Trichostatin A, an epigenetic modifier, mitigates radiation-induced androphysiological anomalies and metabolite changes in mice as evident from NMR-based metabolomics

Teena Haritwala, Kiran Maanb, Poonam Ranab, Suhel Parvezc, Ajay K. Singha, Subash Khushub and Paban K. Agrawalaa
aDepartment of Radiation Genetics and Epigenetics, Institute for Nuclear Medicine and Allied Sciences, Delhi, India; bNMR Research Centre, Institute for Nuclear Medicine and Allied Sciences, Delhi, India; cDepartment of Toxicology, Jamia Hamdard University, New Delhi, India

ABSTRACT
Purpose: Ionizing radiation is known to damage male reproductive system. Current study aims to study the mitigative effects of trichostatin A on male reproductive system and accompanying metabol- ite changes in testicular tissue of mice.
Materials and methods: Eight-week-old male C57 Bl/6J mice were exposed to 2 Gy c-radiation with or without trichostatin A administration. The animals were sacrificed at various time intervals for organ body weight index, sperm head abnormality assay, sperm mobility assay, and study of various metabo- lites in testicular tissue using NMR spectroscopy.
Results: Ionizing radiation induced no significant change in organ body weight index at any time points studied, however a significant increase in sperm head abnormality and significant decrease in sperm mobility was evident on fifth postirradiation week. trichostatin A administration, 1 and 24 h pos- tirradiation, could efficiently mitigate radiation-induced changes studied. NMR metabolome profile also showed prominent changes associated with energy metabolism, osmolytes and membrane metabol- ism at 24 h postirradiation and some of these changes (choline, glycerolphosphoethanol amine, and glycine) were persistent till fifth postirradiation week. Trichostatin A administration resulted in revert- ing metabolic profile of the irradiated animals to normal level suggesting its mitigative role. Conclusion: Results obtained suggest that trichostatin A could restore normal metabolic profile of tes- ticular tissue of irradiated male mice and also restored certain morphological and functional properties of sperms. Trichostatin A thus could further be exploited for its radio-mitigative properties.
ARTICLE HISTORY Received 6 March 2018 Revised 28 August 2018 Accepted 30 August 2018

KEYWORDS
Ionizing radiation; male reproductive system; 1H NMR; metabolome; trichostatin A; mitigation

Introduction
Ionizing radiation has brought several great benefits to man- kind ranging from power generation, medical diagnostics to therapy but with some health risks (Alonzo et al. 2008) that may range up to mortality depending on the extend of exposure. A protector administered prior to exposure or administration of a mitigator immediately or shortly after the exposure may help reduce radiation-induced injuries. To date only amifostin has been approved as a radioprotector for clinical use but under strict medical supervision due to its inherent toxicity and other limitations (Haritwal et al. 2017). Several agents are in advanced stage of approval as investi- gational new drug (IND) by the US FDA for use as protector and/or mitigator. Neupogen and neulasta have recently been approved IND status for use as radiomitigatiors of hemato- pietic acute radiation syndrome (H-ARS) through repurposing (Singh et al. 2018).
Several synthetic, semisynthetic, and herbal compounds have been extensively studied for their radioprotective or

mitigative activity in preclinical model systems with little or no advancement leading to clinical benefit so far. A series of histone deacetylase inhibitors (HDACi) have been discovered and synthesized in the past (Haritwal et al. 2017) and some have been reported to protect or mitigate against radiation injuries (Brown et al. 2008; Tiwari et al. 2017) in mice model. DNA metabolism including gene expression, DNA replication and repair requires access of the respective factors to the chromosomal DNA and the higher level of DNA organization in chromosome poses hurdles in those processes. HDACi results in hyperacetylation of histones and ultimately changes the DNA conformation and alters gene expression pattern (Marks et al. 2001). TSA (trichostatin A) is a highly potent HDACi that acts at nanomolar concentrations (Yoshida et al. 1990). TSA has been shown to enhance the cell growth and has significant effect on apoptosis too (Karagiannis et al. 2005).
Testes are one of the most radiosensitive organs due to presence of rapidly proliferating cells (Luo et al. 2014) and doses as low as 0.2 Gy can damage spermatogonia cells

CONTACT Paban K. Agrawala [email protected] Department of Radiation Genetics and Epigenetics, Institute for Nuclear Medicine and Allied Sciences, Delhi, India
Supplemental data for this article can be accessed here.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/irab.
Copyright ti 2018 Taylor & Francis Group LLC.

(Howell and Shalet 2005). Radiation exposure of testes results in many abnormalities in spermatogenesis, lowers the sperm count, induce abnormalities in spermatozoa and also induce defects in the functional characteristics which ultimately leads to permanent or temporary sterility (Shen and Ong 2000).
In the recent years, metabolomics has been used exten- sively to obtain unique insights to disease and health at both cellular and organism level (Stewart and Bolt 2011; Cheema et al. 2018). Metabolome has been considered to be the best indicator of an organism’s phenotype (Blow 2008). Metabolomic profile allows the simultaneous analysis of various metabolites in a single sample; it gives an over- view of the metabolic status that reflects the effect of any disturbances (stress/exposure) on the phenotype. In the pre- sent study, we have assessed the metabolic responses of the male reproductive system, testes of male mice in par- ticular, to radiation exposure using nuclear magnetic reson- ance (NMR)-based metabolomics and further correlated the mitigation potential of TSA against radiation-induced tes- ticular damage.
We hypothesize that TSA postirradiation administration will lead to a more open DNA conformation and facilitate the accessibility of repair proteins to damaged DNA. Thus it will enhance the repair of damaged DNA and also repair the anomalies caused due to radiation in cells and tissues (Katoch et al. 2013), including that of testes. In this study we aimed to identify potential metabolic changes in tes- ticular tissue following radiation and administration of TSA, an epigenetic modulator. Those results were correlated with the structural changes like testes weight and sperm head abnormality and functional changes like sperm motil- ity. To the best of our knowledge, this is the first study representing metabolomics and epigenetic mechanism of TSA to explain radiation-induced male reproductive sys- tem failure.

Materials and methods
Animals
Eight-week-old inbred C57Bl/6J male mice weighing 20 ± 2 g were used in the present study. The mice were maintained in a controlled environment (temperature20 ± 2 ti C and rela- tive humidity 40–60%) with constant 12 h light and 12 h dark schedule. The animals were fed with standard chow palates and tap water was provided ad libitum. All animal experi- ments were conducted strictly in accordance with the institu- tional animal ethics committee (IAEC) approval.

Irradiation
Whole body irradiation (WBI) was performed at a dose rate of 1 Gy/min using a Cobalt Tele therapy Unit, Bhabhatron II (Panacea Biotech, India). Mice were physically restrained dur- ing exposure in well aerated cages and no anesthesia was used.

Chemicals and reagents
All chemicals and reagents of highest quality and purity used were purchased from Sigma, St Louis, MO.

Experimental design
Mice were divided into four groups: Group 1 (C) served as untreated control, Group 2 (R) mice were exposed to 2 Gy WBI, Group 3 (D) mice were administered i.v. injection of 150 ng/kg TSA, two doses at a time interval of 24 h each, and
Group 4 (D þ R) mice were administered i.v. injection of 150 ng/kg TSA 1 and 24 h post 2 Gy WBI, sample collection were made considering second dose of TSA administration time as 0 h time point. Selection of TSA dose and schedule were based on our other studies that rendered maximum survival advantage (Figure S1, unpublished data). Mice were sacrificed at time points as depicted in figures after various treatments for sample collection and further studies. Three age-matched animal per group and three repeats (n ¼ 9 per group) were used for all assays except for metabolomics studies where no repeat experimentation were performed.

HDAC activity assay
HDAC activity was estimated in total nuclear protein extract of testes tissue from different treatment groups of mice at 2 h after second dose of TSA administration time using HDAC Activity Colorimertic Assay kit from Biovision (USA) as per the manufacturer’s instructions and Tiwari et al. (2017).

Body organ weight index
Body weight of each animal from all treatment groups was recorded before treatment and on the day of sacrifice. The testicle weight was recorded just after sacrifice. Testis body weight index {(testis weight in mg/body weight in mg) ti 100} of each treatment group was plotted.

Sperm head abnormality assay
Sperm head abnormality assay was performed using the method of Aduloju et al. (2008). The caput and cauda epi- didymis were dissected out from mice sacrificed at desired time points, minced in phosphate buffer saline (PBS) with fine scissors and forceps to release out spermatozoa and fil- tered with 70 mm nylon cell strainer (BD falcon) to remove tissue fragments. 1% Eosin Y was added to this suspension in a ratio of 10:1 and kept for 30 min. Air dried smears were prepared on clean, grease free slides and examined at 40 magnification of a light microscope. For each mouse 2400ti sperm cells were scored for morphological aberrations.

Sperm motility assay
Sperm samples prepared as described above without Eosin Y staining were observed under a light microscope at 40 magnification by placing them on a tihemocytometer. The percentage of motile sperms was calculated using the

number of motile sperms over total number of sperm scored.

Metabolomic profiling of testis
Immediately after sacrifice of animals at desired time points (24 h, 3 weeks, and 5 weeks) the testes were excised, weighed and frozen in liquid nitrogen and stored at –80 ti C until further analysis. The sample preparation was conducted according to (Beckonert et al. 2007). Briefly, 20 g% (w/v) tes- ticular tissue was made by homogenization of tissue in acetonitrile/water mixture (1:1 v/v) using a handheld hom- ogenizer (IKA T-10, Germany). The homogenate was centri- fuged at 12,000 g for 10 min at 4 ti C, the supernatant was lyophilized and stored at –80 ti C until NMR analysis. The sam- ple was reconstituted in 550 ml of deuterated water (D2O) containing 1 mM trimethylsilyl -2,2,3,3-tetradeuteropropionic acid (TSP) as internal standard and transferred to 5 mm NMR tubes for NMR spectroscopy. 1H NMR spectra were acquired for each sample at 600.33 MHz on a Bruker 600 MHz NMR spectrometer (Bruker Biospin, Switzerland) at 300 K using 1D pulse sequence with water saturation (NOESYGPPR1D). NMR acquisition parameters included a 90ti pulse, a 6 kHz spectral width, 64 transients, 64 K data points with 4 s. relaxation delay. Fourier transformed spectra obtained were phase and base line corrected and calibrated using TSP with Topspin software (version 3.5, Bruker, Switzerland). The metabolites were assigned on the basis of chemical shifts and signal multiplicity according to literature (Lindon et al. 1999; Griffin et al. 2000) and HMDB database. Identified metabolites were then integrated normalized with total spectral area to calcu- late the relative intensity (in %). Changes in the correspond- ing peak areas among experimental group were further examined using two-way ANOVA.

Statistics analysis
The data are presented as mean ± SE and were analyzed using two-way analysis of variance (ANOVA) with Bonferroni correction using Graph Pad Prism version 7. In all cases, a value of p < .05 was considered significant.

Results
HDAC activity assay
2 Gy radiation exposure significantly (p < .01) enhanced the HDAC activity in testicular tissue of mice as compared to the control mice that was reverted back to normal value by TSA administration in irradiated mice. TSA administration alone at the depicted dosage marked no change in HDAC activity as compared to control animals (Figure 1).

Testis body weight index
Drug or TSA (group 3) showed no apparent changes in testes weight compared to controls (group 1) at any of the time points studied. 2 Gy exposure (group 2) induced a time

dependent reduction in weight of the testes as evident from the testes body weight index that was at its maximum at 3-week time period and returned back to normal level at 5-week time point. TSA administration at 1 and 24 h postirra- diation (group 4) could restore testes body weight index to normal level at all the time (Figure S2).

Sperm head abnormality assay
Different forms of abnormal sperm morphologies were observed such as pin head, long hook, short hook, oval- shaped head, two heads, two tails and hook at wrong angle (Figure 2). The most common abnormality observed was pin- shaped sperm head. The total abnormal sperm head recorded in irradiated alone animals (group 2) were 7, 7.37, and 9.45% in mice for 1, 3, and 5 weeks time points, respect- ively. In control (group 1) mice, the values were 6.16, 6.54, and 6.66% respectively. The sperm head abnormalities recorded thus were significantly (p < .0001) high in irradiated group at fifth postirradiation week time point. TSA alone induced no significant alteration in sperm head morphology

Figure 1. The HDAC activity in testicular tissue homogenates of mice 2 h after various treatments was monitored colorimetrically. 2 Gy irradiation significantly (titip < .01) increased the HDAC activity as compared to untreated controls. TSA postirradiation administration effectively countered such increase. The data rep- resent mean ± SD of three independent experiments (n ¼ 9 per group).

Figure 2. The percent abnormal sperm head observed at various time points. The data presented are mean ± SD of three independent experiments with n ¼ 9 per group. titip < .01 and titititip < .0001. A significant (p < .0001) increase in abnormal sperm head was observed in irradiated animals at 5 weeks postir- radiation time point.

Figure 3. Representative photomicrographs of various sperm head abnormalities observed in irradiated animals, (a) with normal sperm head morphology, (b) with oval-shaped head morphology, (c) with pin head-shaped morphology, and (d) with double head.

at any time points studied. TSA postirradiation administra- tion, though significantly reduced abnormal sperm counts compared to irradiated animals, the values still were higher compared to control and drug alone treated groups (Figure 3).

Motility assay
Alteration in the number of motile sperms among various treatment groups was highly evident at fifth postirradiation week time point. Irradiation significantly reduced the number of motile sperms (p < .001) when compared with the control. The number of motile sperms was comparable to that of control or untreated animals in TSA and TSA plus irradiation

group animals at all time points studied (Figure 4).

Metabolomics analysis
NMR spectra and metabolite assignment of testicu- lar tissue
The representative 1H NMR spectrum of testicular tissue is shown in Figure 5. The peak location (ppm), multiplicity and integrated areas of NMR spectra provided information for peak assignments. The important metabolites observed in 1H NMR spectra of testicular tissue were mainly associ- ated with energy metabolism (creatine, citrate, lactate and adenosine/AMP), Osmolytes (taurine, betaine and myoinosi- tol), antioxidant (glutathione), amino acids (alanine, lysine, glycine, glutamate, glutamine and aspartate), and mem- brane lipid metabolites [phosphotidyl choline (PC),
Figure 4.: Radiation effect on percentage of motile sperms and modulating effects of TSA on it. Irradiation led to significant decrease in percent motile sperms at 3 weeks (p < .05) and 5 weeks (p < .0001) postirradiation time points. The data presented are mean ± SD of three independent experiments with n ¼ 9 per group, tip < .05, titip < .01, and titititip < .0001.

glycerophosphoethanol amine (GPE), choline]. The complete list of metabolites identified is presented in Table 1.

Metabolic response to radiation exposure
The 1H NMR spectra displayed several metabolites that were altered in testis at 24 h postirradiation compared to controls (Figure S3). The changes were followed till 5 weeks postirra- diation. Maximum changes were observed at 24 h postirra- diation time. There were total 18 metabolites identified as associated with metabolic response postradiation exposure. List of metabolites with significant increase or decrease

Figure 5. Representative high resolution 1H NMR spectrum showing different metabolites from testicular tissue. The identified metabolites were as follows: 1. branched chain amino acid, 2. lactate, 3. alanine, 4. acetate, 5. glutamine, 6. methionine, 7. glutamate, 8. glutathione, 9. citrate, 10. aspartate, 11. lysine, 12. creatine, 13. choline, 14. GPE, 15. betaine, 16. taurine, 17. myo-inositol, 18. glycine, and 19. phosphatidylcholine.

Table 1. Summary of NMR spectroscopy-based variation in testicular tissue compared to age-matched controls postirradiation time point.

Metabolites

Chemical shift with multiplicity (bracket)
24 h PI 3 weeks PI 5 weeks PI
R D D þ R R D D þ R R D D þ R

Lactate 1.33 (d), 4.10 (q) – – – – – – – – –

Alanine Acetate
N-Acetyl glutmate (NAG) Glutamate
1.48 (d), 3.79 (q) 1.92 (s)
2.16 (s)
2.10 (m), 2.36 (m), 2.50 (m)
– – "ti
"ti – –
"ti – –
– – –
"ti – "ti
– – –
– – –
– – –
–
–
–
–
–
–
–
–
–
–
–
–

Glutamine Methionine Glutathione Citrate
2.08 (m), 2.41 (m) 2.16 (m)
2.58 (m)
2.52 (d), 2.62 (d)
"ti – –
"ti – "ti
#ti – #ti
– – –
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–

Aspartate 2.63 (dd), 2.81 (dd) – – – – – – – – –
Lysine 2.96 (m) – – – – – – – – –

Creatine Choline
Glycerophosphoethanol amine (GPE) Betaine
Taurine
Myo inositol (mI) Glycine Adenosine/AMP Phosphotidyl choline
3.04 (s)
3.21 (s), 3.52 (m), 4.07 (m) 3.23 (t)
3.28 (s)
3.25 (t), 3.43 (t)
3.28 (t), 3.56 (dd), 3.63 (dd) 3.61 (s)
6.10 (d), 8.24 (s), 8.35 (s) 3.58 (m)
– – –
"ti – "ti
– – #ti
#ti – #ti
"ti – –
#ti – #ti
"ti – "ti
– – –
– – –
#ti – –
"ti – –
#ti – –
– – –
"ti – –
"ti – –
– – –
#ti – –
#ti – #ti
– –
"ti –
#ti –
– –
– –
– –
– –
#ti –
– –
–
–
–
–
–
–
–
–
–

R ¼ 2 Gy irradiated, D ¼ TSA administered, D þ R ¼ 2 Gy irradiated and TSA treated. p value for the changing metabolites were assessed using ANOVA and were less than .05. " indicates increase in the levels of selected metabolites; –, no change; # indicates relative decrease in the level of selected metabolite. Keys: s: singlet; d: doublet; t: triplet; m: multiplet. ti indicates p< 0.05.

based on relative intensities (%) at different time points pos- tirradiation is presented in Table 1 and actual values of sig- nificant metabolites are presented in Table S1. ANOVA-based analysis of relative integrals of identified metabolites showed significant decrease in glutathione, inositol and betaine levels and significant increase in methionine, choline and taurine levels compared to controls at 24 h postirradiation. At 3- week postirradiation, number of metabolite with significant altered levels were lesser compared to 24 h time point. Most of the changes at this time were associated with membrane metabolites like choline, PC, GPE, and myoinositol. Energy metabolites creatine and adenosine/AMP were also
significantly reduced. No significant change was observed in metabolites except adenosine, choline and GPE at 5 weeks postirradiation compared to age-matched controls (Figure 6). The longitudinal changes in radiation group demonstrated continued increase in choline levels till fifth week (p ¼ .033). However, most of the metabolites reverted back to normal level at fifth postirradiation week time point. Myoinositol (p ¼ 10ti 6; p ¼ .001 at 3 and 5 weeks, respectively) and beta- ine levels (p ¼ .001; p ¼ .027 at 3 and 5 weeks, respectively) were significantly increased at third and fifth week compared to 24 h time point (Figure 6). Reduction in energy metabolite (adenosine, creatine) levels were continued at third and fifth

Figure 6. Longitudinal changes in radiation only group till fifth week postirradiation. Data presented as mean ± standard deviation with overall difference using repeated measure ANOVA (p < .05). tisignificant comparison with 0 h, # significant comparison with 24 h, † significant comparison with 3 weeks.

postirradiation week time point as no significant change in levels were observed compared to 24 h time point.

Metabolic changes of TSA response on radiation exposure To look for TSA effect on radiation response, NMR spectros- copy of testicular tissues were carried out in animal group
injected with D only and D þ R group. The analysis showed that animals with drug only group did not show any signifi- cant changes in metabolites levels compared to control but D þ R group rendered profound alterations in metabolite lev- els against radiation exposure when compared. The detailed significant observations are tabulated in Table 1 at different
postirradiation time points. At 24 h postirradiation D þ R group showed more or less similar changes as observed in radiation only group. However, at 3-week time point, there
was profound change in metabolites levels in D þ R group compared to radiation group as most of the changes observed in this group reached normal level (Figure 7). Similar changes were also observed at 5 weeks postradiation as no significant changes observed in any of the metabolites in D þ R group compared to controls.
Discussion
Diallyl sulfide, sulforaphane, EGCG, and trichostatin A are HDAC inhibitors with different levels of HDAC inhibition potency and origin. Our laboratory has been exploring if such HDAC inhibitors can ameliorate radiation-induced dam- ages by reducing acute effects of radiation in cell culture and mouse model (Katoch et al. 2012, 2013; Tiwari et al. 2017). In this study, the ability of TSA in mitigating radiation- induced reproductive system anomalies in male mice has been studied following our earlier observations on the ability of TSA to enhance survival of lethally irradiated mice using the same treatment schedule (unpublished data, Figure S1). Radiation doses as low as 2 Gy can significantly damage reproductive system of mice (Howell and Shalet 2005), par- ticularly the process of spermatogenesis which takes about 5 weeks or nearly 35 days to complete.
In the current study, 2 Gy radiation exposure induced a significant increase in HDAC activity in testes tissue that was effectively neutralized by postirradiation TSA administration. The observation corroborated our earlier findings where an increase in HDAC activity shortly after irradiation was

Figure 7. Relative changes in levels of identified metabolites from 1H NMR spectra in control , radiation Drug only , and Drug plus Radiation group at 24 h, 3 weeks, and 5 weeks postirradiation. Data presented as relative levels with respect to total spectral area ± standard deviation with overall difference
using one way ANOVA with Bonferroni’s comparison test (p < .05). tisignificant comparison with controls and # significant comparison with radiation group.

observed (Tiwari et al., 2017). The lowest testes body weight index recorded at 3 weeks postirradiation time period in group 2 (radiation alone) that may be due to radiation- induced germinal epithelial cells loss, whereas notable recov- ery in testes body weight index was seen in group 4 (Radiation þ TSA) animals at 3 weeks postirradiation time. Similar pattern of weight loss in testis has earlier been reported with different doses of radiation (Jagetia et al. 1998). Sperm head abnormality assay has been used as a reliable short term detection method for genotoxicity of chemical or mutagens (Giri et al. 2002). Various types of defects in the morphology of sperm head arising as a result of radiation exposure were recorded in this study. Highest numbers of abnormal sperm head were found in the radi- ation alone group indicating the affect being induced by irradiation whereas the percentage of abnormal sperm heads were significantly reduced in the irradiated plus TSA treated group. It has been shown earlier that, sperm morphology is one of the best predictor of fertilization potential (Wyrobek et al. 1983). A high proportion of morphologically abnormal spermatozoa in the exposed group therefore indicate the possible association between radiation exposure and defect- ive spermiogenesis that may lead to reduced fertility. Our observations are in agreement with the results from ear- lier studies which have shown increased sperm defects in Chernobyl salvage workers exposed to radiation and health workers with occupational exposure (Fischbein et al. 1997). Significant hypermethylation of sperm DNA has been reported by Kumar et al. (2013) from occupationally exposed individuals. Hypermethylation and other epigenetic changes may lead to higher sperm abnormalities and reduced fertility. In the current study, though no direct study was performed
for DNA hypermethylation or histone acetylation status, an increased level of methionine and choline was observed at early time point which may lead to hypermethylation. The abnormalities observed might have occurred due to point mutations or epigenetic alterations caused by radiation in the spermatogonia and early spermatocytes at the premei- otic stage of spermatogenesis (Hugenholtz and Bruce 1983). This may lead to carcinogenicity in the somatic cells of next generation (Aduloju et al. 2008) as well. Dysfunctional mito- chondria and other mechanism like as oxidation of glycolytic enzyme and DNA fragmentation may contribute to motility changes in spermatozoa (Ramos and Wetzels 2001). Restoration in sperm motility following postirradiation TSA administration, as was observed in this study, thus indicates restoration of cellular integrity with respect to DNA and energy metabolism.
The metabolic response to radiation in testicular tissue provides valuable information regarding possible mechanism of radiation-induced anomalies on male reproductive system. Creatine was observed as the dominant signal in the testis spectra that indicates the requirement of creatine to main- tain sperm cell viability and function (Lee et al. 1988). The spectra also had prominent signals form adenosine whose high concentration along with uridine in testis and seminal vesicle decreased creatine level along with increased alanine and decreased adenosine levels at 3 weeks postirradiation could be correlated with reduced energy levels and per- turbed energy metabolism. It is considered that testicular tis- sue needs a higher energy state to continuously maintain spermatogenesis and reduced energy levels are suggestive of spermatocyte degeneration following radiation exposure that was evident from the observed lower number of viable

and motile sperms (Figure 4). In addition to energy metabol- ism, in male reproductive system, creatine metabolic path- way plays an important role in supply of adenosine as a signaling agent. Since several types of adenosine receptors have been identified in the testis, altered levels of adenosine affect intracellular cAMP levels which in turn disrupt the physiological functions of sperm (Rivkees, 1994). Decreased creatine and adenosine levels in testicular tissue in our study clearly indicate the influence of these metabolites on sperm viability and function following radiation exposure. Another important metabolite that is markedly seen in testis spec- trum is glutathione which is a prevalent antioxidant present in testis (Griffin et al. 2000). Reduced levels of glutathione seen at 24 h time point clearly points toward radiation- induced oxidative stress. However, these changes subsided at later time points. Our findings further illustrated changes in choline, glycerophosphoethanolamine (GPE), and phos- photidyl choline (PC). Choline is the substrate for most abun- dant membrane lipids phosphotidyl choline and sphingomyelin. Increased choline and decreased phospho- tidyl choline levels might be due to loss of spermatozoa membrane integrity as a result of radiation-induced oxidative stress as high lipid content on sperm membrane may pro- vide an ideal site for free radical attack. Likewise, decreased GPE levels in testis may suggest decreased phosphoethanol amine that could limit the sperm growth. Myoinositol, which
plays an important role in spermatozoa maturation (Eisenbergand Bolden 1964), also showed changes in radi- ation group compared to controls. Reduced myoinositol observed in our study extends support for radiation-induced poor maturation of sperms. The higher level of sperm head abnormalities thus can be a result of radiation-induced reduction in testicular membrane metabolites having a role in maintaining structural integrity of cells while radiation- induced reduction in metabolites responsible for energy pro- duction justify a significant reduction in sperm mobility. Taurine, an osmolyte known to maintain cell viability by
maintaining the osmotic balance, showed significant increased level till 3 week postirradiation time point could be suggestive of disturbed osmotic balance following radi- ation exposure and could also be one of the mechanisms
leading to radiation-induced spermatogenesis failure. Metabolomics analysis further revealed that betaine, a methyl donor, decreased in the testicular tissue in animals exposed to radiation. Low betaine and high methionine observed at 24 h are consistent with hypermethylation pat- tern. Ionizing radiation is known to induce DNA hypermethy- lation (Aypar et al. 2011). In a study on occupational hazard workers, DNA hypemethylation along with increased sperm abnormalities and sperm DNA fragmentations was observed (Kumar et al. 2013). Radiation-induced hypermethylation may implicate defective chromatin condensation resulting in mor- phologically abnormal spermatozoa. Trichostatin A, an HDAC inhibitor, is also shown to have profound effect on DNA methylation (Ou et al. 2007). Reduction in abnormal sperma- tozoa thus could be attributed to this effect of TSA. Metabolomics study further affirm the mitigation potential of TSA as in our study we did not find any significant changes

except phosphotidyl choline (PC) and alanine in TSA treated (D þ R) group compared to age-matched controls at 3- and
5-week postirradiation time point (Table 1). However, changes were still persistent in radiation only group com- pared to controls.
In recent times, it is evident that metabolomics can uncover global changes in molecular signatures and meta- bolic pattern for a disease state or any environmental stress along with mechanistic information. The current study com- pares and correlates the metabolic changes resulting from radiation exposure to their alteration as a result of TSA pos- tirradiation administration that can ameliorate radiation induced reproductive damages in mice model.

Conclusion
In the current study, an attempt has been made to correl- ate the structural and functional alterations in mice testes and spermatogenesis following irradiation with the changes in metabolomic profile. Also the effects of an epi- genetic modifier, trichostatin A, in mitigating those altera- tions were studied. The results obtained indicate a good correlation of structural and functional changes in repro- ductive system of mice not only with the metabolomic profile but also TSA as a potential radiomitigator. Further extensive studies, however, are necessary in order to valid- ate the observations.

Notes on contributors
Teena Haritwal is a DST sponsored researcher currently working on radiation effects on mice reproductive system.
Kiran Maan is a UGC sponsored researcher currently working on radi- ation induced metabolomic changes in mice.
Poonam Rana, PhD, is Scientist E in NMR Research Centre, INMAS and currently involved in identification of imaging and metabolic markers for radiation stress using advanced MR techniques. Her area of research interest include MR imaging, metabolomics and NMR spectroscopy. Suhel Parvez, Professor and Head of Toxicology Department of Jamia Hamdard University, is keenly interested in behavioral biology of mice and other model organisms.
Ajay K. Singh is Director of INMAS, Delhi and his main interest is in nuclear medicine.
Subash Khushu is head of NMR research centre, INMAS and is interested in medical imaging and identification of metabolomic markers of stress. Paban K. Agrawala is keenly interested in the epigenetic alterations induced due to stress and radiation countermeasure development.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The work was supported by DRDO, Govt. of India. TH was supported by a Department of Science and Technology (DST, India) fellowship (DST Inspire) and KM was supported by University Grants Commission (UGC), India for carrying out research, [INM 313].

ORCID
Paban K. Agrawala http://orcid.org/0000-0002-7460-0231

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