
==== Front
J Genet Eng Biotechnol
J Genet Eng Biotechnol
Journal of Genetic Engineering & Biotechnology
1687-157X
2090-5920
Academy of Scientific Research and Technology, Egypt

S1687-157X(24)00128-8
10.1016/j.jgeb.2024.100425
100425
Full Length Article
Opuntia ficus indica cladode extract inhibit DNA double-strand breaks and locally multiply damaged sites induced by gamma radiation
Kouass Sahbani Saloua saloua.sahbani@usherbrooke.ca
abc⁎
a Faculty of Applied Medical Science Al Ula branch, Department of Nursing, Taibah University, Kingdom of Saudi Arabia
b Department of Nuclear Medicine and Radiobiology, University of Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada
c Laboratory of Biochemistry and Molecular Biology, Faculty of Sciences of Bizerte, Carthage University, Tunisia
⁎ Address: Laboratory of Biochemistry and Molecular Biology, Faculty of Sciences of Bizerte, Carthage University, Tunisia. saloua.sahbani@usherbrooke.ca
07 9 2024
12 2024
07 9 2024
22 4 1004254 5 2024
29 8 2024
1 9 2024
© 2024 The Author. Published by Elsevier Inc. on behalf of Academy of Scientific Research and Technology.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Graphical abstract

Highlights

• Ionizing radiation can induce the formation of locally multiply damaged sites (LMDS) which considered very toxic.

• The Opuntia ficus indica cladode extract (CE) is a potent radioprotector; it efficiently inhibits the DNA DSBs, and LMDS.

• The Opuntia ficus indica cladode extract (CE) can be a good solution to protect normal cells during radiotherapy treatment.

It is beyond doubt that radiotherapy is extremely effective in treating a wide variety of cancers. The sensitivity of the surrounding normal tissues limits the amount of radiation administered to the tumor. There is an urgent need to develop a treatment that combines pharmacological treatment with ionizing radiation (IR) specifically designed to specifically target cancer cells while protecting the surrounding normal tissue, resulting in an increase in the efficacy of the cancer treatment. IR could cause many types of DNA lesions. Double-strand breaks (DSBs) and locally multiple damaged sites (LMDS) are the main radiotoxic damages. Recently, the identification of new antioxidants from natural sources has attracted the attention of scientists. In this context, the present study aims to determine if the Opuntia ficus indica cladode extract (CE) can be used as a radioprotector.

Materials and methods

The DNA treated by 137Cs γ-radiation (25–700 Gy) in the absence or presence of cactus cladode extract (CCE) was added to the E. coli base excision repair. The amounts of both DNA damages were calculated using the electrophoretic method.

Results

The irradiation of DNA in the presence of CCE induced a dramatic decrease of the yields of purine and pyrimidine-DSB. A decrease of 65 % and 84 % of the purine and pyrimidine-DSB sensitive sites have been calculated, respectively, when the sample added CCE3 during the radiotreatment. Moreover, a reduction of 80 % in the amount of Nth + Fpg-DSB SSs (non-DSB cluster damage) after γ-irradiation in the presence of CCE3 was observed.

Conclusion

Through the present it was found that the CCE can play an important role as a radio protector, maybe by scavenging the ROS formed during radio treatment or by other unknown pathways. The most toxic DNA lesions (DSBs, and LMDS) decreased dramatically. Studies aimed at obtaining more documentation about CCE components with potential radio-preventive activity are desirable because of their protective properties.

Keywords

Gamma radiation
DSBs
LMDS
Cactus extract
Radioprotection
Abbreviations

Bp Base pair

C Cactus

CCE Cactus Cladode Extract

CCE1 Cactus Cladode Extract1 (0.5 µg)

CCE2 Cactus Cladode Extract2 (1 µg)

CCE3 Cactus Cladode Extract3 (1.5 µg)

CE Cladode extract 

137Cs 137Cesium 

DSB Double Strands Breaks

DPPH 2,2-Diphenyl-1-Picrylhydrazyl

ESS Enzyme Sensitive Site

Fpg Formamidopyrimidine-DNA N-Glycosylase

IR Ionizing Radiation

Gy Gray

LMDS Locally Multiple Damages

L Linear

ng Nanogram

Nth Base Excision Repair Endonuclease III

.OH Hydroxyl Radical

ROS Reactive Oxygen Species

S Supercoild

TE Tris-EDTA

TAE Tris-Acetate-EDTA

γ Gamma

µg Microgram

Nth+Fpg-DSB SSs Base Excision Repair Endonuclease III + Formamidopyrimidine-DNA N-Glycosylase-Double Strands Breaks
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pmc1 Introduction

Radiotherapy, a primary method for treating cancer, triggers apoptosis or senescence, in tumor cells by inducing significant DNA damage.1, 2 Ionizing radiation has the aspect of a 'double-edged sword', because it can alter the genetic information in exposed normal tissue, but also reduce tumor cells.3 It activates response transcription factors and signal transduction pathways leading to molecular changes and damage to DNA, lipids, and proteins4. These pathways’ activation induces a series of restorative and reparative processes, leading to cytokine milieu changes, the influx of inflammatory cells, and post-radiation problems5. The consequent activation of various enzymes and transcription factors ultimately contributes to genes coding inflammatory cytokines, such as TNF-, IL-1, and IL-6, including submucosa and basal epithelium, being up-regulated. This inflammation and tissue damage trigger ulceration and subsequent bacterial colonization, further feeding a vicious cycle of cytokine-mediated inflammatory damage.6

In addition to causing damage to the DNA of irradiated tumor tissue, ionizing radiation also causes cell death, which is exploited by radiotherapy with positive results.7, 3 During the passage of an ionizing radiation track of DNA damage sites, discrete energy depositions are observed, confirming that there are clusters of lesions that form over a few nanometers.8 The harmful biological effects of ionizing radiation are caused by these multiple DNA damage sites, which can be thought of as indicators of ionizing radiation..8

In the formation of lesions, clusters of lesions may form against a background of single, endogenous lesions at some DNA damage sites. It is known that radiation can cause different types of DNA damage, including single-strand DNA breaks (SSBs), and double-strand DNA breaks (DSBs). DSBs are the most critical radiotoxic lesions in living cells..7

LMDS, such as DNA DSBs defined by Ward as the creation of two or more DNA damages, within one or two helical turns caused by a single radiation track and local energy deposition, can be induced by low doses of radiation.9, 10 DNA DSBs are a serious type of DNA damage and are known to trigger multiple cell responses. The deleterious biological consequences of IR are attributed to this clustered DNA damage.7 Cluster damage repair is a challenge because of the complexity of DNA lesions caused by IR, such as complex double-strand breaks (DSBs), which the developed concepts exploit.7 Radiation therapy can take advantage of this decreased reparability of LMDS in cancer treatment.7 While radiation treatments can lead to complete tumor regression (80–90 % have been reported), normal tissue experiences both acute and late-stage damage due to radiation exposure. New technologies are being developed to minimize the dose to surrounding normal tissue..7, 8, 9, 10, 11

Food scientists and technologists are increasingly interested in plants because they are rich in nutrients and bioactive compounds. Opuntia ficus indica, is known as cactus, is primarily grown for its fruits and cladodes.12, 13 Opuntia fruits contain bioactive compounds that have been shown to have health-promoting properties. The bioactive constituents are antioxidants, anticancer agents, immunomodulators, anti-inflammatory agents, and renal protective agents..13, 14

The health-enhancing characteristics of Opuntia fruits are primarily due to their high antioxidant content.14, 15 By analyzing the phenols, and the carotenoids of the fruit, skin, seeds, and cladodes of different varieties, interestingly, regardless of the variety, the cladodes contained more antioxidants than the fruit itself. Stem cladodes have been traditionally used to treat different diseases.16, 17.

Furthermore, the extract of cactus fruit has also proved effective in treating other diseases and improving human health. Although, cacti have many medicinal properties and many benefits, they need to be studied more thoroughly for their antioxidant properties.17 The present study aims to show that CCE can protect DNA against gamma radiation, which induces DNA DSBs and non-DSBs lesions. In order to accomplish this, the yield of DSBs and LMDS when irradiating DNA in the two conditions with or without CCE has been calculated.

2 Materials and methods

The present studies employed a variety of experimental methods that have been extensively described elsewhere.18 Each method has been described briefly here.

2.1 Extract of cactus cladodes

Fresh cactus (Opuntia ficus indica) cladodes were collected. The collected cactus cladodes were mature (30–35  cm long, ≈20  cm wide and ≈2 cm thick). They were washed, spines removed, cut into small pieces (0.5  cm side) and then squeezed with a hand press, homogenized with 10 mM Tris–HCl. Then the extract were filtered and centrifuged 30 min at 3500 g at 4 °C.12 The supernatant was collected, concentrated and lyophilized at − 60 °C, until completely dry. Before use, the lyophilized extract was dissolved in water..12

2.2 Irradiation of the extracellular plasmid

The plasmid pGEM-3Zf (–) was purified using a HiSpeed Plasmid Maxi Kit from E. coli JM109.18 DNA concentration and protein concentration were determined using the synergy HT-I spectrophotometer.18 A total of nine samples were prepared in phosphate buffer for each experiment for each treatment at room temperature. Two samples with or without CCE1 (0.5 ng), CCE2 (1 ng), CCE3 (1.5 ng) served as unirradiated controls, and the others were irradiated with or without CCE1, CCE2 and CCE3 with 137Cs γ-radiation. The doses used were 25_700 Gy at a dose rate of 12.5 Gy min−1. The using of these doses help us to see clearly the loss of supercoiled and the formation of linear DNA (DSBs). For each assay, measurements were made in triplicate.

2.3 DNA analysis using agarose gel electrophoresis

The seven microliters have been separated of each sample with an agarose gel in TAE buffer containing 1 % agarose and stained with SYBR Green I.18 The DNA bands were quantified using a Typhoon-Trio laser scanner and Image Quant 5.0 software.18, 19 It is known that the supercoiled form of DNA has a weaker binding of SYBR Green I compared to nicked circular and linear configurations, so it was necessary to correct the obtained values.18, 19 The correction factor was determined to be 1.5. The corrected relative amounts of supercoiled (S), circular (C), and linear (L) plasmid DNA were determined from the integrals of their respective peaks (SG, CG, or LG) recorded in Image Quant 5.0 as follows:(1) S=1.5SG1.5SG+CG+LG

(2) C=CG1.5SG+CG+LG

(3) L=LG1.5SG+CG+LG

SG, CG, or LG are the integral values (i.e., the area under the peak) of the bands corresponding to the supercoiled, circular, and linear forms of DNA, respectively.18, 19

2.4 Enzymatic incubation to determine the base lesions

Base lesion amount was determined by mixing DNA (without or with CCE1, CCE2 and CCE3) irradiated by 137Cs γ-radiation (25–700 Gy) with E. coli base excision repair endonuclease III (Nth) and/or formamidopyrimidine-DNA N-glycosylase (Fpg) (Trevigen Inc.). Nth and Fpg can recognize and delete multiply lesions Cytosine and Thymine and Guanine and Adenine, respectively.20, 21 One hundred ng of DNA was mixed with one unit of Nth or Fpg or a mixture of both and incubated at 37℃ for 60 min. The control samples were also incubated in the presence of an incubation buffer only.21 The enzyme reactions were stopped by adding 10 µl of 0.5 M EDTA buffer, mixed with loading buffer and kept on ice before electrophoresis.21 The amounts of SSBs and DSBs were calculated using the agarose gel electrophoresis.18

2.5 Calculation of the damage yields

The amounts of SSB and DSB were measured by the respective dose–response curves for the generation of circular (C) and linear (L) plasmid.22, 23 The isolated base damage amount was measured by subtracting the amount of SSB; treated with only enzyme, from the amount of SSB after enzyme treatment.24 The net yield of enzyme-sensitive sites, n(ESS), may be obtained using equation (4) (Table 1).(4) nESSSSB=nNthorFpg+SSB-nprompt+heat-labileSSB

Table 1 Yields of DSBs and non-DSBs cluster damage induced in pGEM-3-Zf (−) plasmid DNA by gamma-irradiation in the absence or presence of the CCE (CCE1 (0.5 µg), CCE2 (1 µg), CCE3 (1.5 µg)); followed incubation with Nth or Fpg.

Without ECC	
Frank DSB
(x10-6SSB/Gy/bp)	Heat labile sites
(x10-6SSB/Gy/bp)	Nth-sensitive sites
(x10-6SSB/Gy/bp)	Fpg-sensitive sites
(x10-6SSB/Gy/bp)	Nth + Fpg-sensitive sites
(x10-6SSB/Gy/bp)	

0.023 ± 0.003	
0.006 ± 0.01	
0.46 ± 0.02	

0.29 ± 0.02	

0.72 ± 0.02	

With ECC1	

0.016 ± 0.01
	
0.005 ± 0.01	
0.29 ± 0.02	
0.21 ± 0.02	
0.44 ± 0.02	

With ECC2	

0.016 ± 0.01
	
0.005 ± 0.01	
0.14 ± 0.2	
0.15 ± 0.02
	
0.34 ± 0.02	

With ECC3	

0.01 ± 0.01
	
0.004 ± 0.01	
0.078 ± 0.2	
0.1 ± 0.02	
0.14 ± 0.02	

The incubation of the irradiated plasmid DNA with either Nth or Fpg at 37 °C results in the formation of linear DNA because the breaks induced by these enzymes at the sites of damaged bases are either in close proximity to one another or to a SSB, but on opposite strands. The yield of enzyme-sensitive sites detected as additional DSBs, n(ESS)DSB, are given by the equation (5) (Table 1).(5) nESSDSB=nNthorFpg+DSB-nprompt+heat-labileDSB

2.6 DNA damage modeling

The mathematical model designed by McMahon and Currell was used to calculate the amount of SSB (βS) and DSB (βD).25

This model assumes that:

1 – SSB and DSB occur randomly in plasmids, with a yield that is proportional to the absorbed dose; 2 – there is the probability of DSB formation from two SSBs that are sufficiently close together on opposite strands; 3 – the production of fragments from linear DNA (e.g., the induction of two or more DSB in the DNA) is negligible due to their minimal quantities at the present radiation doses. Based on these assumptions, the percentage of supercoiled, circular, and linear forms of plasmid can be described in the following equations:(6) dSdD=-βS+βDS

(7) dCdD=βSS-βDC-ρβS2DS+C

(8) dLdD=βDS+C+ρβS2DS+C

S, C, and L are the relative percentages of supercoiled, circular, and linear DNA, respectively, and D is the absorbed dose. The probability of two SSBs on a single plasmid becoming a DSB is denoted as ρ and can be revealed as the ratio of the maximum distance between two SSBs, which can still lead to a DSB divided by the length of the plasmid.18 These equations can be integrated analytically to give:(9) SD=S0e-βS+βDD

(10) CD=e-βDDC0e12βS2ρD2+S0e-12βS2ρD2-e-βSD

(11) LD=C0+S01-e-βDD+12βS2ρD2

Where S0 and C0 are the initial amounts of supercoiled and circular DNA before irradiation. The initial amount of linear DNA is set to zero since none is detected following gel electrophoresis.18

2.7 Statistical analysis

For each assay, measurements were made in triplicate, and the experimental outcome was presented as mean SD. One-way analysis of variance was used to compare the mean values between the groups (p ≤ 0.05, n = 3).

3 Results

3.1 The inhibition of the conversion of purine base damage to DSBs in the irradiated DNA by cactus cladodes extract

CCE1, CCE2 and CCE3 showed protection against purine-DSB DNA damage caused by ROS generated during IR. When the irradiated DNA was treated with Fpg at 37 °C without CCE1-CCE3, a further increase in the L form of the DNA was observed, as shown in Fig. 1. That observation was caused by the fact that Fpg induced other breaks at the sites of damaged bases. The yield of Fpg-DSB SSs and clustered DNA damage increases linearly with the increasing radiation dose (Fig. 1). The amount of ESS recognized as further DSBs, n(ESS)DSB, is given by the equation mentioned elsewhere26 (Table 1).Fig. 1 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA with CCE1 (0.5 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Fpg (open rhombus). DNA with CCE1 (0.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg (open circle).

However, a decrease of 27 %, 48 %, and 65 % in the yield of Fpg-DSB SSs was seen when the sample was irradiated with CCE1, CCE2 and CCE3, respectively, Fig. 1, Fig. 2, Fig. 3. A 0.21 ± 0.02 x 10-6 DSB/Gy/bp, 0.15 ± 0.02 x 10-6 DSB/Gy/bp, 0.1 ± 0.02 x 10-6 DSB/Gy/bp have been calculated when the plasmid was irradiated with CCE1, CCE2 and CCE3, respectively. While, without CCE, the amount was 0.29 ± 0.02 x 10-6 DSB/Gy/bp.Fig. 2 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE2 (1 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Fpg (open rhombus). DNA with CCE2 (1 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg (open circle).

Fig. 3 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE3 (1.5 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Fpg (open rhombus). DNA with CCE3 (1.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg (open circle).

3.2 The inhibition of the conversion of pyrimidine base damage to DSBs in irradiated DNA with the cactus cladodes extract

The CCE1, CCE2 and CCE3 exhibit protection of pyrimidine DSB DNA by the ROS generated by γ-radiation. When the irradiated DNA was treated with Nth at 37 °C without of CCE1, CCE2 and CCE3, a further increase in the L form of the DNA was observed, as shown in Fig. 4. The amount of Nth-DSB SSs increases linearly with increasing radiation dose (Fig. 4). Thus, the amount of multiple DNA lesions increases with increasing doses. The yield of n(ESS)DSB identified as extra DSBs, is given by theequation mentioned elsewhere26 (Table 1). While, in the presence of CCE the amount of Nth DSBs SSs has been reduced, Fig. 4, Fig. 5, Fig. 6.Fig. 4 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA in the absence of CCE1 (0.5 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Nth (open rhombus). DNA with CCE1 (0.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Nth (open circle).

Fig. 5 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE2 (1 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Nth (open rhombus). DNA with CCE2 (1 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Nth (open circle).

Fig. 6 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE3 (1.5 µg) was untreated (minus) or incubated at 37 °C (close square) or incubated with Nth (open rhombus). DNA without CCE3 (1.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Nth (open circle).

The inclusion of CCE1, CCE2 and CCE3 to the irradiated sample reduced the Nth-DSB SSs by 37 %, 70 % and 84 % respectively. Without CCE, the amount at Nth-DSB SSs have been 0.46 ± 0.02 x 10-6 DSB/Gy/bp. With CCE1, CCE2 and CCE3, the amount of Nth-DSB SSs has been 0.29 ± 0.02 x 10-6 DSB/Gy/bp, 0.14 ± 0.2 x 10-6 SSB/Gy/bp, and 0.078 ± 0.2 x 10-6 DSB/Gy/bp, respectively.

3.3 Cactus cladodes extract reduce the yield of non-DSB cluster lesion

The amount of multiple DNA lesions increases with increasing doses (Fig. 7). The amount of ESSs recognized as additional DSBs, n(ESS)DSB, is calculated by the equation mentioned elsewhere26 (Table 1).Fig. 7 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE3 (0.5 µg) was untreated (close rhombus) or incubated at 37 °C (close square) or incubated with Fpg + Nth (open rhombus). DNA with CCE1 (0.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg + Nth (open circle).

A reduction of Nth + Fpg DSBs SSs was observed after the addition of CCE to the sample before irradiation (Fig. 7, Fig. 8, Fig. 9).Fig. 8 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE2 (1 µg) was untreated (close rhombus) or incubated at 37 °C (close square) or incubated with Fpg + Nth (open rhombus). DNA with CCE2 (1 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg + Nth (plus).

Fig. 9 Dose response plots showing the fractions of linear DNA. The pGEM-3Zf (−) DNA (10 ng/µl) irradiated with phosphate buffer (10 mM) and with or without CCE with γ-rays. After irradiation DNA without CCE3 (1.5 µg) was untreated (close rhombus) or incubated at 37 °C (close square) or incubated with Fpg + Nth (open rhombus). DNA with of CCE3 (1.5 µg) was untreated (close circle) or incubated at 37 °C (close triangle) or incubated with Fpg + Nth (star).

There was a 39 %, 52 %, and 80 % a reduction of Nth + Fpg-DSB SSs (non-DSB cluster damage) when the plasmid was irradiated with CCE1, CCE2 and CCE3, respectively. Without CCE, the amount of Nth + Fpg-DSB SSs have been 0.72 ± 0.02 x 10-6 DSB/Gy/bp. While, without CCE1, CCE2 and CCE3, a 0.44 ± 0.02 x 10-6 DSB/Gy/bp, 0.34 ± 0.02 x 10-6 DSB/Gy/bp, and 0.14 ± 0.02 x 10-6 DSB/Gy/bp have been calculated the amount of Nth + Fpg-DSB SSs, respectively.

4 Discussion

Ionizing radiation causes a large amount of accumulated DNA lesions, including complex DSBs,27, 28 which kill tumor cells because the accumulated damage sites are not easy to repair.29 When irradiation occurs in the presence of oxygen, almost all DNA lesions are induced by hydroxyl radicals,30 which, can attack DNA bases and sugar units and induce the breakdown of the backbone of DNA.31 DSB can be formed by the conversion of LMDS during the enzymes of the repair process.32 This reduced ability to repair LMDS using different repair pathways could be utilized during the radiotherapy treatment.

The addition of Nth and Fpg to the DNA plasmids induces additional DSBs (Table 1). IR has been shown to have cytotoxicity, mutation, and the formation of tumors, some of which can be related to DNA cluster lesions.8 In eukaryotic systems, it was demonstrated that non-DSB clustered DNA damage with one base lesion and three or more SSBs can impede DNA lesion repair by diminishing the efficiency of Nth and Fpg in excising base damage, which in turn cuts off sites within the AP cluster and reduces damage.33 In addition, it has been discovered that certain non-DSB cluster lesions can become DSBs after irradiation with prokaryotic systems.29 Radiotherapy spares healthy tissue while focusing on the malignancy. Plant extracts were used for the treatment of many diseases before the discovery of new, currently approved drugs.34

The medicinal properties of the plant were shown in different studies.34 Although Opuntia originated in Mexico, it is still widely cultivated there and in different countries worldwide. Opuntia monacantha has been shown to decrease blood glucose and cholesterol. Moreover, it has anti-inflammatory, antioxidant, and detoxifying effects.35, 13 Opuntia monacantha also inhibited gastric ulcers and had neuroprotective effects.36 Opuntia contains vitamins and phenolic compounds, which have very high activity as antioxidants.37, 38 However, regardless of cultivar, the concentration of carotenes and phenolics was higher in the cladodes than in the fruits. The researchers found that the fruits, peels, and seeds of the different varieties contained specific antioxidants, while the cladodes of all varieties contained similar antioxidants..39, 40

Recently, interest in the use of plant components as effective radioprotectors has developed dramatically, replacing synthetic medications with different limitations.34, 41, 42, 43 Plant extracts have reduced oxidative DNA lesions induced by the Fenton reagent.44, 42, 43 Scientists are trying to find efficient and nontoxic radioprotective agents.34, 43 Various radioprotective agents have been produced to prevent radiation damage. These agents are usually considered antioxidants.45, 42, 43 Plant extracts could be the best solution. Polyphenols and flavonoids are potent, easy to find, safe, and economical radioprotective agents, there is an important interest in ethnomedicinal agents worldwide.34 They also have anticancer and antiradiation properties. Recently, in order to identify efficient radioprotective agents, various compounds have been tested.46 Many studies have shown these compounds can be used as potent radioprotective agents. The use of radioprotective compounds before or after radiotherapy reduces tissue lesions caused by IR.34 This study was conducted to confirm the hypothesis that CCE can reduce the amount of LMDS produced by IR. The effect of γ-radiation on plasmid DNA after the addition of CE was examined. Three quantities of CE have been chosen. CCE3 showed that it is suitable for high protection against ROS (LMDS) induced by γ-radiation.

The addition of cactus extract to the DNA sample induced a decrease of a frank DSB yield. The βD was reduced by 27 %, 36 %, and 47 % when the DNA was treated with CCE1, CCE2 and CCE3, respectively. Moreover, a decrease by 39 %, 52 %, and 80 % of the amount of non-DSB cluster damage revealed after enzymatic treatment with Nth plus Fpg as additional DSBs was observed after the addition of CCE1, CCE2 and CCE3, respectively. These observations show that CCE can be a powerful protector of DNA from.OH induced DSB. It has been found that flavonoids is a good DNA protector against ROS induced lesions by DNA duplex induction.47 In addition, Opuntia ficus indica CE is hepatoprotective as it enhances the activities of liver function, as evidenced by the decrease in MDA, protein carbonyl generation and Hsp 70, and Hsp 27 levels.48 The treatment of mice hepatic cells with AFB1 induced genotoxicity markers and decreased the expressions of pro-apoptotic proteins p53 and bax,48 which can be responsible for the DNA fragmentation.48 CCE restored a hepatic cell’s DNA when it was incorporated into the diet of mice.49 My study is consistent with other studies showing that CCE reduces the genotoxicity of cis-diamminedichloroplatinum(II).50 May the CE extract contains flavonoids, which can be responsible for DNA protection42, 51or the extract might have reduced the amount of damage by other pathways.

In addition, Brahmi et al. found that chromosomal aberrations were reduced dramatically when animals were given a CE before or after treatment with aflatoxin B1.50

Flavonoids can decrease the DNA lesions induced by oxidative stress by forming complexes with ROS. Delphinidin, hesperetin, and naringenin have been shown to be good stabilizers of DNA more strongly than quercetin and kaempferol.52, 53 In addition, the small quantity of green tea polyphenols gives good protection to DNA by transferring the electrons from polyphenols to DNA.54 In conclusion, CCE also protects DNA highly efficiently against gamma radiation, especially from DSB and non-DSB cluster damage. My finding is consistent with other studies that have found that the stem segments (cladode) of Opuntia monacantha are used in the treatment of inflammation and tumors due to their positive therapeutic effects.55 In addition, the cactus cladode can be used to microencapsulate chemotherapeutic agents. As encapsulating agents, cactus cladode mucilage and maltodextrin can be used for microencapsulation by spray drying.37 This can be a good way to transport the chemotherapeutic agents to the target organ during concomitant therapy with ionizing radiation.

5 Conclusion

The DNA DSBs and LMDS are considered the most devastating damage caused by IR.54, 55 The fact that lesions within double-stranded and tandem clusters heal less effectively than lesions alone is now commonly known.7 Biological responses to radiation can be altered by radioprotective agents. In this context, the present study showed that the CE extracts included antioxidants in addition to the fruits that are normally present in other cactus species. Radiation damage can be prevented with high efficiency by CE antioxidants. The CCE reduces the induction of DSBs and non-DSB cluster damage. Therefore, it will be good to use the CCE in healthcare facilities as a strong radioprotective agent to protect normal tissue instead of sulfur-containing compounds, which are mainly synthetic radioprotective drugs, but their application is limited by side effects. Moreover, it was recently discovered that CCE exhibited strong anti-breast cancer and anti-prostate cancer properties.56 As a result, this extract has practical and exceptional anticancer and radioprotective properties. It will be very interesting to find out the mechanisms through which CCE exerts its protective and anticancer effects and to conduct more experiments in vivo. Moreover, testing if it has a synergistic effect when we combine CCE extract with other radioprotective agents will be very important.

CRediT authorship contribution statement

Saloua Kouass Sahbani: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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References

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