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 Table of Contents 
ORIGINAL ARTICLE
Year : 2016  |  Volume : 39  |  Issue : 4  |  Page : 183-189  

Radioprotective potential of Decalepis hamiltonii: A study on gamma radiation-induced oxidative stress and toxicity in Drosophila melanogaster


1 Department of Studies in Zoology, University of Mysore, Mysuru, Karnataka, India
2 Department of Studies in Physics, Microtron Centre, Mangalore University, Mangalore, Karnataka, India

Date of Web Publication13-Feb-2017

Correspondence Address:
S R Ramesh
Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore - 570 006, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-0464.199977

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  Abstract 

Radiation-induced damage to normal tissues restricts the therapeutic use of radiation in clinical application for cancer treatment and thereby limits the efficacy of the treatment. The use of chemical compounds as radioprotectors is a desirable strategy to improve the therapeutic index of radiotherapy. However, most of the synthetic radioprotective compounds studied have shown to have undesirable properties of toxicity. There is a need for safer, natural radioprotective agents without compromising efficacy of the treatment. We have investigated the radioprotective potential of Decalepis hamiltonii (Dh) root extract which is rich in natural antioxidants by employing Drosophila melanogaster as a model. Irradiation of Drosophila with 100, 200, and 400 Gy of gamma radiation induced dose-dependent mortality. Elevation in the levels of thiobarbituric acid reactive substances (TBARS), the activities of catalase (CAT) and superoxide dismutase (SOD), and depletion of glutathione (GSH) content suggested radiation-induced oxidative stress. Pretreatment of flies with Dh root extract protected them from radiation-induced mortality and oxidative stress as evidenced by reduction in TBARS and restoration of the antioxidant enzymes, SOD and CAT, and GSH to control levels. This is the first report of radioprotective action of Dh root extract in D. melanogaster.

Keywords: Antioxidants, comet assay, Decalepis hamiltonii, Drosophila melanogaster, gamma radiation, oxidative stress


How to cite this article:
Pasha M, Sanjeev G, Shivanandappa T, Ramesh S R. Radioprotective potential of Decalepis hamiltonii: A study on gamma radiation-induced oxidative stress and toxicity in Drosophila melanogaster. Radiat Prot Environ 2016;39:183-9

How to cite this URL:
Pasha M, Sanjeev G, Shivanandappa T, Ramesh S R. Radioprotective potential of Decalepis hamiltonii: A study on gamma radiation-induced oxidative stress and toxicity in Drosophila melanogaster. Radiat Prot Environ [serial online] 2016 [cited 2022 Jan 19];39:183-9. Available from: https://www.rpe.org.in/text.asp?2016/39/4/183/199977


  Introduction Top


Antioxidants are desirable choices to mitigate oxidative stress-mediated toxicity of radiation. The physiological role of antioxidants includes prevention of damage to cells as a consequence of chemical reactions involving free radicals. Borek has shown that administration of antioxidants prevented radiation-induced short- and long-term injury to healthy cells, tissues, and oncogenic transformation.[1] Radiotherapy is used in cancer management and its success is limited by the undesirable toxic effects on the normal cells. Protection of the normal cells from radiation-induced damage by radioprotectors is important in radiation therapy. Natural radioprotectors also have potential application during nuclear emergency situations.

Drosophila melanogaster is an excellent model organism for toxicological studies.[2],[3] It is being used as a model organism in various fields of biology. Lamb has shown that gamma radiation caused reduction in the life span of Drosophila.[4] Whereas Parashar et al. have shown that there is a significant age-dependent decline in the radiation resistance in both males and females of Drosophila.[5] A 5000 roentgen gamma radiation exposure resulted in mutations in D. simulans.[6] Evangelia et al. have shown that a daily 6-min exposure of D. melanogaster to GSM-900 MHz mobile phone radiation-induced DNA fragmentation and cytoskeleton disorganization.[7]

Tuberous roots of Decalepis hamiltonii (Dh), a shrub found in the forests of southern India, are consumed as pickle and as a health drink. Several compounds isolated from its roots have been shown to possess free radical scavenging ability in vitro[8],[9] and provide protection against oxidative stress-induced cellular damage.[10],[11] Further, we have shown that Dh protects against oxidative stress induced by ethanol and paraquat in Drosophila.[12],[13] In view of the strong free radical scavenging activity of the natural antioxidants of roots of Dh, we have investigated the in vivo radioprotective potential in D. melanogaster.


  Materials and Methods Top


Chemicals

1,1-diphenyl-2-picrylhydrazyl and thiobarbituric acid (TBA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA), nicotinamide adenine dinucleotide reduced, hydrogen peroxide (H2O2), 5,5-dithiobis (2-nitrobenzoic acid) (DTNB), pyrogallol, bovine serum albumin (BSA), reduced glutathione (GSH), and other chemicals were purchased from Sisco Research Laboratories (Mumbai, India). All the chemicals used were of the highest purity grade available.

Drosophila stocks

D. melanogaster (Oregon K) strain was obtained from Drosophila Stock Center, Department of Studies in Zoology, University of Mysore, Mysore. The flies were raised and maintained on standard wheat cream agar medium supplemented with dry yeast at 22°C ± 1°C and 70%–80% relative humidity in a vivarium. Adult flies of the same age were used for the experiments.

Preparation of the extract and treatment

The roots of D. hamiltonii were washed with water, followed by crushing with a roller to separate the inner woody core from outer fleshy layer. The fleshy portion was collected, dried at 40°C in a hot-air oven until they lost complete moisture (4–6 h) and finely powdered, and used for aqueous extraction as described earlier.[14]

Two hundred synchronized Drosophila eggs were placed in each culture bottle containing the medium with 0.5% and 1.0% (w/v) of Dh extract for feeding. After eclosion, the adults were transferred to vials containing Dh supplemented medium for 5 days with a change after 3 days. Similarly, the adults obtained from eggs raised in medium without Dh extract formed the corresponding control group. This batch of adult flies after eclosion was transferred to fresh food vials (without Dh) every 3 days.

Feeding assay

To know whether the presence of Dh in the media affects feeding of the flies, their food intake was visually examined by feeding them on media containing the dye FD and C Blue No. 1 which showed that the feeding was comparable to the control diet.[12]

Irradiation

Twenty-five adults of 5-day–old Drosophila from each group were transferred to 2 mm thick polypropylene tubes of 65 mm × 25 mm size and exposed to 60 Co gamma radiation (Gamma 5000 chamber, source strength of 14,000 Ci that delivers about 9 kGy/h) at 100, 200, and 400 Gy at the Center for Application of Radioisotopes and Radiation Technology, University Science Instrumentation Centre, Mangalore University, Mangalagangotri, Konaje, Karnataka, India. In earlier set of experiments, we irradiated the flies by exposing them to 60 Co gamma radiation ranging from 2 to 200 Gy. However, we could not notice any mortality up to 100 Gy; hence, for further studies, we chose dosages above 100 Gy. After irradiation, the flies were transferred into fresh media bottles containing control and Dh diet.

Treatment groups

Following treatment groups were set up for the present study

  • Group I: Flies with normal diet (control)
  • Group II: Flies fed on diet containing 1% Dh extract
  • Group III: Gamma irradiated flies (with normal diet)
  • Group IV: Gamma irradiated flies fed on diet containing 0.5% Dh
  • Group V: Gamma irradiated flies fed on diet containing 1% Dh.


D. melanogaster adults exposed to different doses of gamma rays (100–1200 Gy) were monitored for survival. Mortality of the flies after 24 h of exposure was recorded, and the LD50 was calculated using the probit analysis.

Lipid peroxidation

Lipid peroxidation (LPO) in the fly homogenate was measured by thiobarbituric acid reactive substances (TBARS) method.[15] Fifty flies were homogenized in 1 ml of 50 mM phosphate buffer (pH 7.4). 250 µl of the homogenate was mixed with 1.5 ml of each trichloroacetic acid (TCA) (20%) and TBA (0.6%). The mixture was incubated in boiling water bath for 30 min, cooled, and 2 ml of butanol was added, mixed by vortexing, and centrifuged. The color of the butanol layer was read at 535 nm in a spectrophotometer (Shimadzu, Japan).

Glutathione assay

Twenty-five flies were homogenized in 1 ml of 5% TCA containing ethylenediaminetetraacetic acid (EDTA) (1 mM) and centrifuged at 15000 rpm for 10 min at 4°C. 200 µl of supernatant was mixed with 2.8 ml of 0.2M Tris-HCl buffer and 50 µl of 10 mM DTNB, and incubated for 10 min at room temperature. The absorbance of the color was read at 412 nm.[16] Glutathione contents were calculated using a standard curve.

Antioxidant enzymes

Catalase (CAT) activity was measured by adding 50 µl of the supernatant to 3 ml reaction mixture containing 8.8 mM H2O2 (1.5%) and 0.1 M sodium phosphate buffer (pH 7.4) and the absorbance at 240 nm was monitored for 3 min. The decrease in H2O2 concentration was expressed as mmol of H2O2 decomposed/min/mg of protein.[17]

Superoxide dismutase (SOD) activity was measured by monitoring the inhibition of pyrogallol auto-oxidation in a total volume of 3 ml reaction mixture containing pyrogallol (2 mM) in 0.1 M Tris buffer (pH 8.2). The reaction was initiated by adding 50 µl of the homogenate and rate of change in absorbance was monitored for 3 min at 412 nm. Enzyme activity was expressed in units as the amount of protein required to inhibit 50% of pyrogallol auto-oxidation.[18]

Protein estimation

Total protein was estimated by the method of Lowry et al.[19] using BSA as the standard.

Comet assay

Cell preparation from the adult gut was made according to the method of Howell and Taylor with some modifications.[20] Midgut from adult control and treated flies were explanted in Poels' salt solution (14.72 mM NaCl, 42.0 mM KCl, 7.89 mM CaCl2·2H2O, 7.34 mM NaH2 PO4, 1.8 mM KHCO3, 20.81 mM MgSO4·7H2O, pH 7.0)[21] and digested with collagenase (0.5 mg/ml in phosphate-buffered saline [PBS], pH 7.4) for 15 min. Cell suspension was then filtered through a nylon mesh (60.0 µm) to remove the debris. Collagenase was removed by washing the cell suspension with 1X PBS (pH 7.4) and was finally suspended in 120 µl of PBS (pH 7.4).

Comet assay was performed according to the method of Siddique et al.,[22] 120 µl of cell suspension was mixed with equal volume of 1.5% low-melting point agarose. 75 µl of this mixture was immediately layered on the top of an end frosted slide that was precoated with 1.0% normal melting point agarose, and the slide was placed on a chilled plate for 10 min to allow solidification of agarose. The slides were immersed in freshly prepared chilled lysing solution (2.5 M NaCl, 100.0 mM EDTA, 10.0 mM Tris, and 1.0% Triton X-100, pH 10.0) for 2 h. After lysis, the slides were kept in electrophoretic buffer (300.0 mM NaC2H3O2 and 100.0 mM Tris–HCl, pH 8.5) for 1 h and then transferred to fresh buffer and electrophoresed for 1 h at 4°C at 14 V and 6 mA. After electrophoresis, the slides were stained with ethidium bromide (20.0 µg/ml) for 20 min following which they were dipped once in chilled distilled water to remove the excess stain and coverslips were placed over the slides. All the steps were carried out under very dim light to avoid any light-induced DNA damage. The slides thus prepared were examined using an image analysis system on Leica DMLB microscope with a fluorescence attachment (Wetzlar, Germany) and photographed using a digital camera. These images were analyzed using Comet score software version 1.5 (Tri Tek Corp., Sumerduck, VA, USA). Fifty cells/slide × 4 replicates were examined.

Statistical analysis

The data were statistically analyzed by one-way ANOVA followed by Duncan's multiple range test using the SPSS version 14 software (Chicago). The data of comet assay were analyzed by Student's t-test.


  Results Top


Radiation toxicity

There was no mortality of the flies exposed to 100 Gy of gamma radiation after 24 h postexposure. However, at higher dosage, dose-dependent mortality was observed [Figure 1]. In our preliminary studies, we found that feeding Dh at larval stages, rather than adult flies, provide better protection against radiation toxicity. Therefore, larval stage fed flies was chosen for further studies [Table 1].
Figure 1: Protective effect of D. hamiltonii against radiation-induced mortality in Drosophila melanogaster. LD50/24h for control flies was (830 Gy), 890 Gy for 0.5% Decalepis hamiltonii flies and 940 Gy for 1% Decalepis hamiltonii fed flies. Values are given as mean ± standard deviation (n = 4)

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Table 1: Protective effect of D. hamiltonii root extract against radiation induced mortality in D. melanogaster adults and larvae

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Lipid peroxidation

Flies exposed to gamma radiation showed elevated levels of LPO which was dose-dependent. Flies fed on control diet showed highest TBARS levels [Figure 2]. Lower levels of LPO (TBARS levels) were seen in flies pretreated with Dh at the 0.5% and 1% dietary levels.
Figure 2: Protective effect of Decalepis hamiltonii root extract against radiation-induced lipid peroxidation in Drosophila melanogaster. Group I: Control, Group II: 1% Decalepis hamiltonii fed without irradiation, Group III: Irradiation without Decalepis hamiltonii, Group IV: Irradiation + Decalepis hamiltonii (0.5%) and Group V: Irradiation + Decalepis hamiltonii (1%). Values are given as mean ± standard deviation. aP < 0.05 when compared to the control group. bP < 0.05 when compared to the corresponding irradiated group (n = 4)

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Glutathione

Flies exposed to radiation showed depletion of GSH in a dose-dependent manner. Pretreatement with Dh protected the flies from GSH depletion and their GSH levels were comparable to control flies [Figure 3]. Maximum protection against radiation-induced GSH depletion was observed in flies fed on 1% Dh.
Figure 3: Effect of Decalepis hamiltonii root extract on radiation-induced changes in glutathione levels in Drosophila melanogaster. Group I: Control, Group II: 1% Decalepis hamiltonii fed without irradiation, Group III: Irradiation without Decalepis hamiltonii, Group IV: Irradiation + Decalepis hamiltonii (0.5%) and Group V: Irradiation + Decalepis hamiltonii (1%). Values are given as mean ± standard deviation. aP < 0.05 when compared to the control group. bP < 0.05 when compared to the corresponding irradiated group (n = 4)

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Antioxidant enzymes

Irradiated flies showed elevated levels of CAT and SOD in a dose-dependent manner. Pretreatment with Dh maintained the CAT and SOD activities and that was comparable to their control flies. Dh protected flies from the elevation of CAT and SOD [Figure 4]. Overall, 1% Dh pretreatment shows higher protection in maintaining antioxidant enzyme levels closer to that of control flies.
Figure 4: Effect of Decalepis hamiltonii root extract on radiation-induced changes in the antioxidant enzymes, catalase (a) and superoxide dismutase (b) in Drosophila melanogaster. Group I: Control, Group II: 1% Decalepis hamiltonii fed without irradiation, Group III: Irradiation without Decalepis hamiltonii, Group IV: Irradiation + Decalepis hamiltonii (0.5%) and Group V: Irradiation + Decalepis hamiltonii (1%). Values are given as mean ± standard deviation. aP < 0.05 when compared to the control group. bP < 0.05 when compared to the corresponding irradiated group (n = 4)

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Comet assay

Our results revealed that exposure of Drosophila flies to different doses of gamma radiation resulted in dose-dependent increase in the DNA damage as evident from the increase in the number of comet cells, length of comet tail, and concentration of DNA present in the comet tail. In contrast, the flies pretreated with different concentrations of Dh (0.5% and 1%) show dose-dependent decline in the gamma radiation-induced DNA damage, i.e., decline in number of comet cells, reduction in comet tail length, and comet DNA content. Maximum radiation damage was observed in flies which were fed on control diet whereas flies fed on 1% Dh showed maximum protection against gamma radiation-induced DNA damage [Figure 5].
Figure 5: Effect of different doses of gamma radiation in comet parameters in gut cells of Drosophila melanogaster. Group I: Control, Group II: Decalepis hamiltonii fed (1%) without irradiation, Group III: Irradiation without Decalepis hamiltonii, Group IV: Irradiation + Decalepis hamiltonii (0.5%) and Group V: Irradiation + Decalepis hamiltonii (1%). Values are given as mean ± standard deviation. aNot significant, *P < 0.05 and **P < 0.01 versus control (n = 4)

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  Discussion Top


D. melanogaster has been used as a model for radiation toxicity.[23],[24],[25],[26],[27] In Drosophila, radiation shortens life by accelerating aging resulting in reduction in life span.[4] Irradiation of D. melanogaster with low dose (60 cGy) affected the lifespan and neuromuscular activity in D. melanogaster[28] whereas irradiation with 1MHz ultrasound intensities of 0.05–2 W/cm 2 produced genetic damage i.e., point mutations through deletion and rearrangements of genes in the chromosomes.[29] A 5000 roentgen (43.85 Gy) of gamma radiation-induced Drosophila mutants and their genetic effects have been studied by Hussain.[6] Seong observed that Curcumin reversed the shortened lifespan of irradiated Drosophila flies and attenuated oxidative stress and DNA alterations caused by ionizing radiation,[30] While Vitamin C pretreatment protected Drosophila larvae against gamma radiation and chromium (VI) oxide induced mutation/recombination.[31]

Information on influence of radioprotective agents in Drosophila is scanty. Parashar et al.[5] reported the exposure of 1 day old D. melanogaster to gamma radiation between 200 and 1500 Gy produced LD50 around 1238 Gy and 1339 Gy for males and females. Our study showed that Dh pretreatment extended the survivability against gamma radiation-induced mortality. The LD50 of the control flies was found to be 830 Gy whereas it was 890 Gy and 940 Gy, respectively to 0.55 and 1% Dh. The LD50 values clearly show that radiation resistance of flies fed on Dh containing diet.

Radiation causes membrane damage by LPO. LPO leads to progressive loss of membrane integrity, impairment of membrane transport system that disrupts cellular ion homeostasis.[32] Marked increase in the levels of LPO in the irradiated flies indicates extreme membrane damage due to induction of free radicals. Pretreatment of flies with 0.5% and 1% Dh minimized the free radical damage leading to LPO. The LPO in the Dh fed flies were comparable to control flies. Thus, our results suggest that Dh pretreatment protects flies from radiation-induced membrane damage. Feeding of 1% Dh provided maximum protection against radiation-induced free radicals and LPO.

GSH, the ubiquitous natural thiol in cells is a powerful natural antioxidant which protects protein thiol groups from oxidation.[33] Administration of various thiols has been reported to protect the cells and animals against the effects of radiation.[34],[35] Our earlier studies have reported that Dh protects against oxidative stress-induced GSH depletion in Drosophila.[12] Present results [Figure 3] showed depletion of GSH levels in irradiated flies, whereas the depletion was lower in flies treated with Dh.

CAT, is an inducible enzyme that degrades H2O2 to water, it is an important antioxidant defense.[36] Higher CAT activity seen in irradiated flies indicates enzyme induction as a consequence of increased H2O2 formation which could be a natural adaptative response. Our results show that the activity of CAT in Dh pretreated flies was restored to that of control flies which could be due to scavenging of H2O2 by Dh extract and/or lower levels of oxidative stress. This study suggests that Dh protects Drosophila flies from gamma radiation-induced oxidative stress by controlling H2O2 generation and consequently CAT activity.

SOD is a vital biochemical defense against oxygen radicals generated during natural metabolism.[37] According to Peng et al., SOD is involved in the resistance of D. melanogaster against radiation and plays a vital role in the adaptive response against radiation-induced oxidative stress.[38] From the present study, it is evident that increased activity of SOD in irradiated flies is caused by reactive oxygen species induction by radiation which was restored to control level in Dh fed flies.

Excessive cellular oxidative stress leads to alterations in DNA.[39] Alkaline comet assay has been extensively used in genetic toxicology to assess DNA damage.[40],[41],[42] Comet assay in Drosophila has been used to asses in vitro genotoxicity potential of xenobiotics in cells such as hemocytes,[43] gut cells [22],[44] and brain cells.[40] Three parameters that are generally used to indicate DNA damage are (a) comet tail DNA, (b) comet tail length, and (c) percentage of cells with tail. It is expressed as percentage and indicates the ratio of the DNA present in the tail to the total DNA content. Comet tail length is the distance from the nuclear core to the end of DNA migration and percentage of cells possessing tails. Our results show that there is a dose-dependent increase in the comet cells, comet length (µm), and percentage tail DNA in all the dosages. Radiation exposed flies showed higher number of comet cells, tail length, and tail DNA. In the Dh fed flies, these effects were greatly reduced, suggesting protective effect of Dh feeding [Figure 5]. Maximum DNA damage was observed in irradiated flies fed with control diet (Group III) while the DNA damage was found to be reduced in 0.5% and further reduced in 1% Dh fed flies (Groups IV and V).


  Conclusions Top


Our study has demonstrated the radioprotective potential of Dh root extract against gamma radiation toxicity for the first time. The protection involves suppression of radiation-induced oxidative stress, prevention of GSH depletion, and oxidative damage to DNA. The Dh extract was effective in restoring the impaired antioxidant enzymes, SOD, and CAT. Our study shows the potential application of natural antioxidants from edible plants in radiation therapy to minimize the damage caused by the radiation to normal tissues.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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