B

Day 1_0Gy Day 1_8Gy

DIM 1

DIM

2

-0.0

6-0

.04

-0.0

2-0

.00

0.02

0.04

0.00 0.02 0.04-0.02-0.04

Top 50 Ions From RF – 100% Accuracy

A C

-0.2 -0.1 0.0

Upregulated in 8Gy GroupS Plot

p[1]P(Loadings)

p(c

orr)

[1]P

(C

orre

lati

on)

0.1 0.2Downregulated in 8Gy Group

0.0

0.2

0.4

0.6

0.8

1.0

-1.0

-0.8

-0.6

-0.4

-0.2

Day1_0Gy Day1_8Gy0.2 0.60.2 1

Colour Key

Supplementary Figure S1: UPLC-ESI-TOFMS GI metabolomics 1 day post-radiation exposure: CD2F1 mice were either sham irradiated or exposed to study 8Gy of γ radiation. The mice were euthanized 1 day, post-radiation exposure for blood and organ collection. Comparative metabolomic profiling of GI tissue was performed as described (see methods). Panel A. OPLS loadings S-plot comparing features from control GI (sham) with irradiated group. Panel B. Two dimensional accuracy plot for top 50 features interrogated using Random Forests. The X-axis denotes the interclass separation while the Y-axis displays the intra-class variability. Panel C. Heat map visualization of the feature rankings comparing relative levels in control and irradiated GI samples. Each row represents a unique feature with a characteristic mass to charge and retention time value.

Supplementary Figure S2. Determination of chemical structure of metabolite (m/z 203.11) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the ion with m/z 203.11 from GI tissue extract, while the bottom panel shows the fragmentation for standard D-Tryptophan.

Tissue Extract TOF MS/MS 203.11 ES-

D-Tryptophan TOF MS/MS 203.11 ES-

Supplementary Figure S3. Determination of the chemical structure of metabolite (m/z = 150.10) in GI tissue by tandem mass spectrometry in electrospray positive mode. Top panel shows MS/MS fragmentation spectrum of the parent ion GI tissue extract while the bottom panel shows the fragmentation for standard methionine.

Tissue extract

standard

Tissue Extract TOF MS/MS 150.10 ES+

Methionine TOF MS/MS 150.10 ES+

Supplementary Figure S4. Determination of the chemical structure of metabolite (m/z=148.10) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion from GI tissue extract while the bottom panel shows the fragmentation for standard Glutamic acid.

Tissue Extract TOF MS/MS 148.10 ES+

Glutamic acid TOF MS/MS 148.10 ES+

Supplementary Figure S5. Determination of the chemical structure of metabolite (m/z= 179.049) in GI tissue by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion from GI tissue extract while bottom panel shows the fragmentation for standard Cys-Gly.

Tissue Extract TOF MS/MS 179.049 ES+

Cys-Gly TOF MS/MS 179.049 ES+

Supplementary Figure S6. Determination of the chemical structure of lipid (PS(16:0/0:0)) with m/z 496.275 and retention time of 1.0176 minutes in GI tissue extracts by matching the fragmentation pattern with that obtained from the standard.

Matched Unmatched

m/z

Inte

nsi

ty

255.2361

409.2421

496.2751

140.0131

196.0404

Matched Unmatched

m/z

Inte

nsi

ty

Supplementary Figure S7. Determination of the chemical structure of lipid (PE(16:0/0:0)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 452.285 and RT 1.2883.

452.2851

Supplementary Figure S8. Determination of the chemical structure of lipid (PE(20:1/0:0)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 506.334 and RT 1.3263.

140.0131 196.0404

Matched Unmatched

m/z

Inte

nsi

ty

506.3342

S. No m/zRT

(minutes)ESI

ModeFold Change (Rad/Sham)

Radiation Dose

P-Value

1 425.9190 0.30 POS ↑ 4 Gy 0.01

2 299.2801 4.86 POS ↑ 4 Gy 0.01

3 564.8312 9.39 POS ↑ 4 Gy 0.05

4 507.8622 8.43 POS ↓ 4 Gy 0.002

5 682.7991 0.25 POS ↓ 4 Gy 0.001

6 332.0132 0.31 NEG ↑ 4 Gy 0.002

7 835.5733 9.24 NEG ↑ 4 Gy 0.04

8 632.4750 7.25 NEG ↑ 4 Gy 0.03

9 469.7843 9.48 NEG ↓ 4 Gy 0.05

10 411.7872 9.52 NEG ↓ 4 Gy 0.05

11 614.2880 2.94 POS ↑ 8Gy 0.03

12 804.5525 7.54 POS ↑ 8Gy 0.01

13 198.1003 1.61 POS ↑ 8Gy 0.01

14 527.3984 8.42 POS ↓ 8Gy 0.03

15 493.3199 6.17 NEG ↑ 8Gy 0.04

16 761.5066 8.87 NEG ↑ 8Gy 0.002

17 189.9880 1.18 NEG ↑ 8Gy 0.004

18 809.1861 1.21 NEG ↑ 8Gy 0.05

19 371.1052 1.40 NEG ↓ 8Gy 0.05

20 436.8698 9.39 NEG ↓ 8Gy 0.01

Supplementary Table ST1: Unidentified putative markers of IR exposure at Day 1.

Supplementary Figure S9. Box-and-whisker plots of putative biomarkers of radiation injury of GI tissue in CD2F1 mice. The features that were significantly altered in the irradiated GI tissue (4 & 8Gy) at day 1 were shortlisted via multivariate data analysis, putatively identified by accurate mass based search using Madison Metabolomics Consortium Database (MMCD) and confirmed by matching fragmentation pattern with standard compounds.

Day 1_0Gy Day 1_8Gy

LMGP01011254 (502.315_0.6916)

Day 1_0Gy Day 1_4Gy

LMGP01010612 (538.387_2.2977)

Day 1_0Gy Day 1_8Gy

LMGP03050002 (496.275_1.0176)

Day 1_0Gy Day 1_4Gy

LMGP02050002 (452.285_1.2883)

Day 1_0Gy Day 1_8Gy

Methionine (150.059_0.3822)

Glutamic acid (148.061_0.3308)

Day 1_0Gy Day 1_8Gy

Cys-Gly (179.049_0.3636)

Day 1_0Gy Day 1_4Gy

D-Tryptophan (203.082_1.4182)

Day 1_0Gy Day 1_8Gy

LMGP02050020 (506.334_1.3263)

Day 1_0Gy Day 1_8Gy

B

Day 4_8Gy Day 4_0Gy

DIM 1

DIM

2

-0.0

6-0

.04

-0.0

2-0

.00

0.02

0.04

0.00 0.02 0.04-0.02-0.04

Top 50 Ions From RF – 100% Accuracy

A C

-0.2 -0.1 0.0

Upregulated in 8Gy GroupS Plot

p[1]P(Loadings)

p(c

orr)

[1]P

(C

orre

lati

on)

0.1 0.2Downregulated in 8Gy Group

0.0

0.2

0.4

0.6

0.8

1.0

-1.0

-0.8

-0.6

-0.4

-0.2

Day4_0Gy Day4_8Gy

Supplementary Figure S10: UPLC-ESI-TOFMS based GI metabolomics at 4 days post-IR exposure: CD2F1 mice were either sham irradiated or exposed to study 8Gy of γ radiation and euthanized after 4 days. Comparative metabolomic profiling of GI tissue was performed as described (see methods). Panel A. OPLS loadings S-plot comparing features from sham with irradiated group. Panel B. Two dimensional accuracy plot for top 50 features interrogated using Random Forests. The X-axis denotes the interclass separation while the Y-axis displays the intra-class variability. Panel C. Heat map visualization of the feature rankings comparing relative levels in control and irradiated GI samples. Each row represents a unique feature with a characteristic mass to charge and retention time value.

Supplementary Figure S11. Determination of the chemical structure of metabolite (m/z = 146.20) by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion from GI tissue extract while the bottom panel shows the fragmentation for standard spermidine.

Tissue Extract TOF MS/MS 146.20 ES+

Spermidine TOF MS/MS 146.20 ES+

Supplementary Figure S12. Determination of the chemical structure of metabolite (m/z = 311.16) by tandem mass spectrometry. Top panel shows MS/MS fragmentation spectrum of the parent ion with m/z 311.169 while the bottom panel shows the fragmentation for standard Eicosenoic acid.

Tissue Extract TOF MS/MS 311.16 ES+

Eicosenoic acid TOF MS/MS 311.16 ES+

Supplementary Figure S13. Determination of the chemical structure of metabolite (m/z = 606.0739) by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion while the bottom panel shows the fragmentation pattern for standard UDP-N-Acetyl-glucosamine.

Tissue Extract TOF MS/MS 606.06 ES-

UDP-N-Acetyl-glucosamine TOF MS/MS 606.06 ES-

Supplementary Figure S14. Determination of the chemical structure of metabolite (m/z = 514.27) by tandem mass spectrometry. Top panel shows MS/MS fragmentation of the parent ion while the bottom panel shows fragmentation pattern of standard Taurocholic acid.

Tissue Extract TOF MS/MS 514.27 ES-

Taurocholic acid TOF MS/MS 514.27 ES-

Supplementary Figure S15. Determination of the chemical structure of lipid (PE(O-20:0/20:5)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 778.547 and RT 5.0338.

Matched Unmatched

m/z

Inte

nsi

ty

742.5486

778.5473

241.0149

255.5363

279.237

553.2868

833.5335

Matched Unmatched

m/z

Inte

nsi

ty

Supplementary Figure S16. Determination of the chemical structure of lipid (PI(18:2/16:0)) with m/z 833.534 and retention time of 4.3 minutes in GI tissue extracts by matching the fragmentation pattern with that obtained from the standard.

Supplementary Figure S17. Determination of the chemical structure of lipid (PI(18:1/18:1)) in GI tissue by SimLipid 3.0 (Waters). tandem mass spectrometry with m/z 861.565 and RT 4.7912.

241.0153

281.2524

Matched Unmatched

m/z

Inte

nsi

ty

861.5652

Supplementary Figure S18. Determination of the chemical structure of lipid (PI(18:0/0:0)) with m/z 599.331 and retention time of 1.4 minutes in GI tissue extracts by matching the fragmentation pattern matching with the standard.

283.2679

437.2751

599.3305

Matched Unmatched

m/z

Inte

nsi

ty

S. No m/z RT ModeFold Change (Rad/Sham)

Radiation Dose

P-Value

1 83.0215 0.28 POS ↑ 4 Gy 0.05

2 441.0753 0.45 POS ↑ 4 Gy 0.03

3 111.1175 1.90 POS ↓ 4 Gy 0.001

4 231.0298 7.08 POS ↓ 4 Gy 0.05

5 736.5543 8.25 POS ↓ 4 Gy 0.05

6 834.2472 0.48 NEG ↑ 4 Gy 0.03

7 162.0214 0.44 NEG ↑ 4 Gy 0.002

8 364.1799 3.13 NEG ↑ 4 Gy 0.05

9 227.9867 0.30 NEG ↓ 4 Gy 0.001

10 445.0260 0.45 NEG ↓ 4 Gy 0.05

11 197.1203 2.14 POS ↑ 8Gy 0.05

12 811.5382 9.41 POS ↑ 8Gy 0.001

13 562.0088 0.36 POS ↓ 8Gy 0.03

14 431.0908 0.36 POS ↓ 8Gy 0.003

15 71.05067 0.32 POS ↓ 8Gy 0.02

16 630.1026 0.40 NEG ↑ 8Gy 0.04

17 708.1268 1.17 NEG ↑ 8Gy 0.05

18 457.0717 0.42 NEG ↑ 8Gy 0.003

19 112.0393 0.34 NEG ↓ 8Gy 0.001

20 469.7843 9.48 NEG ↓ 8Gy 0.004

Supplementary Table ST2: Unidentified putative markers of IR exposure at Day 4.

Supplementary Figure S19: Box-and-whisker plots of putative biomarkers of IR injury of GI tissue in CD2F1 mice. The features that were significantly altered after 4 days of IR exposure were shortlisted via multivariate data analysis, putatively identified by accurate mass based search using Madison Metabolomics Consortium Database (MMCD) and confirmed by matching fragmentation pattern with standard compounds.

Day 4_0Gy Day 4_8Gy

LMGP02050011 (476.287_1.0965)

Day 4_0Gy Day 4_8Gy

LMGP06010002 (861.565_4.7912)

Day 4_0Gy Day 4_8Gy

Taurocholic acid (514.285_3.1882)

Day 4_0Gy Day 4_4Gy

Spermidine (146.166_0.2437)

Day 4_0Gy Day 4_4Gy

LMGP02020084 (778.574_5.0338)

Day 4_0Gy Day 4_4Gy

LMGP06010847 (833.534_4.2999)

UDP (606.077_0.3992)

Day 4_0Gy Day 4_8Gy

Eicosenoic acid (311.169_0.1542)

Day 4_0Gy Day 4_4Gy

LMGP06050004 (599.331_1.4042)

Day 4_0Gy Day 4_8Gy

Supplementary Figure S20. Global metabolome visualization of GI tissue in sham or IR treated CD2F1 mice. Panels A and B. Self-organizing Maps (SOMs) reveal the effect of IR exposure after 1 and 4 days respectively. The data were acquired by UPLC-QTOF MS, pre-processed using XCMS and the feature intensities were normalized to internal standards and to total protein concentration. Top 100 features were used to construct SOMs which create a series of coherent mosaic heat maps representing overall ion profile in each sample. Tiles containing the highest-abundance ions are shaded in deep red, while those containing low abundance ions are shaded deep blue. The areas highlighted in black boxes show the change in the intensity of metabolites in GI tissue in a time and dose dependent manner.

Panel A: Day 1

Panel B: Day 4

0GY 4GY 8GY

0GY 4GY 8GY

Time and radiation dose Top Functions -log(p-value) Ratio Molecules Involved in each pathway

Day 1_4Gy

Arachidonic Acid Metabolism 1.71E01 7.28E-02 Prostaglandin J2,prostaglandin E2,epoprostenol,20-hydroxy-leukotriene B4,prostaglandin D2,prostaglandin C2,lipoxin A4,5,6-epoxytetraene,thromboxane A2,prostaglandin B2,prostaglandin A2,prostaglandin J2,prostaglandin h2,12-keto-leukotriene B4,lipoxin B4

Eicosanoid Signaling 1.44E01 8.86E-02 prostaglandin h2,prostaglandin E2,epoprostenol,prostaglandin D2,lipoxin A4,thromboxane A2,lipoxin B4

MIF-mediated Glucocorticoid Regulation

1.68E00 2.38E-02 prostaglandin E2

MIF Regulation of Innate Immunity

1.58E00 2E-02 prostaglandin E2

Day 1_8GyGlutathione Metabolism 1.82E+00 1.12E-02 Glutathione oxidized, glutathione reduced, microsomal glutathione S-

transferase 3Metabolism of Xenobiotics by Cytochrome P450

1.53E+00 5.10E-03 microsomal glutathione S-transferase 3

Arachidonic Acid Metabolism 1.49E+00 4.85E-03 prostaglandin J2,prostaglandin E2,epoprostenol,20-hydroxy-leukotriene B4,prostaglandin D2,prostaglandin C2,lipoxin A4,5,6-epoxytetraene,thromboxane A2,prostaglandin B2,prostaglandin A2,prostaglandin J2,prostaglandin h2,12-keto-leukotriene B4,lipoxin B4

Aryl Hydrocarbon Receptor Signaling

1.45E+00 6.29E-03 microsomal glutathione S-transferase 3

NRF2-mediated Oxidative Stress Response

1.33E+00 5.24E-03 microsomal glutathione S-transferase 3

Day 4_4GyEIF2 Signaling 2.4E00 9.9E-03 Eicosenoic acidFatty Acid Elongation in Mitochondria

2.03E00 2.17E-02 PE(16:0/0:0), PS(16:0/0:0)

β-alanine Metabolism 1.65E00 1.12E-02 AlanineBile Acid Biosynthesis 1.55E00 9.52E-03 Taurocholic acidLysine Degradation 1.51E00 7.3E-03 LysineValine, Leucine and Isoleucine Degradation

1.47E00 9.35E-03 Valine, isoleucine

Day 4_8GyRiboflavin Metabolism 1.94E00 2E-02 hydroquinonePyruvate Metabolism 1.49E00 7.19E-03 pyruvaldehyde, pyruvic acidGlycine, Serine and Threonine Metabolism

1.38E00 6.76E-03 pyruvaldehyde

Tyrosine Metabolism 1.37E00 5.05E-03 hydroquinone

Supplementary Table ST3: Functional pathway analysis of canonical pathways enriched in response to IR exposure

B

Day 1_4Gy_KidneyDay 1_4Gy_GI

DIM 1

DIM

2

-0.0

6-0

.04

-0.0

2-0

.00

0.02

0.04

0.00 0.02 0.04-0.02-0.04

Top 50 Ions From RF – 100% Accuracy

A C

Day1_4Gy_GI Day1_4Gy_Kidney0.2 0.60.2 1

Colour Key

Number of variables

RF

Acc

ura

cy [

%]

020

4060

8010

0

60 80 1004020

Supplementary Figure S21: UPLC-ESI-TOFMS based comparative metabolomic profiling of GI and Kidney tissue at 4 days, post-IR exposure: CD2F1 mice were either sham irradiated or exposed to 4Gy of γ radiation and euthanized after 4 days. Panel A. Two dimensional separation plot for top 50 features interrogated using Random Forests. Panel B. Normalized accuracy plot showing unambiguous separation of the two groups Panel C. Heat map visualization of feature rankings comparing relative levels in the irradiated GI and Kidney tissue. Each row represents a unique feature with a characteristic mass to charge and retention time value.

Supplementary Table ST4. Putative serum biomarkers of radiation injury in C57BL6 mice irradiated with 4 & 8 Gy gamma radiation after 1 and four days respectively.

4 Gy 8 Gy 4 Gy 8 Gy

Day 1 1 Glutamic acid 148.068 0.4703 POS ↑ ↑ 0.04 0.012 D-Tryptophan 203.083 1.3468 NEG ↓ ↓ 0.03 0.033 PC(8:2/8:2) 502.327 2.2362 POS ↑ ↑ 0.02 0.024 PE(20:1/0:0) 506.324 4.9385 NEG ↓ ↓ 0.04 0.05

Day 4 5 Spermidine 146.164 0.2553 POS ↓ ↑ 0.01 0.046 PI(18:1/18:1) 833.564 6.5222 NEG ↑ ↑ 0.01 0.04

Fold Change p-valueS. No Metabolite m/z RT ESI Mode