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Genoprotective effects of Chaga mushroom (Inonotus obliquus) polysaccharides in
UVB-exposed embryonic zebrafish (Danio rerio) through coordinated expression of
DNA repair genes
Authors:
Jehane Ibrahim Eid*1, Swabhiman Mohanty2, Biswadeep Das**2
1Department of Zoology, Cairo University, Egypt 12613.
2School of Biotechnology, KIIT University, Bhubaneswar 751024, India
*Correspondence:
Department of Zoology, Cairo University, Egypt 12613
Ph: +201011439568
jehaneeid@sci.cu.edu.eg
**Co-correspondence
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Abstract
Background: Chaga mushroom (Inonotus obliquus) is one of the most promising antioxidants
with incredible health-promoting effects. Chaga polysaccharides (IOP) have been reported to
enhance immune response and alleviate oxidative stress during development. However, the
effects of IOP on the genotoxicity in model organisms are yet to be clarified. Methods:
Zebrafish embryos (12 hours post fertilization, hpf) were exposed to transient UVB (12 J/m2/s,
310 nm) for 10 secs using a UV hybridisation chamber, followed by IOP treatment (2.5 mg/mL)
at 24 hpf for up to 7 days post fertilization (dpf). The genotoxic effects were assessed using
acridine orange staining, alkaline comet assay, and qRT-PCR for screening DNA repair genes.
Results. We found significant reduction in DNA damage and amelioration of the deformed
structures in the IOP-treated zebrafish exposed to UVB (p < 0.05) at 5 dpf and thereafter. In
addition, the relative mRNA expressions of XRCC-5, XRCC-6, RAD51, P53, and GADD45
were significantly upregulated in the IOP-treated UVB-exposed zebrafish. Pathway analysis
demonstrated coordinated regulation of DNA repair genes, suggesting collective response
during UVB exposure. Conclusions. Overall, IOP treatment ameliorated the genotoxic effects
in UVB-exposed zebrafish embryos, which eventually assisted in normal development. The
study suggested the efficacy of Chaga mushroom polysaccharides in mitigating UV-induced
DNA damage.
Keywords: Chaga polysaccharides; Zebrafish; UVB light; DNA-repair genes; Genotoxicity
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Introduction
The National Cancer Institute (NCI), United States has recently intensified its focus on natural
products such as plants, marine organisms, and certain microorganisms for use in drug and
biosimilar discovery [1] In this regard, Chaga (Inonotus obliquus) mushroom is one such
biologically relevant product, which has gained increasing attention worldwide because of its
high nutritional and medicinal values, such as antioxidant, anti-inflammatory, immune booster,
antidiabetic, antiviral and several others [2–5]. In particular, Inonotus obliquus polysaccharide
(IOP) represents the most active ingredient of Chaga mushroom, known for treating and
preventing various diseases such as tumors, metabolic disorders, and many chronic diseases in
folk medicine. Such powerful beneficial effects are largely due to the immunomodulatory, anti-
inflammatory and anti-oxidative properties of Inonotus obliquus polysaccharide. Chaga tea is
popular and consumed in many countries for several remedies and health promoting effects
[5,6].
Because DNA mutations are considered as the prime reason for developing cancer and genetic
disorders, it is important to maintain the integrity of DNA in the cell for proper functioning.
Large-scale DNA damage can be deleterious to the cell and are caused mainly by alkylating
agents (transform a functional base into a mutagenic one), hydrolytic deamination (lead to base
alterations), dyes (ethidium bromide), reactive oxygen species (intrinsic) and ionizing
radiations (ultraviolet light, UV) that render maximum DNA damage [7–9]. UV light
comprises three broad categories based on their wavelength: UVA (320–400 nm), UVB (290–
320 nm), and UVC (240–290 nm). UVC is the most harmful to living organism due to its
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exorbitantly high frequency and is mostly absorbed by the stratospheric ozone [10,11]. A part
of UVB radiation is also absorbed in the stratosphere, though majority of UVB radiation
reaches the earth’s surface, and is responsible for serious health comes, such as skin cancer by
inducing DNA damage. UVB results in two major types of mutagenic DNA lesions:
cyclobutane–pyrimidine dimers, and pyrimidine adducts 6–4 photoproducts, in addition to
their Dewar valence isomers [11].
Although there are in vivo repair mechanisms to manage and correct DNA mutations and
maintain is integrity such as photo-reactivation, base/nucleotide excision repair, DNA
replication related methyl mismatch repair, in addition to large scale and nonspecific repair
systems (SOS response and apoptosis), the degree of repair depends on the extent of damage
and the status of the repair systems of the human body [10]. Therefore, intake of natural anti-
oxidants and immune boosters are highly recommended for maintaining a healthy state of the
DNA and eventually the whole body [4,6]. In this regard, Chaga mushroom is a promising
supplement whose benefits have been assessed in several metabolic and chronic disorders.
However, it is imperative to understand the effect of Chaga mushroom on the genotoxic profile
in vivo to understand if it has any effect on DNA damage reversal.
Recently, many international toxicity researches have utilized zebrafish (Danio rerio) as an in
vivo model organism because it possesses several measurable indicators in ecotoxicology,
such as small size, high fecundity, well-characterized embryonic ontogenesis, transparent
embryos, and rapid development [12]. Besides, zebrafish has more than 70% genome
similarity with the humans that render it to effectively model any human disease with high
phenotypic similarity and convenience [13]. Zebrafish is an ideal model for assessing DNA
damage and its counter mechanisms because zebrafish genomic DNA contains DNA repair
genes orthologues that participate in DNA repair mechanisms [12,13], as well as zebrafish
DNA is amenable to genetic manipulation using morpholinos, shRNA, or Crisprs to assesses
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the role of genes during DNA repair response [14]. Zebrafish has been used as in vivo model
organism for testing a plethora of exogenous agents comprising drugs, nanoparticles, organic
and inorganic compounds, besides being exposed to different stress environments. UV-
induced zebrafish models have also been developed to study the expression of DNA repair
genes and tumor suppressor genes regulation [15–17]. Furthermore, transparent zebrafish
casper mutants have been consistently used for tumour-related and toxicological studies for
assessing the phenotypic manifestations during development [18]. Such avenues will be
worthy to explore the specific DNA repair genes that might play additional roles in
embryological development. The objective of this study was to assess the molecular
mechanism of IOP in UVB-exposed zebrafish during early development.
Results
Development analysis upon IOP exposure in zebrafish embryos
Embryo development varied across the three groups; the UVB-exposed zebrafish showed
severe structural aberrations, such as yolk sac edema, loss of vital structures, and slow
development compared to control and IOP treated UVB-exposed groups (Fig 1). Average
mortality was high in the UVB-exposed group (80%) during the course of development
compared to IOP-treated (15%) and control (10%). Interestingly, IOP-treated UVB exposed
group showed structural deformations like pericardial edema and spinal curvature in the early
days of development (3 dpf), which were significantly ameliorated during late development (³
5 dpf). The embryos remained healthy without showing any signs of mortality at the later stages
of development in the IOP-treated group. Most vital statistics such as development time,
hatching rate and heart rate was similar in the control and IOP-treated UVB exposed group,
which indicated that IOP promoted the development of zebrafish embryos as they would have
developed under normal conditions.
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Fig. 1. Bright field images of morphological analysis of zebrafish embryos (1 dpf to 4 dpf) in the
three different zebrafish groups (control, UVB-exposed, IOP treated UVB-exposed).
Acridine orange staining
The level of DNA damage was assessed by the interaction of DNA binding AO dye, which
was observed to be significantly uptaken by the UVB-exposed zebrafish embryos (5 dpf)
compared to control and IOP-treated UVB-exposed groups. The uptake was visualized as
intense green fluorescence (Fig 2A), which was graphically represented as a histogram (Fig
2B). Moreover, IOP-treated UVB-exposed group showed remarkably low green fluorescence
demonstrating less DNA interactions with the dye. The results showed that IOP assisted in
maintaining the integrity of the DNA, owing to less penetration of AO into the nucleus, which
was similar to that in control embryos. These results indicated significantly increased apoptosis
(p < 0.05) in the UV-exposed group without IOP exposure compared to IOP-treated UVB
exposed group, which implied that IOP conferred protection against DNA damage.
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Fig. 2. A) Acridine orange staining of zebrafish embryos (5 dpf) for the three zebrafish (groups
control, UVB-exposed, IOP treated UVB-exposed), showing intensive green fluorescence as
significant uptake of AO dye due to cellular-compartment and DNA damage. B) shows the histogram
generated by Image J analysis showing the percentage of apoptotic cells in the three groups.
Genotoxicity assessment using alkaline comet assay
Genotoxic assessment using the alkaline comet assay by analyzing the tail intensity in the three
zebrafish groups revealed varied genotoxic effects, exhibiting distinct comet heads and tail
regions (Fig 3). On 5 dpf, the tail intensity was significantly increased in the UVB-exposed
zebrafish embryos by almost three-fold and four-fold compared to IOP-induced UVB exposed
and control groups, respectively (Fig 4). On 7dpf, the tail intensity was further significantly
increased in the UVB-exposed zebrafish embryos (86.6 %) by almost four-fold compared to
both IOP-induced UVB exposed (32.1%) and control groups (26.1%), respectively (Fig 4).
These results indicated that IOP acted as an anti-genotoxic compound and assisted in the
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amelioration of genotoxicity caused due to UVB damage, thereby allowing the normal
development of the zebrafish embryos.
Fig. 3. Alkaline comet assay showing distinct comet head and tail after ethidium bromide staining and
fluorescent microscopy in the three zebrafish (groups control, UVB-exposed, IOP treated UVB-
exposed) at 5dpf.
Fig. 4. Comet assay histogram at 5 dpf and 7 pdf generated by analyzing the tail intensity (DNA
fragmentation) using Image J in the three zebrafish (groups control, UVB-exposed, IOP treated UVB-
exposed) at 5dpf.
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Gene expression analysis upon IOP exposure in zebrafish embryos
qRT-PCR analysis showed that the relative expression of DNA repair genes XRCC5, XRCC6,
RAD51, GADD45, P53, and BAX was significantly up-regulated in the IOP-treated UVB-
induced zebrafish compared to UVB-exposed and control groups (p < 0.05). The mean
expression of XRCC5, XRCC6, RAD51, GADD45, and P53 in the IOP-treated UV-exposed
zebrafish was significantly increased by 1.87, 1.73, 2.41, 2.55, and 1.65 times in comparison
to control fish. In the UVB-exposed group, the mean expression of all the above-mentioned
genes, except BAX was lower than or equal to control zebrafish (Fig. 5), indicating that UVB
exposure induced substantial damage to the DNA repair system. The increased expression of
BAX gene in the UVB-exposed group could attribute to the increased apoptosis, leading to
rapid cell death. These findings demonstrated that IOP enhanced the expression of DNA repair
genes upon UVB exposure, thereby promoting DNA protective effects and eventually assisting
in the development of zebrafish.
Fig. 5. qRT-PCR bar graphs showing the fold change in the gene expression in UVB-exposed and
IOP-treated UVB exposed groups compared to that of control.
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Discussion
Mushrooms possessing medicinal values, such as Ganoderma, Chaga, and many others are
currently being explored significantly because of their tremendous benefits without inducing
any undesirable effects, besides possessing beneficial attributes such as anti-inflammatory,
anti-oxidant, and anti-tumor [12–17]. Given the wide benefits of Chaga mushroom, the effects
of Chaga mushroom on the genotoxicity profile of an organism will be intriguing to explore.
In this study, we found that hot water extracted-Chaga polysaccharides significantly mitigated
genotoxicity by enhancing the expression of DNA repair genes in UVB-induced zebrafish
embryos. The major polysaccharide found in Chaga mushroom was b-glucan, a polymer of b-
D glucose, which is documented to possess several health benefits [18]. b-glucan has been
reported to have anti-diabetic, anti-proliferative properties, and anti-tumorigenic properties
[19]. Such beneficial effects could be attributed to the ability of b-glucan to scavenge free
radicals generated due to endogenous or exogenous agents, such as ionizing UVB radiation,
thereby assisting the cells to repair DNA [20]. Several studies have reported that b-glucan
readily facilitates the reduction of both simple and complex chromosomal aberrations, and
confer protection to the DNA against single-strand and double-strand breaks [21], in addition
to enhancing the DNA repair system [22]. b-glucan thus confers protection to DNA through
its antioxidant activity and by enhancing the DNA repair system, besides being water soluble
that allows it to be readily uptaken by the cell without any perturbations in the cellular
processes. Recently, several bioactive polysaccharides isolated from natural sources have been
given much attention in clinical pharmacology [23]. Such polysaccharides can be modified by
chemical methods to improve the antitumor activity of polysaccharides and their clinical
qualities, such as water solubility. Therefore, IOP could be regarded as a potential adjunct
along with conventional chemotherapy for several ailments and can have wide application in
health clinics [23,24].
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The present study revealed that continuous exposure to IOP reversed the structural
deformations that were induced due to UVB exposure in zebrafish embryos. Severe organ
damage, and loss of vital structures that occurred in the UVB-exposed zebrafish were
significantly ameliorated in the IOP-treated UVB induced groups. This suggested that IOP
conferred protection to the zebrafish embryos against exogenous agents. During early
development, DNA damage results in severe structural deformations, which was evidently seen
in the UVB-exposed zebrafish. The reversal of such damage and eventual normal development
of the zebrafish embryos could be solely attributed to continuous exposure to IOP that can be
further explained due to the presence of high amount of beta-glucans [24–27]. Moreover, we
observed the reversal of DNA damage by comet assay; and DNA fragmentation, which was
measured as the comet tail intensity was significantly reduced in the IOP-treated UVB-exposed
group. This suggested that IOP was anti-genotoxic and conferred protection to DNA by
maintaining its integrity. Furthermore, IOP also reduced the number of apoptotic cells in the
zebrafish embryos as demonstrated by acridine orange staining, whereas the apoptosis was
significantly increased in the UVB-exposed zebrafish. This could also be correlated with
increased expression of the Bcl2 associated X gene, Bax that promoted apoptosis in the UVB-
exposed zebrafish. However, the Bax gene was downregulated in IOP-treated group compared
to UVB exposed group, which suggested that IOP was inhibited apoptosis and increased
cellular longevity.
Continuous exposure to IOP significantly reduced the amount of damaged DNA and thus, the
embryos developed normally [28]. Such DNA protective effects could be explained by the
enhanced expression of several DNA repair genes. In this study, we observed significantly
elevated expression of xrcc5, xrcc6, rad51, and gadd45 genes in the IOP-treated UVB-exposed
group compared to UVB-exposed group. Xrcc5 and xrcc6 encode Ku70 and Ku80 DNA repair
proteins that function collectively during non-homologous end joining during DNA damage.
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Overexpression of these genes have also been reported in several disorders involving DNA
damage to correct the DNA [29,30]. Rad51 encodes a protective enzyme that facilitates the
repair of double strand DNA breaks by catalyzing strand transfer between a broken sequence
and its undamaged homologue to allow re-synthesis of the damaged region [31,32]. It has a
major role in homologous recombination, and assists in recombination repair during extensive
DNA damage [32]. The substantial increase in the expression of these genes indicated prompt
recruitment of DNA repair proteins and processing of damaged DNA for the correction of
damaged DNA, in addition to maintaining its integrity. We also found increased expression of
GADD45 gene, which encodes the growth arrest and DNA damage proteins that have a critical
role during embryogenesis by regulating differentiation (by inducing the zygotic gene
expression) along with protecting DNA from spontaneous damage, mainly by efficient
recognition and repair of spontaneous DNA damages by ten-eleven translocation
methylcytosine dioxygenase 1 (TET)-mediated DNA demethylation [33–36]. This gene was
the most highly expressed in the IOP-treated UVB-exposed group, which suggested that IOP
greatly enhanced the expression of GADD45 proteins, which eventually assisted the embryos
in normal development. Network analysis showed that all the DNA repair genes encoding
proteins acted in a coordinate manner during cellular signaling against DNA damage.
Furthermore, tp53 acted as the central player regulating the coordinate response of all the genes
involved in DNA repair (Fig 7 6). These findings demonstrated that all the genes need to act
in a coordinate manner during DNA repair under the control of tp53.
In vivo developmental analyses showed that continuous exposure to IOP extract did not exhibit
any significant developmental deformities in embryonic zebrafish, which developed in line
with control embryos. This finding suggested that Chaga mushroom aided in normal
development of the zebrafish and reduced DNA damage in the developing embryos.
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Therefore, Chaga mushroom could be a potent natural therapeutic for ameliorating disorders
linked to DNA damage.
Fig. 6. Network analysis using String software demonstrating the coordinated association of the DNA
repair genes involved during UV induced DNA repair.
Methods
Extraction of Chaga mushroom polysaccharides and GC-MSMS analysis
Extraction of Chaga mushroom polysaccharides (IOP) was performed using hot water-
ethanolic extraction method according to Eid et al., 2020 [12]. Briefly, 10 g of Siberian grade
Chaga chunks were dried, ground to fine powder and dissolved in 150 mL followed by
refluxing at 70 °C, and vacuum dried and concentrated using 3 volumes of 95% ethanol. The
solution was then centrifuged at 5000 rpm for 10 min, and the supernatant was dried and treated
with Sevag reagent (chloroform:butanol in the ratio 4:1) to remove the proteins. The solution
was then oven-dried and mixed with distilled water (5 g in 250 mL w/v), followed by
chromatographic analysis and spectrophotometric confirmation [12].
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GC-MS analysis was performed using trimethylsilylation reagent, TMS (N-
Trimethylsilylimidazole) as per previous protocol [12]. The area normalization method was
employed to estimate the molar ratio of the monosaccharides present in the polysaccharide
extract and the corresponding histogram was plotted accordingly (Fig 7)
Fig. 7. GC-MS chromatogram depicting the retention time peaks for different monomers of hot
water-extracted Chaga mushroom polysaccharides.
Zebrafish genotoxic experiments
Zebrafish rearing and exposure
All the methods were approved and performed in accordance with the relevant guidelines and
regulations of the Institutional Ethical Committee (IEC) of KIIT University. Zebrafish embryos
were obtained from mating adult wild type zebrafish using a 2:1 female: male ratio, and the
fertilized embryos were reared in embryo water (0.06% sea salt). Culture density of the
fertilized embryos per mating was averaged to 100. The fertilized embryos of 6-hour post-
fertilization (hpf) were observed under microscope and pipetted in 6-well microplates for the
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exposure experiments. Zebrafish embryos at 12 hpf (n = 120), in the shield stage, were divided
into three groups of 40 embryos each: Control, UVB-exposed, and IOP treated UVB-exposed
groups. For UVB exposure and IOP treatment, the embryos (n = 80, 12 hpf) were exposed to
UVB dose of 12 J/m2/s using a UV cross linker hybridization chamber that emitted a range of
310–312 nm UVB light for 10 secs. Then, the embryos were reared in the embryo water at 28
°C. IOP treatment (2.5 mg/mL) was applied to half of the UVB-exposed embryos (n = 40, 24
hpf) that were labelled IOP-treated-UVB exposed group. The IOP concentration was selected
based on our previous study that showed that the adopted dose did not affect the normal
development of zebrafish embryos [12]. The embryos were then grown for upto 7 days in the
same medium and the fetal embryo toxicity and genotoxicity was assessed. The control group
embryos were reared without any treatment in the embryo water for the same duration. The
assays were performed in triplicates. Morphological deformities based on phenotypic
observations were recorded microscopically during embryonic development. The three
zebrafish embryo groups were further assessed for genotoxicity assays using acridine orange
staining, alkaline comet assay and qRT-PCR.
Acridine orange staining
Fluorescent acridine orange dye that binds to DNA and emits green fluorescence was used to
stain the all the three groups at 5 dpf for qualitative assessment of the DNA damage and
apoptosis. Briefly, control, UVB-exposed and IOP-treated (2.5 mg/mL)-UVB-exposed
zebrafish embryos of 5 dpf (n=10/group) were stained with 5 μg/mL acridine orange for 20
min, followed by washing the excess stain with embryo water. The corresponding images were
captured in the GFP (green channel) of inverted fluorescent microscope (EVOS, Thermo
Fischer Scientific, USA) to assess DNA damage (green dots represented apoptotic cells). The
assays were performed in triplicates.
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Alkaline comet assay for DNA damage analysis
DNA fragmentation is considered as one of the endpoints of genotoxicity. Genotoxicity was
assessed by evaluating the DNA breaks using the alkaline comet assay and assessing the
amount of DNA in the tail region (tail intensity). For this, control zebrafish, UVB-exposed
zebrafish, and zebrafish treated with IOP (2.5 mg/mL) after UVB exposure were homogenized
thoroughly in a micropestle in two independent experiments at 5 dpf and 7 dpf (n = 5/group).
Then, 1 mL Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS was added, and
centrifuged for 5 min at 700 g at 15 °C. The pellet was retrieved and diluted in 1X PBS buffer
pH 7.4 to form the cell suspension. Cell homogenate (10 μL) was mixed with 0.8% low melting
agarose and were dropped onto a glass slide precoated with normal melting agarose (1.5%),
followed by covering with a cover slip. The slides were kept at 4°C for 15 minutes for drying,
followed by incubation in a cold lysis solution containing 100 mM EDTA, 2.5M sodium lauryl
sulphate, 1% Triton X-100, and 10% DMSO (pH 13.0) in the dark at 4°C for overnight.
Following incubation, the slides were dipped in a neutralizing solution containing 400 mM
Tris at pH 7.4 for 30 minutes. Then, for assessing the unwinding of the DNA, electrophoresis
was performed in a cold alkaline buffer (12 g/L NaOH and 0.37 g/L EDTA, pH 11) at 25V for
30 minutes. Post electrophoresis, the slides were washed in distilled water, and fixed with 70%
ethanol for 5 minutes. Finally, the slides were stained with ethidium bromide (5 mg/mL) for 5
minutes and analyzed by fluorescence microscopy at 4X and 40 X magnifications
(fluorescence at emission (500 nm) and excitation (530 nm) with an inbuilt image system
(EVOS M5000, Thermo Fisher, USA). The images were analyzed using Image J analysis
software for assessing the tail intensity in the three groups. All the assays were conducted in
triplicates.
Gene expression analysis using qRT-PCR
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Relative expression of DNA repair genes, XRCC5, XRCC6, RAD51, GADD45, BAX, and P53
were assessed using Syber Green qRT-PCR. Beta actin was used as the reference gene. Briefly,
total RNA was extracted from 5 dpf zebrafish larvae from the 3 groups [control, UVB-exposed
and IOP-treated (2.5 mg/mL) UVB exposed] by using Trizol and converted to cDNA following
standard protocol. cDNA was amplified using a Sybr-green based qPCR master mix and
specific primers (Supplementary table 1). The PCR thermal profile consisted of an initial
denaturation of at 95 °C for 2 mins, followed by 33 cycles of 30 seconds at 95 °C, 30 seconds
at 55-62°C (varied as per primers), and 30 seconds at 72°C, and final extension at 72°C for 3
mins. Qauntification and data analysis of the respective genes were assessed using the CFX
manager of real-time PCR (Bio-Rad). The Ct values were calculated for each gene and were
compared with the reference gene beta-actin, followed by estimation of DDCt and fold change
(2-DDCt) to assess the relative gene expression among the different groups.
Statistical analysis
The data obtained on the developmental characteristics and alkaline comet assay attribute, tail
intensity (%) were reported as the mean ± SD for all experiments independently. The relative
gene expression for all the DNA repair genes was estimated with reference to the beta-actin
gene in all the three groups. The relative gene expression and the tail intensity of UV exposed
zebrafish and IOP-treated UVB exposed group were compared with respect to control zebrafish
larvae using one-way analysis of variance (ANOVA), followed by multiple comparison tests
in GraphPad Prism software. A p < 0.05 was considered statistically significant for assessing
the association of the variables in context to genotoxicity.
Ethical statement All the methods were approved and carried out in accordance with relevant
guidelines and regulations of the Institutional Ethical Committee (IEC) of KIIT University.
.CC-BY 4.0 International license(which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprintthis version posted June 19, 2020. . https://doi.org/10.1101/2020.06.19.161182doi: bioRxiv preprint
Author Contributions JIE conceived the experiment design, performed experiments and
data analysis and was involved in the manuscript drafting. BD conceived performed and
designed the experiments and also involved in writing and editing the manuscript. Both
authors reviewed and discussed their views on the manuscript.
Competing Interests: The authors declare no competing interests.
Funding: No funding support was received for this study.
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