Chironomus ramosus larvae exhibit DNA damage control in response to gamma radiation.

PURPOSE
Chironomus ramosus is one of the recently reported radiotolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a lethal dose for 20% of the population (LD(20)) of gamma radiation (2200 Gy, at a dose rate 5.5 Gy/min).


MATERIALS AND METHODS
Genomic DNA preparations were subjected to competitive ELISA (Enzyme linked immunosorbent assay) for detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation-induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis (ASCGE), comet assay and pulsed field gel electrophoresis (PFGE) to check for DNA double-strand breaks.


RESULTS
Results from all four experimental procedures confirmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Some 48 h after radiation exposure, modified 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed significantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradiated DNA.


CONCLUSION
Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.


Introduction
Tolerance to high doses of ionizing radiation makes nonbiting midges (Diptera: Chironomidae) an extraordinary group of stress-tolerant insects (Watanabe et al. 2006, Datkhile et al. 2009a, Jorgelina and Karsten 2010. Members of this primitive group thrive under diverse ecological and climatic conditions (Ferrington 2008), sustain a wide variety of biotic and abiotic stresses (Choi 2004, Warrin et al. 2012 including accumulation of uranium in simulated laboratory conditions (Muscatello and Liber 2009). So far, tolerance to very high doses of ionizing radiation has been reported in rare life forms like the radioresistant tardigrades (Harikawa and Higashi 2004), bdelloid rotifers (Gladyshev and Meselson 2008), or radioresistant bacteria Th ermococcus gammatolerans (Jolivet et al. 2003) and Deinococcus radiodurans (Slade and Radman 2011). Among the dipteran insects, Polypedilum vanderplanki , is radiotolerant. Resistance to radiation here is suggested to be a consequence of their evolutionary adaptation to survive desiccation as both radiation and desiccation are DNA damage stressors (Gusev et al. 2010). Gamma radiation doses aff ecting the developmental stages of Indian tropical midge Chironomus ramosus are remarkably high compared to other known insects (Datkhile et al. 2009a). One of the hallmarks of ionizing radiation on biological systems is damage caused to cellular DNA. Damage can be direct through the generation of single-or double-strand breaks in DNA or indirectly by the generation of reactive oxygen species (ROS), which gives rise to an additional stress leading to oxidatively damaged biomolecules. Th e most commonly formed adduct in nuclear DNA, 8-oxo-7,8-dihydro-2 ′deoxyguanosine (8-oxodG) residues has been a ubiquitous biomarker for measurement of DNA oxidation (Cooke et al. 2003, Cadet et al. 2012. Th e presence and repair of modifi ed base 8-oxodG has emerged as a quantifi able biomarker for the detection of oxidatively damaged and repaired DNA (Rosen et al. 1996, Kasai 1997, Elliott et al. 2000, Shen et al. 2007. Th e extent and nature of radiation-induced damage however, depends on the type of radiation, rate of exposure, eff ective cumulative dose to specifi c cellular and sub-cellular compartments and lastly, upon tolerance threshold of each organism. In midges, salivary gland cells (SGC) are the most metabolically active larval organs containing giant polytene chro-

Abstract
Purpose : Chironomus ramosus is one of the recently reported radiotolerant insects. Salivary gland cells of fourth instar larvae respond to ionizing radiations with increases in the levels of antioxidant enzymes and chaperone proteins. Here we made an attempt to study the state of nuclear DNA after exposure of larvae to a lethal dose for 20% of the population (LD 20 ) of gamma radiation (2200 Gy, at a dose rate 5.5 Gy/min). Materials and methods : Genomic DNA preparations were subjected to competitive ELISA (Enzyme linked immunosorbent assay) for detection of 8-oxo-7,8-dihydro-2 ′ -deoxyguanosine (8-oxodG) and dynamic light scattering (DLS) to monitor any radiation-induced damage. Single salivary gland cells were subjected to alkaline single cell gel electrophoresis (ASCGE), comet assay and pulsed fi eld gel electrophoresis (PFGE) to check for DNA double-strand breaks. Results : Results from all four experimental procedures confi rmed damage of nucleobases and fragmentation of nuclear DNA immediately after radiation. Some 48 h after radiation exposure, modifi ed 8-oxodG residues returned to basal level, homodispersity of genomic DNA reappeared, the length of comet tail regressed signifi cantly (ASCGE) and PFGE pattern matched with that of high molecular weight unirradiated DNA. Conclusion : Chironomus ramosus larvae showed control of DNA damage as observed over 48 h in post irradiation recovery which could be attributed to their ability to tolerate gamma radiation stress.
Keywords: Chironomus ramosus , alkaline comet assay , gamma radiation stress , DNA damage response , Pulsed fi eld gel electrophoresis , dynamic light scattering , September 2015; 91(9): 742 -74 mosomes. High transcriptional activity, content of abundant proteins and genomic DNA make it the most widely used tissue for cytotoxicity, genotoxicity and molecular studies (Nath et al. 2005, Th orat and Nath 2010 ). Our earlier study on radiation tolerance of Indian tropical midge, C. ramosus showed that the larvae contained relatively high antioxidant enzymatic activities concomitant with an elevated level of 70 kDa Heat Shock Protein (HSP 70) expression in the irradiated SGC during recovery from radiation stress (Datkhile et al. 2009b(Datkhile et al. , 2011. Increased DNA repair capabilities of tissue cultured cells have been reported from other insects in the past (Koval 1994, Chandna et al. 2004. In this study, biochemical and biophysical methods were implemented to observe eff ects of gamma radiation on SGC nuclear DNA of C. ramosus larvae.

Rearing of Chironomus and gamma radiation exposure
Th e inbred line of laboratory culture of C. ramosus was originated from a single egg mass (isofemale line) from a water body of Pune city, India (Lat. 18 ° 33 ′ 17.73 ′ ′ N, Long. 73 ° 51 ′ 48.06 ′ ′ E) inhabited by the natural population of C. ramosus . Culture was maintained by mass rearing technique (Nath and Godbole 1998) at 25 Ϯ 2 ° C temperature, relative humidity of 50 Ϯ 10%, and a photoperiod of 14 h light and 10 h darkness in the insectary of Bhabha Atomic Research Centre (BARC), Mumbai. Th irty-day-old fourth instar larvae were put in a glass beaker containing tap water and exposed to a dose of 2200 Gy a lethal dose for 20% of the midge population (LD 20 ) determined by dosimetric studies for the determination of percent mortality of irradiated larvae followed by Probit analysis (Datkhile et al. 2009a) from a cobalt 60 Co source (dose rate 5.5 Gy/min, Gamma Cell 220, Atomic Energy of Canada Ltd, Ottawa, Canada). Another 60 Co source (dose rate 55.6 Gy/min, Gamma Cell 5000 BRIT, Mumbai, India) was used to check the eff ect of shorter exposure time for the same dose of radiation. Equal numbers of fourth instar larvae from the same generation were kept outside the gamma chamber for the same duration of time in a beaker containing tap water. All subsequent experiments were done with larvae that survived radiation exposure. Fifty larvae each from control and test groups were dissected to obtain 100 intact salivary glands. An average of 20 cells/gland was separated with fi ne needle and single intact cells were used for electrophoresis and extraction of genomic DNA.
All observations were made at three diff erent time-points, i.e., immediately after radiation exposure [referred here as 0 hour (0 h)], and at 24 h and 48 h during post irradiation recovery (PIR).

Alkaline single cell gel electrophoresis (ASCGE)
Single cell suspensions of salivary glands were mixed with 1.5 ml of 0.8% agarose solution (Sigma, St. Louis, MO, USA) at 42 ° C and carefully poured over pre-chilled frosted slides (Fisher Scientifi c, Carlsbad, CA, USA) to make a uniform layer.

Competitive enzyme linked immunosorbent assay (ELISA) for detection of oxidatively damaged DNA
Levels of 8-oxodG were estimated by competitive enzyme linked immune-sorbent assay (Modak et al. 2009). Genomic DNA was extracted from 100 SGC per sample (i.e., control larvae and larvae at diff erent recovery phases post exposure to radiation) following standard organic phase separation method (Sambrook et al. 1989).

Pulsed fi eld gel electrophoresis (PFGE)
DNA agarose plugs containing either salivary gland cells or cell nuclei were prepared under sterile conditions and were stored at 4 ° C after complete removal of lysis buff er containing additional 1 mg/ml Pronase-A (MBI Fermentas, Vilnius, Lithuania) (Cantor et al. 1988).

Restriction endonuclease digestion of DNA in agarose plugs
Agarose plugs were transferred to microfuge tubes (Tarson, Kolkata, India) containing 10 volumes of 1 ϫ restriction enzyme buff er-O (MBI Fermentas) for 30 min at room temperature. Th is was replaced with 3 volumes of 1 ϫ buff er-O containing 20 units of Nru-I restriction endonuclease (MBI Fermentas) and incubated at 37 ° C for 12 h to obtain complete restriction of embedded genomic DNA in plugs.

Migration of DNA from restricted and non-restricted DNAagarose plugs
High molecular weight DNA fragments resolvable in agarose gel showed diff erences between the migration patterns of Nru I restricted DNA in control and irradiated larvae. Low molecular weight DNA fragments observed in SGC of irradiated larvae at 0 h shifted towards high molecular weight DNA on the gel after 24 h PIR and this was more pronounced by 48 h PIR ( Figure 3A). Shift of fragment sizes were compared with molecular weight markers ( Figure 3B). Salivary gland cell nuclei plugs prepared from larvae exposed to higher dose rate radiation showed similar eff ects of radiation on high molecular weight DNA even without restriction digestion ( Figure 3C). Immediately after removal from radiation environment DNA damage was maximal.

Dynamic light scattering (DLS)
Ten whole larvae each of irradiated and non-irradiated groups from 0, 24 and at 48 h PIR were homogenized in 500 μ l of DNAzol solution (Molecular Research Centre, Cincinnati, OH, USA), centrifuged at 15,000 g for 30 min at 4 ° C and genomic DNA was precipitated from the supernatant. After RNase (Sigma) treatment, 1 μ g/ml of DNA in Milli Q water (Millipore, Darmstadt, Germany) was subjected to DLS (Malvern ZP, Worcestershire, UK) at 20 ° C fi xed scattering angle. Size by volume variations of DNA in solution was measured.

Statistical analysis
Alkaline comet assay and DLS results were analyzed using MicroCal, Origin Version 6, (Northampton, MA, USA). Mean, standard error ( Ϯ SE) for each group is depicted in fi gures. Statistical analysis was carried out using one-way analysis of variance (ANOVA) with p Ͻ 0.05 for alkaline comet assay. For DLS data, Student ' s t -test was used for analysis with p Ͻ 0.05.

Measurement of comets
Percent DNA in comet tail (% T-DNA), tail moment (TM) and tail length (TL) of comets were evaluated in radiation exposed SGC at 0, 24 and 48 h PIR to study the fate of DNA in individual cells ( Figure 1A). Total fl uorescence intensity of DNA in head and tail of comets showed wide variations among the control and irradiated groups immediately after exposure and up to 24 h PIR. Th is diff erence was not signifi cant among samples taken at 48 h PIR. Comparative values of percent DNA estimated from the tail moment and tail length clearly indicated that maximum damage occurred immediately after exposure to radiation stress and during PIR ( Figure 1B) these values came down to normal levels.

Estimation of 8-OxodG
A two-fold increase in the level of 8-oxodG 34,600 Ϯ 2200 nmoles to 73,800 Ϯ 1730 nmoles per μ g of DNA was observed immediately after exposure to radiation stress. Th e amount of 8-oxodG reduced signifi cantly (25,800 Ϯ 1220 nmoles) in DNA extracts prepared from SGC of larvae after 48 h post exposure to radiation stress indicating effi cient removal of modifi ed bases from the system by this time period (Figure 2).

Estimation of DNA fragmentation by dynamic light scattering (DLS)
Th e size by volume distribution of total DNA molecules in solution showed additional peaks from fragmented DNA in irradiated samples. Hydro dynamic diameter of control DNA sample, i.e., 122.42 d nm (diameter in nanometers) with 19.16% volume distribution (Figure 4Aa) changed to two peaks of 78.8 d nm with 12.7% volume distribution and 615.1 d nm with 8.7% volume distribution after gamma radiation (Figure 4Ab). After 24 h PIR, hydro dynamic diameter of DNA was 396.0 d nm with 15.0% volume distribution (Figure 4Ac). By 48 h post radiation, average size of larval DNA was 220.2 d nm with 13.9% volume distribution showing a reverse trend as it was found in the non-stressed condition (Figure 4Ad). A histogram plotted from three independent observations showed maximum polydispersity of DNA (i.e., larger hydrodynamic diameter) immediately after removal from stress conditions ( Figure 4B). Over a period of 24 and 48 h, the hydrodynamic diameter reduced gradually towards that found in control larvae. Importantly this is a trend where we did not fi nd a complete reversal to baseline levels unlike what was observed in the experimental results with 8-oxodG and the alkaline comet assay.

Discussion
DNA damage is the critical target of any ionizing radiationinduced biological endpoint. Extent of damage and inherent capacity to repair determines fate of survival of an organism under radiation stress. Some prokaryotes have evolved extremely effi cient mechanisms of DNA repair; allowing restoration of DNA structures even after very high doses of gamma radiation (Battista 1998, Billi et al. 2000. DNA repair mechanisms are well documented in radiotolerant eukaryotes K. D. Datkhile et al. 746 (Koval 1994, Elliott et al. 2000, but it is not considered as the primary mechanism for radiation tolerance in multicellular animals (Clegg 2001, Chandna et al 2004. Radiation tolerance in C. ramosus larvae was demonstrated by us in SGC in earlier studies (Datkhile et al. 2011), hence the current investigation was focused on the same tissue. Also, DNA content is higher in SGC compared to other diploid tissues of the midge larvae (Daneholt and Edstr ö m 1967, Macgregar and Varley 1988, Nath and Godbole 1997. Detailed dosimetry studies of C. ramosus had established that 2200 Gy dose was well tolerated by 80% larvae over a period of 24 h observation, hence this LD 20 dose was chosen for studying eff ect on DNA. Comet formation and PFGE have been chosen as preferred experimental methods to demonstrate DNA damage (Metzger and Iliakis 1991, Chaubey et al. 2001, Speit and Hartmann 2006, Herschleb et al. 2007). Differences in the tolerance dose could be noticed in the literature with reference to DNA damage experiments. Chandna et al. (2004), compared lepidopteran Sf-9 cell line with the threshold dose used for human BMG-1 glioma cell line. In our case, where whole organism is exposed to radiation, tolerance data from the cell lines would not be appropriate for comparison. Inclusion of other insect model systems for the comparison of the parameters between radioresistant and radiosensitive individuals at the whole organismal level would be useful in future. Here, we introduced DLS, a biophysical technique for measuring the size of molecules in suspension in order to assess radiation stress induced changes in DNA (Banerjee et al. 2014). Th e method is presently gaining momentum for evaluation of DNA and chromatin damage (Jain et al. 2014). To our knowledge this is the fi rst report of this method using an insect model organism, as well as optimization of PFGE for separation of large polytene DNA of Chironomid midge salivary gland cells . Resistance to migration in agarose gel was observed over time of PIR suggesting rejoining of damaged SGC DNA to higher molecular weight. Experimental conditions for PFGE studies (diff erent dose rates of gamma source) were designed to achieve the same dose of 2200 Gy in a shorter exposure time in order to confi rm that DNA fragmentation were indeed an eff ect of radiation and not any other physiological parameters that may prevail during the longer exposure time for the same dose. Indirect evidence for existence of oxidative damage control system was also shown for the fi rst time in C. ramosus by ELISA. Lower than basal levels of modifi ed 8-oxodG bases present in irradiated larvae (after 48 h in PIR) could be transient due to diff erences in the metabolic state of the two groups. Nevertheless, further studies are warranted to elucidate the mechanism of this remarkable system(s) of control of DNA damage caused by gamma radiation.

Conclusion
Evidence obtained in the present study in conjunction with our earlier fi ndings encourage us to hypothesize that in C. ramosus , tolerance to gamma radiation could be an integrative cellular defense mechanism comprising of control of DNA damage, role of antioxidant enzymes and radiation responsive stress proteins. Evolutionarily, the ancient history of existence of insects may be an appropriate justifi cation for such adaptability seen in Chironomus species.
Gamma radiation : DNA damage control in midges 747