Madhu Bala

*Radiation Biology Department, Institute of Nuclear Medicine and Allied Sciences Brig. S K Mazumdar Marg, Delhi, India* 

### **1. Introduction**

The bio-positive effects of exposure to small doses of environmental stressors such as radiation, chemicals and mutagens have been reported since long. A number of studies and reviews (Bala & Mathew, 2000; Luckey, 2008; Pandey et al., 2006; Sasaki et al., 2002) document that exposure to small doses of ionizing radiation enhanced the tolerance towards the detrimental effects of lethal doses of ionizing radiation given subsequently. Such phenomenon was observed in prokaryotes as well as in eukaryotes. Some of the laboratory studies with human lymphocytes are summarized in Table 1. The information on beneficial effects of low dose irradiation also poured in from the epidemiological studies (reviewed by Bala & Mathew, 2000; Dasu & Denekamp, 2000; Luckey, 2008). The populations exposed to high background radiation showed long term beneficial effects such as increased life span, enhanced immune system, decreased cancer mortality and cancer risk (Calabrese et al., 2001; Cohen, 1999; Nambi & Soman, 1987, UNSCEAR, 2000). Among the A-bomb survivors from Hiroshima and Nagasaki, those, who received doses lower than 200 mSv, showed no increase in cancer deaths. Further, the population which received doses below 100 mSv, showed decrease in the mortality caused by leukemia in comparison to the age-matched control cohorts (UNSCEAR, 1994).

Often in epidemiological studies the exposure to low levels of radiation was for longer duration, while in laboratory studies the exposure to low level radiation was for a shorter duration (sometimes even a pulse exposure). Nonetheless, beneficial effects were observed in short as well as in prolonged exposures. This strongly suggested that the low dose radiobiological studies could have bearing in diverse and important applications such as radiation protection, risk assessment and radiotherapy. It was, therefore, considered important to initiate investigations for understanding more about the mechanisms of radioprotective effects caused by pre-exposure to low doses of radiation. It was reported that the resistance to lethal doses of ionizing radiation could be induced not only by low doses of radiation but also by variety of agents other than radiation, *viz.* heat, pH, nutrients, UV rays, though, the genes and the molecular pathways affected in these cases differed with the inducing agent. (Bala & Goel 2007; Boreham & Mitchel, 1991; 1994; Boreham et al.,2000). Further, it was believed that the induced resistance to lethal doses of radiation by short term pre-exposure to low doses of radiation was transient in nature and the radiation doses

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 151

were conducted with cultured human lymphocytes to investigate some of the key events

The diploid strain D7 of *Saccharomyces cerevisiae* with genotype a/: *trp5-12/trp5-27, ilv1- 92/ilv1- 92* (Zimmermann *et al.*, 1975) was used because it allowed quick detection of mutants, recombinants and survivors. The strain D7 of *S. cerevisiae* had functional defects in *TRP* gene (heteroallelic) and *ILV* gene (homoallelic) making it auxotrophic for tryptophan and isoleucine. Presence of two different inactive alleles within tryptophan locus (*trp5- 12/trp5-27*) caused nutritional requirement for tryptophan which could be recovered by gene conversion to form fully active wild type gene, thereby alleviating the need for tryptophan requirement. The resultant colonies after gene conversion could be scored on synthetic medium lacking tryptophan. The presence of two defective copies of alleles at isoleucine locus *(ilv1-92/ilv1-92)* caused the nutritional requirement of isoleucine which could be corrected by reverse mutation. The resultant colonies could then be scored on the synthetic medium lacking isoleucine. The usefulness of this organism to understand radiation responses and their modification has been demonstrated (Bala & Jain, 1994, Bala & Jain,

Cultures of *S. cerevisiae* were grown on yeast extract peptone dextrose medium (YPD; 1% yeast extract powder, 2% peptone, 2% dextrose, HiMedia, India) at 30+1 0C. The cells were harvested, washed and suspended in phosphate buffer (PB, 67 mM, pH 6.0; 4x107cells/ml). The cell suspension was cooled to 4 0C, and irradiated with 60Co- gamma- radiation using Gamma Cell-220 (Atomic energy, Canada; dose rate 0.0078 Gy/s) or Gamma Cell-5000 (BRIT, India; dose rate 1.26 Gy/s). After low dose irradiation, cell suspension was maintained at 30+1 0C till the subsequent exposure to lethal doses of radiation (LD50, 400 Gy). The survivors, gene convertants and revertants were estimated using defined synthetic complete (DSC), tryptophan omission (TRP-) and isoleucine omission (ILV-) medium, respectively as described (Bala & Goel, 2007). While survivors were expressed as a fraction of unirradiated controls, the gene convertants and revertants were expressed as fraction of CFUs on respective omission medium to the CFUs on DSC medium after the corresponding treatment. The RIR was calculated in terms of percent changes in survivors, convertants and

s / c /r pre irradiated s / c /r non pre irradiated X 100 % Change s / c /r from non pre irradiated cells 

Where, s=survivors; c=convertants/106 survivors; r=revertants/106 survivors (after 400 Gy) RNeasy Mini Kit and OneStep rt-PCR Kit (Qiagen, Germany) was used to isolate total RNA and carry out rt-PCR respectively (Bala & Goel, 2007). Table 2 enlists the gene specific primers (synthesized from IDT, Coralville). Reverse transcription was at 50 oC for 30 min, followed by incubation at 95 oC for 15 min. The amplification was for 25 cycles. The denaturation was at 94 oC for 45 s; annealing at 60 oC for 45 s; extension at 72 oC for 60 s and the final extension was at 72 oC for 10 min. The PCR products were separated on 1% agarose gel, stained with ethidium bromide and quantified using Lab Works software, version 4.0

that were observed in *S. cerevisiae.*

**2.1 Studies with** *Saccharomyces cerevisiae*

1996; Bala & Goel 2004; Bala & Goel 2007).

revertants as below:

(s,c,r)

required to induce beneficial effects varied qualitatively as well as quantitatively from organism to organism. To understand the genetic basis of the phenomenon of low dose induced radioresistance in a comprehensive manner, we chose two different model systems i.e. the microbe *Saccharomyces cerevisiae* and cultured human peripheral blood lymphocytes. The unicellular eukaryote *Saccharomyces cerevisiae* was used to carry out the basic studies and perform the genome wide search for identifying the affected genes. The cultured human peripheral blood lymphocytes were employed to study the role of select genes. In order to minimize the load of mutations, it was considered important to select the smallest possible doses of ionizing radiation for pre-exposure. The term radiation-induced radioresistance (RIR) was introduced (Bala & Goel, 2007) to explain the phenomenon of radioresistance to lethal doses of ionizing radiation, which was (i) specifically induced by a single preexposure to sub-lethal doses (causing not more than 10% death of population, < LD10 ) of ionizing radiation, and (ii) was transient in nature. The conventional term 'radio-adaptive response' was avoided because the term 'adaptation' in one of the several senses referred to the evolutionary transformations, where new stable behavioral patterns evolved due to prolonged exposure to the environmental stress. This review chapter presents some of our important findings.


Table 1. Summary of some important studies performed to understand low dose response in human lymphocytes.

#### **2. Materials and methods**

The studies were performed sequentially with two different types of cells. Initial studies were executed with the unicellular eukaryotic microbe, *Saccharomyces cerevisiae*, where the focus was to identify the effects of inducing radiation doses and dose rates; the beneficial effects induced in terms of survival, mutagenesis and recombinogenesis; for conducting the genome wide search to identify the affected genes; and reconfirming the role of cell cycle, DNA repair and mitochondrial genes in inducing beneficial effects. The subsequent studies were conducted with cultured human lymphocytes to investigate some of the key events that were observed in *S. cerevisiae.*

### **2.1 Studies with** *Saccharomyces cerevisiae*

150 Gamma Radiation

required to induce beneficial effects varied qualitatively as well as quantitatively from organism to organism. To understand the genetic basis of the phenomenon of low dose induced radioresistance in a comprehensive manner, we chose two different model systems i.e. the microbe *Saccharomyces cerevisiae* and cultured human peripheral blood lymphocytes. The unicellular eukaryote *Saccharomyces cerevisiae* was used to carry out the basic studies and perform the genome wide search for identifying the affected genes. The cultured human peripheral blood lymphocytes were employed to study the role of select genes. In order to minimize the load of mutations, it was considered important to select the smallest possible doses of ionizing radiation for pre-exposure. The term radiation-induced radioresistance (RIR) was introduced (Bala & Goel, 2007) to explain the phenomenon of radioresistance to lethal doses of ionizing radiation, which was (i) specifically induced by a single preexposure to sub-lethal doses (causing not more than 10% death of population, < LD10 ) of ionizing radiation, and (ii) was transient in nature. The conventional term 'radio-adaptive response' was avoided because the term 'adaptation' in one of the several senses referred to the evolutionary transformations, where new stable behavioral patterns evolved due to prolonged exposure to the environmental stress. This review chapter presents some of our

> First *in vitro* experiment with human lymphocytes, reduction in chromosomal aberration was greater at higher pre-irradiation dose.

Pre-exposure of human lymphocytes to [3H] dThd reduced the number of mutations at *hprt* locus by 1.5 or 3.0 Gy of X-rays.

Only stimulated and not G0 lymphocytes, pre-irradiation with low dose of X-rays showed survival benefit against high doses of X-rays.

The beneficial effects of low dose depended upon total dose, dose rate of pre-irradiated dose but not on dose rate of challenge dose in

Elevated level of PCNA, cyclin D1, cyclin A in human cell line preirradiated with gamma-rays, may play a role in cell cycle regulation

Implication of PBP74 in low dose irradiated human tumour cell lines HT29 and MCF-7 with gamma-rays within 30 min after irradiation.

Seo *et al.*, 2006 Role of p27Cip/Kip in the induction of radio-adaptive response in

Table 1. Summary of some important studies performed to understand low dose response in

The studies were performed sequentially with two different types of cells. Initial studies were executed with the unicellular eukaryotic microbe, *Saccharomyces cerevisiae*, where the focus was to identify the effects of inducing radiation doses and dose rates; the beneficial effects induced in terms of survival, mutagenesis and recombinogenesis; for conducting the genome wide search to identify the affected genes; and reconfirming the role of cell cycle, DNA repair and mitochondrial genes in inducing beneficial effects. The subsequent studies

important findings.

Olivieri *et al.,* 1984

1987

Shadley & Wiencke, 1989

Boothman *et al.*, 1996

Carette *et al.*, 2002

Sanderson & Morley, 1986

Shadley & Wolff,

human lymphocytes.

**2. Materials and methods** 

**References Important findings**

human lymphocytes.

gamma-irradiated RIF cells.

and DNA repair.

The diploid strain D7 of *Saccharomyces cerevisiae* with genotype a/: *trp5-12/trp5-27, ilv1- 92/ilv1- 92* (Zimmermann *et al.*, 1975) was used because it allowed quick detection of mutants, recombinants and survivors. The strain D7 of *S. cerevisiae* had functional defects in *TRP* gene (heteroallelic) and *ILV* gene (homoallelic) making it auxotrophic for tryptophan and isoleucine. Presence of two different inactive alleles within tryptophan locus (*trp5- 12/trp5-27*) caused nutritional requirement for tryptophan which could be recovered by gene conversion to form fully active wild type gene, thereby alleviating the need for tryptophan requirement. The resultant colonies after gene conversion could be scored on synthetic medium lacking tryptophan. The presence of two defective copies of alleles at isoleucine locus *(ilv1-92/ilv1-92)* caused the nutritional requirement of isoleucine which could be corrected by reverse mutation. The resultant colonies could then be scored on the synthetic medium lacking isoleucine. The usefulness of this organism to understand radiation responses and their modification has been demonstrated (Bala & Jain, 1994, Bala & Jain, 1996; Bala & Goel 2004; Bala & Goel 2007).

Cultures of *S. cerevisiae* were grown on yeast extract peptone dextrose medium (YPD; 1% yeast extract powder, 2% peptone, 2% dextrose, HiMedia, India) at 30+1 0C. The cells were harvested, washed and suspended in phosphate buffer (PB, 67 mM, pH 6.0; 4x107cells/ml). The cell suspension was cooled to 4 0C, and irradiated with 60Co- gamma- radiation using Gamma Cell-220 (Atomic energy, Canada; dose rate 0.0078 Gy/s) or Gamma Cell-5000 (BRIT, India; dose rate 1.26 Gy/s). After low dose irradiation, cell suspension was maintained at 30+1 0C till the subsequent exposure to lethal doses of radiation (LD50, 400 Gy). The survivors, gene convertants and revertants were estimated using defined synthetic complete (DSC), tryptophan omission (TRP-) and isoleucine omission (ILV-) medium, respectively as described (Bala & Goel, 2007). While survivors were expressed as a fraction of unirradiated controls, the gene convertants and revertants were expressed as fraction of CFUs on respective omission medium to the CFUs on DSC medium after the corresponding treatment. The RIR was calculated in terms of percent changes in survivors, convertants and revertants as below:

$$\% \text{ Change}\_{\{\text{s,c,r}\}} = \frac{\text{s / c / r \, pre-irradiated} - \left(\text{s / c / r \, non \, pre-irradiated}\right) \times 100}{\text{s / c / r \, from \, non-pre-irradiated \, cells}}$$

Where, s=survivors; c=convertants/106 survivors; r=revertants/106 survivors (after 400 Gy)

RNeasy Mini Kit and OneStep rt-PCR Kit (Qiagen, Germany) was used to isolate total RNA and carry out rt-PCR respectively (Bala & Goel, 2007). Table 2 enlists the gene specific primers (synthesized from IDT, Coralville). Reverse transcription was at 50 oC for 30 min, followed by incubation at 95 oC for 15 min. The amplification was for 25 cycles. The denaturation was at 94 oC for 45 s; annealing at 60 oC for 45 s; extension at 72 oC for 60 s and the final extension was at 72 oC for 10 min. The PCR products were separated on 1% agarose gel, stained with ethidium bromide and quantified using Lab Works software, version 4.0

Radiation Induced Radioresistance – Role of DNA Repair and Mitochondria 153

for first 13 h and 90 sec pulse for next 7h) at 200 V, using CHEF DRII (BioRad, USA), to

Heparinized vacutainers (Griener, Astria) were used to draw 3-5 ml venous blood from healthy, non-smokers, non-alcoholic male donors (age 25-30 years). The blood was layered on the ficoll-histopaque column (Sigma Aldrich Chemicals, USA) and centrifuged at low speed at 26+2°C, the interface between plasma and histopaque comprising PBMCs was collected and washed three times with serum free RPMI-1640 (HiMedia, India). The washed cells were suspended @1×106 cells/ml in complete RPMI-1640 containing 10% fetal bovine serum, 100 units/ml penicillin sodium salt, 100 μg/ml streptomycin sulphate, 2 mg/ml sodium bicarbonate. Phytohemagglutinin (PHA, Difco, Hamburg, Germany) was added to stimulate the cells proliferation. The cultures were setup in 96 well flat-bottomed micro titer plates (Tarson, India) at 37°C, 5% CO2. Each well had 150 μl volume containing 1.5×105 cells. The 22-24 hour old cultured PBMCs were irradiated first with low dose of 60Co-γ-radiation (0.07 Gy, using Gamma Cell GC 220, Canada dose rate 0.0078 Gy/s) and then after suitable time interval with lethal dose of 60Co-γ-radiation (5.0 Gy, using Gamma Cell-5000, BRIT,

The cell proliferation was quantified using Hoechst 33342. The cells were washed at least three times with saline in microtiter plate, freshly prepared Hoechst 33342 solution in serum free RPMI (10 μg/ml) medium was added and the suspension was incubated at 37°C for 30 min. (Blaheta et. al., 1991). Fluorescence was measured at ex 355 nm and em 460 nm in

To score the micronuclei, cytochalasin B (Sigma Aldrich Chemicals, USA) was added at 44 hour after initiation of human PBMC culture and the cells were harvested at 72 hour. 1×106 cells were washed, the cell pellet was suspended in 200 µl carnoy solution (methanol: acetic acid, 3:1) and incubated at 4°C for 2 hour. This cell suspension was laid on the chilled slides, dried overnight at 26 2°C and stained with hoechst 33342 (10 μg/ml) at 26 2°C for 30 min. in dark. Micronuclei were counted at ex 355 nm and em 460nm as per criteria described (Fenech, 1993). At least 1000 cells per sample were scored at 1000× magnification

Protein extraction and Western blotting was as per procedures standardized in our laboratory (Bala & Goel, 2007). Briefly, 4x107 cells/ ml of *S. cerevisiae* were lysed and treated with 160 ml of 50% trichloroacetic acid (TCA), washed with 1.5 ml of chilled acetone, resuspended in 100 ml of extraction buffer (4% SDS; 0.16 M Tris-Cl, pH 6.8; 20% Glycerol; 0.38 M b-mercaptoethanol) and heated for 4 min at 95 oC. For extracting proteins from PBMCs, standard protocol was used. Briefly 5x106 cells were suspended in PB containing protease inhibitors for 1.5 hours at 4 oC. The cells were ruptured by sonication and soluble proteins were collected after centrifugation in cold. Total soluble proteins were quantified by using Bradford's reagent and resolved by one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using Mini-PROTEAN II (BIO-RAD, US). Gels were stained with Coomassie brilliant blue R-250. Electro-blotting was on nitrocellulose membrane

**2.2 Studies with cultured human Peripheral Blood Mononuclear Cells (PBMC)** 

resolve genomic DNA into a number of chromosomal bands.

India; dose rate 1.26 Gy/s).

under oil immersion.

**2.3 Western blotting** 

fluorescence spectrophotometer (Varion, Australia).

(UVP Inc., U.K.). Real-time one-step rt-PCR kit with SYBR green as flourophore (Qiagen, Germany) was used as per manufacturer's protocol to perform quantitative rt-PCR using iCycler (Bio-RAD, US, software version 2.1). The fold changes were determined by calculating the fold change in threshold cycle (Δ Ct').



Where Ct: threshold cycle

Table 2. Primers for genetic studies with *Saccharomyces cerevisiae* 

For microarray studies, the labeled cDNA was synthesized from total RNA by using CyScribeTM First-Strand cDNA Labeling Kit (Amersham Biosciences). Either Cy3-dUTP or Cy5-dUTP was incorporated into the cDNA of samples under comparison. The cDNAs were dried in a vacuum trap. Pre-printed DNA microarrays with complete set of 6400 Open Reading Frames (ORFs) of *S. cerevisiae* genome (Microarray Centre of the University Health Network, Toronto, Canada), were used in this study. The slides were first hybridized in prehybridization solution (6x SSC, 0.5% SDS, 1% bovine serum albumin) for 1 h and then hybridized overnight with labeled probe at 42 °C in a water bath. Before using as a hybridization probe, the labeled cDNA was re-suspended in 40 µl of hybridization solution (50% Formamide, 6x SSC, 5x Denhardt's, 0.5% SDS, 20 µg of poly(A) and salmon sperm, Invitrogen). For each test, cDNAs from the un-irradiated control and from the stress dose irradiated samples were together hybridized on to one chip. Further, for each test, two different hybridizations were performed by swapping the fluorochromes to cross check the transcriptional changes, if any, due to experimental procedures. At least two DNA microarrays were analyzed for each test condition. The chips were scanned at a resolution of 10 µm and data was analyzed using GenePix Pro 4.0 analysis software (Axon Instruments, Union City, CA).

To study the DNA damage in individual chromosomes by pulsed field gel electrophoresis, the samples were prepared as described earlier (Bala & Jain 1996, Bala & Mathew, 2002). In brief, the cell suspension was washed with PB, centrifuged; pellet was treated with lyticase enzyme and then immobilized in low melting agarose plugs using the mould provided by BioRad USA. The plugs were first treated with LET buffer [0.5 *M* EDTA pH 8.0, 0.01 *M* Tris(hydroxymethyl)-aminomethane pH 7.0, 7.5% Mercaptoethanol] for 20 h at 37 oC. The LET buffer was removed, plugs were washed two times with NDS buffer [0.01 *M*  Tris(hydroxymethyl)-aminomethane pH 7.0,7.5% EDTA pH 8.0, 1% n-luaryl sarcosine] . The plugs were then treated with NDS buffer containing 2mg/ml Proteinase K for 20 h at 48 oC. Sufficient washings were given in EDTA (0.5 *M,* pH 8.0) thereafter. The plugs were stored at 4 oC before electrophoresis. The pulsed-field gel electrophoreses (PFGE) was for 20 h (60 sec for first 13 h and 90 sec pulse for next 7h) at 200 V, using CHEF DRII (BioRad, USA), to resolve genomic DNA into a number of chromosomal bands.
