light emitted from renal cells stained with 2,7 Dichlorofluorescein diacetate dye consistent with the previous suggestion of oxidative stress induction through ROS generation as an accepted mechanism of acrylamide or TiO 2 NPs induced toxicity 13,20,22,44,45 .Therefore, the increased DNA damage induction observed in the renal tissues of mice orally given low doses of acrylamide and TiO 2 NPs together may result from the observed higher ROS generation that attacks cellular proteins, lipids, carbohydrates, and DNA causing cell damage and necrosis making renal cells more susceptible to DNA damage than that induced after administration of acrylamide or TiO2NPs alone.These results are consistent with our recent study showing increased TiO 2 NPs genotoxicity when TiO 2 NPs coadministered with acrylamide through increased ROS generation in mice brain tissue 13 .
Oxidizing cellular macromolecules and depleting antioxidants by over ROS generation also promotes mitochondrial DNA damage and triggers apoptosis 46,47 .Our finding of Loss of mitochondrial membrane potential integrity and remarkable reduction in number of mitochondrial DNA copies demonstrated the induction of mitochondrial dysfunction by multiple administrations of the tested low doses of TiO 2 NPs or acrylamide.Moreover, dramatic reduction of mitochondrial DNA copies and impairment of mitochondrial membrane potential noticed in the mice renal cells after simultaneous coadministration of TiO 2 NPs with acrylamide can be attributed to the Table 3. Expression level of p53 and β-Catenin genes in the renal tissue of negative control mice and mice given orally acrylamide or/and TiO 2 NPs.Six mice were used for each group.Results are expressed as mean ± SD. Results were analyzed using one-way analysis of variance followed by Duncan's test to test the similarity between the control and three treated groups.According to Duncan's test means with different letters indicates statistical significant difference between the compared groups in the same column at a significant level of p < 0.001.www.nature.com/scientificreports/induced increased extra ROS generations that highly disrupted mitochondrial membrane and mitochondrial DNA integrity hampering mitochondrial DNA biogenesis 43,48,49 .
Apoptosis occurs naturally during aging and development to preserve cell populations in the tissues.However, inappropriate apoptosis can be induced by excessive ROS generation and DNA damage induction 50 .Consequently, our findings on the marked induction of DNA damage, ROS generation and mitochondrial dysfunction after chronic administration of TiO 2 NPs or/and acrylamide may serves as a signal triggering apoptosis of renal cells because even a single double-stranded DNA break is sufficient to induced cellular death 51 .Moreover, the remarkable elevations in the expression level of the apoptotic p53 gene and significant reductions in the expression level of β-Catenin gene noticed after administration of TiO 2 NPs or/and acrylamide confirmed the induction of apoptosis by TiO 2 NPs or/and acrylamide administration because overexpression of the p53 gene mediates and stimulates apoptosis by reducing the expression of β-Catenin as well as accelerated degradation of the β-Catenin protein itself 52,53 .

Conclusion
Based on the above findings, it was concluded that the coadministration of TiO 2 NPs with acrylamide increased the genomic DNA damage and mitochondrial dysfunction induced by administration of TiO 2 NPs or acrylamide alone by significantly increasing the generation of ROS and the expression of p53 and β-catenin genes.Therefore, further studies are recommended to understand the biological and toxic effects of coadministration of TiO 2 NPs with acrylamide.

Figure 1 .
Figure 1.Representative photo for the seen electrophoresed pattern of renal genomic DNA extracted from negative control mice (C) and mice given orally acrylamide (A) and TiO 2 NPs (T) separately or simultaneously together (AT).M: Marker.Three samples were analyzed per each group.

Figure 2 .Figure 3 .
Figure 2. Representative photos for the scored Comet nuclei in the renal cells of the negative control mice and mice given orally acrylamide and TiO 2 NPs separately or simultaneously together.

Figure 4 .
Figure 4. Variation in the copy of mitochondrial DNA in the renal cells of the negative control mice and mice given orally acrylamide and TiO 2 NPs separately or simultaneously together.Six mice were analyzed for each group.Results are expressed as mean ± SD and analyzed using one-way analysis of variance followed by Duncan's test to test the similarity between the control and three treated groups.Means with different letters indicates statistical significant difference between the compared groups at a significant level p < 0.001.

Figure 5 .
Figure 5. Representative photos for the ROS level generated within the renal cells of the negative control mice and mice given orally acrylamide and TiO 2 NPs separately or simultaneously together.