Changes In Gene Expression Induced by Tienilic Acid and Sulfamethoxazole: Testing the Danger Hypothesis

Figures & data

FIG. 1 Tienilic acid and sulfamethoxazole structures.

TABLE 1 Primer sequences for qRT-PCR

Gene	Forward Primer	Reverse Primer
SerDH	5′-TGCAAGATGAAGGCAAAACA-3′	5′-CAGCATCTCTCCCACCACTT-3′
Caspase-12	5′-CATCAGAATCCAGGGGAAAAG-3′	5′-CTGGATTCCCTGAGGAACTG-3′
Cadherin-17	5′-CCCAGAAGGTTCTCAGGTATTG-3′	5′-CATTCCACTGCACCTGAGTG-3′
Spectrin β II	5′-AAGAGCTGTCCCTCTGGATC-3′	5′-AGCTGCATTCCTTCCTTCTC-3′
DUSP6	5′-TCTTTGGCTCCACTATACGCAA-3′	5′-ATCCAGGCAATAGGTTTGCTTC-3′
p27^Kip1	5′-CGACCTGCGGCAGAAGAT-3′	5′-ATTCGGAGCTGTTTACGTCTGG-3′
IGFBP-1	5′-TCTCAGCATGAAGAGGCAAA-3′	5′-AGGGCTCCTTCCATTTCTTG-3
Inhibin βA	5′-AGATCATCACCTTTGCCGAG-3′	5′-GTTCACTCCTCTCCCCCTTC-3′
AKR1B7	5′-CCCAGACAGGCCTTATGCTA-3′	5′-CATCGCTTGTGACAAACAGG-3′
Yc2/GSTA5	5′-CCTGCCTATGAGAAGGTGTTG-3′	5′-TCTCAGGGCCTTTAGCAGAG-3′
GCLC	5′-CGAGGTGGAGTACATGTTGGTG-3′	5′-AGCCTAGCCTGGGAAATGAAGT-3′
AKR7A3	5′-AGCGGCTGCAAAGTAAAAAT-3′	5′-ATGCCCTGGTACACAGTTGG-3′
IgGFcRIIβ	5′-CACCATCACTGTCCAAGAGC-3′	5′-GGAGTGTCTGGAACCTGCTT-3′
SGK1	5′-CACCTCCTCCAAGTCCCTC-3′	5′-TAGAACAGCTCTCCGCCATT-3′
TrxR	5′-AGCTAAGGAGGCAGCCAAAT-3′	5′-GCCATAATTTTGTGAGGACCAA-3′
MST1	5′-CCCCAATGACCACAAGTACA-3′	5′-GGAACTTTTCAGGGTGGAAA-3′
IRF-1	5′-AGCTGCAACAGATGAGGATGA-3′	5′-CTATCTCGATGTCCCCTCCA-3′
IL-1β	5′-TCGACAGTGAGGAGAATGACC-3′	5′-AGGACGGGCTCTTCTTCAA-3′
Epoxide hydrolase	5′-AGATCGAAGGGCTTGACATC-3′	5′-AGGCCTTTCACGTGGTTG-3′
p57^Kip2	5′-CCAATGCGAACGACTTCTTC-3′	5′-CTGGCCGTTAGGCTCTAAACTA-3′
SOCS2	5′-AACAGGATGGTACTGGGGAAG-3′	5′-CCGTTTGTCCTTGCACATCT-3′

FIG. 2 (A) Changes at 6 hr of genes differentially regulated at both 6 and 24 hr. The top bar for each gene is the data from the microarray, the middle bar is the data from the same mRNA analyzed by quantitative PCR, and the bottom bar represents the data from an independent experiment involving four treated and four control animals analyzed by quantitative PCR. By microarray, it was found that 42 genes were significantly differentially regulated at both 6 hr and 24 hr. In this figure, the focus is on 9 of these genes that are involved in cell stress and the promotion of liver regeneration or cell growth. (B) Changes at 24 hr of genes differentially regulated at both 6 and 24 hr. Graphed are the same 9 genes graphed in the same manner but at the 24 hr timepoint. Data for both graphs are expressed as log₂ values, and all gene expression relative to GAPDH. All qRT-PCR were repeated 2 to 3 times, and run in triplicate. Standard error bars indicate variability between PCR runs.

TABLE 2 Brief description of genes involved in liver regeneration, Cell-cycle promotion, intercellular adherence, and cell polarization

Gene name	Function
IGFBP-1 (Insulin-like growth factor binding protein-1)	Binds to IGF-I and regulates IGF bioavailability; conflicting findings, as some data show enhanced action while other data suggest diminished action of IGF-I upon IGFBP-1 binding; known to be transcriptionally mediated, and one of most abundant early genes in regenerating liver
SerDH (serine dehydratase)	Catalyzes deamination of L-serine and threonine to pyruvate and α -ketobutyrate, respectively; inhibited in regenerating liver
DUSP6 (Dual-specificity phosphatase 6; also called MKP3)	Dephosphorylates (inactivates) extracellular signal-regulated kinase (ERK); ERK1/2 promotes cell survival by preventing Bcl-2 from ubiquitination; decreased DUSP6 promotes cell survival
Inhibin βA	Activin A (inhibin βA:βA dimer) autocrine inhibitor of DNA sy5nthesis; decreased activin A levels promote liver regeneration
Spectrin β II	Maintain cell shape, establish polarity, involved in formation of distinct membrane domains; crucial for hepatocyte differentiation and intrahepatic bile duct formation in murine embryonic liver
p27^Kip1	Down-regulation promotes hepatocyte entry into the cell cycle, allowing activation of genes coding for proteins required for S phase of cell cycle
p57^Kip2	Plays role in developing liver; expression has negative effect on cell growth and serves to inhibit hepatocyte proliferation; acts to curb cell proliferation
Cadherin-17	Mediates intercellular adhesion, has role in morphogenesis during embryogenesis and polarization of cells; also called liver-intestine cadherin in rats and thought to play role in morphological organization of adult liver and intestine
SOCS2 (Suppressor of cytokine signaling 2)	Represses activities of growth hormone, thought to act by competitive inhibition of STAT5- and SHP2-binding sites on growth hormone receptor.

TABLE 3 Brief description of genes involved in oxidative stress, inflammation and Cytotoxicity, and Liver regeneration

Gene name	Function
AKR7A3 (Aldo-keto reductase 7A3; also called aflatoxin B1 aldehyde reductase or AFAR)	8,9-AFB1-epoxide can hydrolyze to AFB1 dihydrodiol, which can rearrange to a dialdehyde; AKR7A3 very efficient at catalyzing reduction of AFB1 dialdehyde to dialcohol
GCLC (glutamate-cysteine ligase catalytic subunit)	Major determinant of overall GSH synthesis capacity by catalyzing glutamate-cysteine reaction (first step in GSH formation); transcriptionally regulated by Nrf1 and Nrf2
GSTA5/Yc2 (glutathione-S-transferase α Yc2 subunit; also called GSTA5)	Along with AKR7A3, very effective transferase activity towards 8,9-AFB1-epoxide
TrxR (thioredoxin reductase 1)	TrxR reduces thioredoxin, making it available for disulfide reduction; TrxR can also reduce disulfides itself. Along with the GSH pathway, a major intracellular disulfide-reducing system
SGK1 (serum and glucocorticoid inducible protein kinase 1)	Involved in a number of cellular stress pathways; under acute transcriptional control; controls cellular responses to a variety of extracellular signals
Epoxide hydrolase	Cleave oxiran derivatives to yield the corresponding diols; major role to control chemically reactive and destructive epoxides that arise from xenobiotic metabolism in vivo
IL-1β (interleukin-1β)	Many roles, notably acute phase response and pro-inflammatory; produced by Kupffer cells and sinusoidal endothelial cells during liver regeneration; stimulates proliferation of thymocytes and B cell maturation and proliferation
IgGFcRIIβ (component of low-affinity Fc receptor)	Component of low-affinity Fc receptor of immunoglobulin; expressed only by macrophages, NK cells and neutrophils; mediates phagocytosis and antibody-dependent cell-mediated cytotoxicity
IRF-1 (interferon regulatory factor 1)	Mediates the effects of interferon; crucial to NK cell development; pro-apoptotic role; mediates lymphocyte infiltration in vivo
MSP1 (macrophage stimulating protein 1)	Controls growth, movement and morphogenesis; expression peaks early in liver regeneration. Also an inflammatory mediator

FIG. 3 Genes differentially regulated at 6 hr only. Shown here are the data on 12 genes of > 300 genes that were significantly different by microarray from control at 6 hr. These genes were chosen because they are associated with oxidative stress, inflammation, cytotoxicity or are promoters of liver regeneration. Similar to Figure 2, the top bar for each gene is the data from the microarray, the middle bar is the data from the same mRNA analyzed by quantitative PCR, and the bottom bar represents the data from an independent experiment involving 4 treated and 4 control animals analyzed by quantitative PCR. Data are expressed as log₂ values, and all gene expression relative to GAPDH. All qRT-PCR were repeated 2 to 3 times, in triplicate or duplicate. Standard error bars indicate variability between PCR runs.

TABLE 4 Genes differentially regulated at 6 hr that were of interest to our study objective, but on which qRT-PCR was not performed

Gene	Probe set #	T1	T2
Protein tyrosine phosphatase 4a2	1367971_at	−0.4	−0.3
Protein tyrosine phosphatase 2E	1368087_a_at	−0.4	−0.3
Nucleolar protein 5	1368173_at	0.5	0.3
Glycoprotein (transmembrane) nmb	1368187_at	0.5	0.6
Baculoviral IAP repeat-containing 2	1368291_at	−0.9	−0.7
Apoptosis antagonizing transcription factor	1368330_at	0.4	0.5
Pyruvate kinase, liver and RBC	1368651_at	−0.8	−0.4
PRKC, apoptosis, WT1, regulator	1368702_at	−0.5	−0.3
Orosomucoid 1	1368731_at	−0.4	−0.7
mutL homolog1 (E. Coli) hypothetical gene supported by NM_031053	1369965_at	0.8	0.7
Laminin, alpha 3	1370538_at	0.8	0.7
Cyclin D1	1371150_at	−1.1	−0.9
Thrombospondin 2 (predicted)	1374529_at	1.5	1.4
Vinculin (predicted)	1375538_at	0.6	0.4
Hematopoietically expressed homeobox	1387270_at	−1.4	−0.4
NAD(P)H dehydrogenase, quinone 1	1387599_a_at	0.5	0.7
Retinoblastoma 1	1388185_at	−0.9	−0.5
Aldo-keto reductase 1D1	1398310_at	0.6	0.5
Programmed cell death 4	1383328_x_at	−0.3	−0.4
Carbonic anhydrase	1394551_at	−0.6	−0.9
CD36 antigen (collagen type I receptor, thrombospodin receptor)-like 2	1395327_at	1.6	2.6
Neural precursor cell expressed, developmentally down-regulated gene 9 (predicted)	1396053_at	−1.0	−0.8
Enoyl-coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase	1368283_at	0.6	0.4
Aldehyde dehydrogenase family 1, subfamily A4	1368718_at	0.8	0.3
Alcohol dehydrogenase 4 (class II), pi polypeptide	1371470_at	0.4	0.3
One cut domain, family member 1	1371034_at	−2.5	−1.6
Similar to Glutathione S-transferase, theta 3	1371942_at	−0.8	−0.5
Coenzyme Q6 homolog (yeast) (predicted)	1372774_at	0.5	0.5
A disintegrin and metalloprotease domain 23 (predicted)	1374635_at	1.0	0.9
Osteoglycin (predicted)	1385248_a_at	1.3	1.6
Interleukin 6 receptor	1386987_at	−0.3	−0.6
Hepcidin antimicrobial peptide	1387396_at	−1.2	−1.6
Protein kinase (cAMP dependent, catalytic) inhibitor beta	1387459_at	0.8	0.7
Fibroblast growth factor receptor 4	1389801_at	−0.5	−0.4
HIV-1 Tat interactive protein 2 (predicted)	1390208_at	0.7	0.5
Translocase of inner mitochondrial membrane 8 homolog a (yeast)	1390237_at	0.4	0.5
Solute carrier family 25, member 30	1390416_at	−2.1	−0.8
Autophagy 7-like (S. cerevisiae) (predicted)	1390436_at	−1.3	−0.9
Interferon (alpha and beta) receptor 1 (predicted)	1382769_at	−0.5	−0.3
RT1 class I, A3	1393108_at	−1.3	−0.6
Myosin X (predicted)	1393639_at	−0.7	−0.5
Eukaryotic translation elongation factor 1 alpha 1	1395586_at	1.4	1.8
Aspartate-beta-hydroxylase (predicted)	1395745_at	0.5	0.5

Data are expressed in log₂ space. T1, treated animal #1; T2, treated animal #2.

TABLE 5 Fold-changes in Hepatic gene expression induced by treatment with SMX, either 6 or 24 hr after SMX treatment

Gene name	SMX 6 hr	SMX 6 hr	SMX 6 hr	SMX 24 hr	SMX 24 hr
Prkar2a	−02.08	−01.84	−01.84	1.17	−01.03
Nope	1.40	1.76	2.28	1.19	1.12
Zfp36	−02.14	−01.58	−01.71	1.01	−01.00
Aggf1	1.22	1.13	1.06	1.00	1.14
Cirbp	−06.52	−07.36	−05.96	−01.08	1.10
LOC433800	−01.93	−02.02	−01.79	1.00	−01.00
Cd52	−09.37	−011.35	−08.20	−01.07	−01.10
Ddx5	−02.27	−02.32	−01.81	1.05	1.11
Rpl12	−02.06	−02.38	−01.83	1.03	1.03
S100a9	−02.45	−02.78	−02.88	−01.03	1.00
LOC433432	−02.22	−02.58	−01.67	−01.11	−01.08
Mbnl1	−02.26	−02.46	−02.31	1.01	−01.02
Gng10	−01.97	−02.20	−01.83	1.11	1.03
Rpl9	−01.60	−02.28	−01.51	−01.24	−01.02
Adh4	1.27	1.45	2.12	1.37	−01.05
LOC545288	2.46	2.01	2.54	1.40	1.26
Mrps24	−01.83	−01.62	−01.93	1.12	1.03
2410012M04Rik	−02.02	−02.99	−01.92	−01.80	−02.22
Mt1	1.05	1.00	1.12	1.01	−01.11
1700020I14Rik	−02.65	−02.08	−01.76	−01.02	−01.01
Ucp2	−03.38	−03.68	−02.47	1.07	−01.21
Rnf170	−02.03	−01.27	−01.25	1.22	1.08
Becn1	−01.49	−01.04	−01.18	−01.02	−01.06
1110038D17Rik	−01.36	−01.19	−01.24	1.03	−01.02
6720463E02Rik	−08.98	−011.37	−09.07	1.04	−01.06
Crry	−01.58	−02.10	−01.65	−01.06	−01.03
Got1	−08.79	−08.55	−09.54	−01.45	1.03
Atrx	−03.27	−03.55	−03.02	−01.01	−01.18
Ganab	−029.17	−022.6	−023.86	−01.22	−01.01
LOC545783	−03.42	−02.89	−02.64	1.00	−01.03
Syngr2	1.05	1.08	−01.16	−01.02	1.16
Zfp1	−02.34	−02.74	−02.23	−01.02	−01.04
D5Ertd593e	−01.86	−01.96	−02.01	1.02	1.10
B430110G05Rik	−024.48	−024.75	−018.75	−01.52	1.00
Cope	−01.92	−02.06	−01.73	1.05	−01.04
Saa1	−08.73	−07.16	−05.85	−01.74	−03.76
Slc4a1	−03.80	−03.86	−02.63	1.00	−01.03
Saa2	−05.79	−05.52	−03.77	−01.68	−03.16
Haptoglobin	−03.24	−02.58	−03.10	−01.84	−02.62
HectD2	−01.13	1.00	−01.08	−01.04	−01.12
Saa3	−022.40	−023.93	−012.72	−04.88	−06.59
Agp	−03.36	−03.42	−02.56	−01.92	−03.38
Temt	1.92	1.25	1.67	−01.06	1.14
Orm1	−03.51	−03.63	−02.58	−01.77	−02.95
Igfbp1	−021.27	−014.26	−011.85	−01.59	−01.17
1110012L19Rik	−01.06	−01.00	−01.07	1.15	1.16
Cyp4a10	2.28	1.21	1.93	−01.03	1.09
Prdx3	1.08	1.05	1.10	1.01	−01.04
MGI:2672795	−01.74	−02.30	−01.65	1.00	−01.02
Bola3	1.02	1.00	1.12	−01.05	−01.05
Rpl5	−01.85	−02.26	−01.62	1.00	1.07
Etnk2	−01.29	−02.26	−01.44	1.04	−01.06
Amy1	1.57	1.33	1.31	−01.06	−01.11
Scd1	−02.00	−01.01	1.03	1.75	1.21
Chrna1	−02.15	−01.79	−01.78	1.07	1.02
Slc25a25	−01.20	−01.14	−01.07	−01.01	−01.08
RIBO 16S	−01.24	−01.10	−01.10	−01.01	−01.03
S100a8	−017.55	−09.60	−012.61	−01.01	−01.01
Hspa5	−01.19	−01.08	−01.16	1.32	−01.12
Tat	−02.01	−02.20	−01.78	1.03	−01.13
Sms	−014.22	−018.43	−014.22	−01.44	−01.07
Rps16	−01.72	−02.13	−01.83	−01.06	−01.10
Rnf10	−02.05	−01.45	−01.33	1.01	−01.00
Rps3	−01.47	−02.14	−01.43	−01.11	−01.17
Ap4e1	−01.62	−02.09	−01.43	1.01	−01.07
Atpif1	−02.04	−01.69	−01.82	1.09	−01.17
Nnmt	−04.21	−07.53	−05.40	1.58	−01.28
Lcn2	−03.28	−02.25	−02.55	−01.33	−01.28
9030611N15Rik	1.46	1.40	1.96	1.26	1.08
Exosc10	−02.13	−01.14	−01.47	1.10	1.04
Rps15	−02.35	−01.83	−01.90	1.14	−01.05
Fabp5	−01.69	−01.04	−01.04	1.27	1.07
Fga	−02.36	−02.10	−02.36	1.00	−01.19
Rps9	−02.15	−02.12	−01.79	−01.06	−01.08
Apob	−01.16	−01.52	2.11	−01.69	1.22
Cyp2c29	1.58	1.32	1.41	1.43	1.22
Hpxn	−02.04	−01.78	−01.61	−01.42	−01.44
Cyp2a4	1.17	−01.00	1.02	1.01	1.03
Itih4	−01.73	−01.69	−01.71	−01.21	−01.47
Atp5f1	−04.72	−04.44	−03.85	−01.13	−01.02
Fgg	−02.06	−01.48	−01.95	1.14	−01.08
Cyp2c44	1.19	1.11	1.40	1.15	1.06
Cyp2c40	1.46	1.34	1.92	1.24	1.23
Cyp2c37	1.65	1.41	1.58	1.19	1.18
Cyp2c50	1.55	1.31	1.40	1.15	1.17
Cyp2b10	1.30	1.16	1.01	1.10	1.12
Cyp1a2	1.43	1.49	1.21	1.41	1.23
Gstm1	1.11	1.20	1.13	1.13	−01.11
Comt	1.58	1.60	1.59	1.25	1.05