An evaluation of the effect of graphene oxide on Saccharomyces cerevisiae

1 Department of Biology, College of Science, Engineering and Technology, Texas Southern University, USA. 2 Center for Biological and Environmental Science Research, College of Science Engineering and Technology, Texas Southern University, USA. 3 Department of Environmental and Interdisciplinary Science, College of Science Engineering and Technology (SURP), Texas Southern University, USA. 4 Department of Interdisciplinary Health Sciences, College of Allied Health Sciences, Augusta University, Augusta, Georgia, USA. 5 Department of Public Health, School of Basic Medical Sciences, Kwara State University, Kwara State, Nigeria. 6 Department of Science Laboratory Technology, School of Technology, Benue State Polytechnic, Ugbokolo, Nigeria. 7 Department of Chemistry, College of Science Engineering and Technology, Texas Southern University, USA.


INTRODUCTION
Two-dimensional materials such as graphene oxide (GO) are currently a subject of investigation due to their possession of properties that are fundamental in the production of innovative devices. Extensive use of GO in *Corresponding author. E-mail: ayodotun.sodipe@tsu.edu. Tel: 713-313-7108.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License drug delivery and cancer therapy has been reported. Loading of more amount of anticancer drugs on GO, excellent drug release and enhanced photo thermal and photo acoustic effect, efficient and accelerated killing of tumor cells, inhibition of tumor growth due to sequential drug release, etc. have been achieved in cancer treatment. Hydrophobic drugs can be delivered to tumor cells via GO (Santhosh et al., 2017;Li et al., 2012). It has also been used in cancer therapy as a photo thermal agent with encouraging therapeutic outcomes due to its high, intrinsic near-infrared (NIR) absorbance for cancer treatment (Li et al., 2012). GO has a wide scope of potential as a raw material for several applications. Unique properties such as excellent aqueous processability, amphiphilicity, surface enhanced Raman scattering (SERS), fluorescence quenching ability, biocompatibility and solubility make GO a candidate for biological applications (Chung et al., 2013;Loh et al., 2010;Liu et al., 2008).
The oxide has antibacterial and anti-tuberculosis potential and showed glucose sensitivity, mutagenesis, scaffold generation, aromatic hydrocarbon extraction from food samples, and triggered growth in plants when treated with it (Santhosh et al., 2017).
It displays interesting optical characteristics such as effectively quenching the fluorescence of other fluorescent dyes (Liu et al., 2008) and fluorescing over a wide range of wavelength from near-infrared to ultraviolet (Loh et al., 2010). These make it a suitable material in fabricating fluorescence resonance energy transfer (FRET) sensors, in which an excitation on another molecule transfers nonradiatively to the oxide. It is used in immunosensing as an energy donor or acceptor (Lim et al., 2015).
Due to its excellent biocompatibility, flexible chemical modifications, ready cellular uptake and unique optical properties, GO has been explored for biological imaging. The protection of DNA from enzymatic digestion by the self-assembly of ssDNA on GO sheets was reported by Tang et al. (2010).
In vivo tumor uptake and photothermal therapy with PEGylated GO using xenograft tumor mouse models study was carried out for the first time by Yang et al. (2010) and it was observed that there is very high tumor uptake of the PEG-modified GO due to highly efficient tumor passive targeting of GO caused by enhanced permeability and retention (EPR) effect.
Graphene derivatives, including pristine graphene, GO, doped and chemically reduced GO (rGO) have been intensively studied for their widespread applications in biosensing and detection of biomolecules such as oligonucleotide, thrombin, adenosine triphosphate (ATP), amino acid, and dopamine graphene (Chang et al., 2010;Dong et al., 2010;Tang et al., 2009Tang et al., , 2011Wang et al., 2009Wang et al., , 2010a. Several types of GO-based biosensors have been built by making use of super-efficient fluorescence quenching ability of grapheme; some novel fluorescence resonance energy transfer (FRET) based biosensors have been developed (Chang et al., 2010;Wang et al., 2010a). Field-effect transistor (FET) type GO-based biosensors are based on the unique electronic property of graphene (He et al., 2010). Controllable self-assembling of graphene-biomolecules allows for the building of highly ultrasensitive biosensors for detection of DNA and other molecules (Tang et al., 2011;Zhang et al., 2011;Wang et al., 2011b;Zeng et al., 2010). As a matrix for detection of molecules, graphene-based nanoplatform for matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported. GObased novel biosensors via electrochemical principle have been built by using its huge surface area, good electrical conductivity, and excellent capability of loading various biomolecules via chemical or physical interactions (Wang, 2011a;Wan et al., 2011). Macroscopic freestanding GO and rGO paper has been prepared from suspension by vacuum filtration technique, and these papers exhibit strong antibacterial effect . Studies on the toxicity and biocompatibility of GO have shown potential accumulation of the oxide in the lung, liver and spleen resulting in acute liver injury and chronic inflammation of the lung, liver and spleen of mice (Ou et al., 2016;Jasim et al., 2015;Wen et al., 2015;Guo and Mei, 2014;Gurunathan and Kim, 2016).
Saccharomyces cerevisiae, commonly called brewers or baker's yeast has as many uses as GO. It is a source of B vitamins and minerals such as chromium and selenium which aid the metabolism of fats, proteins and carbohydrates (Yu-hui et al., 2016). It is widely used as a veterinary probiotic and have exhibited probiotic potential in humans with the strain Saccharomyces boulardii licensed for use as a human probiotic (Suarez and Guevara, 2018;Fakruddin et al., 2017;Moslehi-Jenabian et al., 2017;Romanio et al., 2017;Palma et al., 2015;Abu-Elala et al., 2013).
S. cerevisiae is used in the manufacture of nutritional yeast which is a rich source of folate used to enrich pastries. It aids the absorption of electrolytes in the body by degrading complexed phytates and has the ability to bind and degrade mycotoxins through fermentation (Shehu et al., 2016). Studies have shown that it reduces oxidative stress and maintains integrity of the mucosal barrier in mice and may be beneficial in gastrointestinal mucositis, a serious side effect of cancer therapy (Jumanah et al., 2017;Bastos et al., 2016;Ognjenovic et al., 2013). It is used in the formulation of cosmetics (Gaspar et al., 2008).
More studies on the standardization of GO and its biofunctionalization to clearly understand how cells respond to it are a requirement for the development of therapeutics. Toxicity and biocompatibility are also usually a concern for biomedical and other areas of application. Therefore, interaction of GO with living systems should be of relevance and stimulated the interest in this study. The versatility of the yeast, S. cerevisiae and the fact that GO is known to elicit toxicity calls for interest on the outcome of their interaction. The objective was to investigate the effect of GO on the yeast, S. cerevisiae.

Materials
GO was obtained from Angstron Materials (Dayton, Ohio, USA). Pierce® fluorescein isothiocyanate (FITC) Antibody Labeling Kit and anti-RAD17 were obtained from Thermo Scientific (Waltham, Massachusetts , USA). Saccharomyces cerevisiae cell line was obtained from ATCC. Nikon Eclipse Ti inverted microscope

Reagents
GO Solution: 0.5% aqueous solution of the oxide was prepared and stored as stock solution.
Step down dilution of the stock solution was effected to obtain four aliquots of 500, 750, 1000 and 1250 µg/ml concentrations.

S. cerevisiae cell culture
S. cerevisiae cell line was cultured in an aqueous Yeast Peptone Dextrose (YPD) broth. The liquid media was autoclaved for 60 min to make it sterile. A colony of S. cerevisiae was introduced into the media and incubated at room temperature (25°C) for 72 h.

Labeling antibodies
A buffer solution composed of 0.67 M borate buffer and diethyl pyrocarbonate (DEPC) treated water with 1 mg of RAD17 was incubated and passed through purification resin in spin columns pre-treated with phosphate buffered solution (PBS) and stored in the dark at 4°C. Antibody RAD17 was labeled with fluorescent dye FITC to aid in the detection of any form of phenotypic expression in the DNA of the yeast. If DNA damage occurred, FITC labeled antibody RAD17 would fluoresce under the FITC filter in the form of foci.

Exposure to graphene oxide
Cultured Saccharomyces cell line was plated in three different well microplates labelled A, B and C. To determine the effect of the concentration of GO on the cell as a function of time, microplate wells were treated with 0, 500, 750, 1000 and 1250 µg/ml of GO and incubated for 24, 48, and 72 h. The wells were labeled A0, A1, A2, A3, A4; B0, B1, B2, B3; B4; C0, C1, C2, C3, C4 for 24, 48 and 72 h incubation periods respectively (Table 1). Three replicates of microplate wells were used to determine the effect of the oxide on the cell line.

Microscopy
At the end of each incubation period, the media was aspirated after centrifuge; microscope slides were stained with samples and fixated by flame and washed in a 10% Triton X solution for 10 min. They were subsequently washed in PBS three times and viewed under microscope.
The procedure was replicated twice independently and viewed using a Nikon Eclipse Ti inverted microscope with and without FITC filter after exposure of the cell line to GO. This filter makes it possible to visualize any DNA damage that might have occurred (in the form of fluorescent foci) to Saccharomyces from exposure to GO.

Ethical considerations
All facets of the research were approved by Texas Southern and Augusta Universities, USA Ethical review Boards Table 2 shows the microscopic observation of the cell line following exposure to varying concentrations of GO. Samples were observed under a monochrome image device and viewed for any appearance of foci in the sample with an FITC filter. No foci were detected. The yeast shape and cell wall integrity were maintained despite the concentration of GO and length of incubation. The control cells, samples A 0 , B 0 , and C 0 looked similar as shown in Figure 1. The cells were oval-shaped with a clear outer cell wall and intact nucleus. However, looking at the result obtained after 72 h exposure (Figure 2), there is a slight morphological deformity of some of the

Sample
Observation A0 Cells are cylindrical with normal cell wall A1 Cells shape and size intact A2 Cells showed no change in shape and size A3 No observable change in the shape and size of cells A4 Cells remained cylindrical B0 Cells looked the same, cylindrical B1 Shape of cells remained the same B2 No change was observed in cells B3 Cells showed no sign of change B4 Cell structure remained the same C0 No observable change in cells C1 No change was observed in the shape and size of the cells C2 Cells looked the same C3 No change was observed in cells C4 Cells structure remained unchanged  affected by GO, eliminating any chance of toxic effects. Although RAD17 is a protein that is expressed in response to DNA damage in Saccharomyces, it may not be expressed as well as its other counterparts. Only the antibody RAD17 was labeled and this may have made it difficult to see any form of foci or reactive oxygen species that may have been induced in reaction to GO.
Graphene oxide had no perceived interaction with S. cerevisiae, This may have been due to the thick cell wall of the yeast which may have prevented the cell from undergoing any changes. In addition, the concentration of GO used, 500-1250 μg/ml may have been too weak to cause the expected toxic or adverse effect. GO concentrations used by Zhu et al. (2017) and Yu et al. (2017) were in the mg/ml range.
The protein RAD17 is one of the proteins expressed in response to DNA damage in the yeast S. cerevisiae (Paulovich et al., 1998). The antibody RAD17 was labeled with FITC, making any DNA damage visible under a FITC filter. There were no demonstrable foci seen with FITC, thus it can be inferred that RAD17 may not express well in response to reactive oxygen species.

Conclusion
Yeast showed no apparent effect on the cell after exposure to the various concentrations of GO used. Higher concentration in the mg/ml level may elicit toxicity.
To confirm the findings in this study other DNA damage sensors are to be investigated and GO concentration is to be increased to the mg/ml range.