Hepatic effects of aminoglutethimide: A model aromatic amine

Figures & data

Figure 1. Histological changes in livers of AMG-treated rats. Representative H&E staining of (A) control and (B) AMG-treated (125 mg/kg/day, 14 days) rats. Evidence of Zone 3 hepatocyte hypertrophy was found in rats treated with AMG as compared to controls; 20× magnification. (C) Rats treated with AMG had an increase in liver weight that was most significant on Day 7 of treatment. Values shown are mean (±SEM) for each group (n = 4). *p < 0.05 compared to control (Mann--Whitney U-test).

Figure 2. Comparison of hepatocyte proliferation in control and AMG-treated animals. Representative Ki-67 staining in tissue from (A) control and (B) AMG-treated (125 mg/kg/day, 14 days) rats; 20× magnification. (C) A possible increase in Ki-67 was observed at Day 7 of AMG treatment. Although some evidence of hepatocyte proliferation was found in the AMG-treated rats, the main effect was hypertrophy rather than hyperplasia. Open bars = control rats; solid bars = AMG-treated rats. Values shown are mean (±SEM) number of Ki-67-positively-stained nuclei per 10× field of view (n = 4). No results were statistically significant (two-way ANOVA).

Figure 3. AMG-induced changes in serum levels of liver enzymes. (A) ALT and (B) GLDH in rats treated with AMG (125 mg/kg/day) compared to control rat values. Open bars = control group; solid bars = AMG-treated group. Values shown are mean (±SEM) (n = 4). **p < 0.01 as compared to control (two-way ANOVA).

Figure 4. Pathway analysis of significantly changed hepatic genes in the apoptotic pathway in AMG-treated rats. Only genes with changes >2-fold (up-regulation) after 14 days of AMG treatment were included in analysis for trends in the apoptotic panel of genes. Arrows indicate known associations and do not represent how genes are regulated by AMG. Solid lines = up-regulation; dashed lines = down-regulation; dotted lines = physical interaction. Analysis was performed using Gene Network Central Pro software (SABiosciences).

Table 1. Hepatic changes in gene expression induced by AMG in the apoptosis pathway.

Gene symbol	Gene name	Day 1	Day 7	Day 14
Aven	Apoptosis, caspase activation inhibitor	−1.16	−1.05	2.39*
Bad	BCL2-associated agonist of cell death	−2.01*	−1.29	2.53
Bag1	BCL2-associated athanogene	−1.10	1.13	2.43*
Bax	Bcl2-associated X protein	−1.25	1.03	2.28*
Bcl2l1	Bcl2-like 1	−2.95	−2.07	2.39*
Bcl2l2	Bcl2-like 2	−2.24*	−1.09	1.14
Bid	BH3 interacting domain death agonist	−1.08	1.21	2.65*
Hrk	Harakiri, BCL2 interacting protein (contains only BH3 domain)	−3.26*	−1.71	−1.27
Birc3	Baculoviral IAP repeat-containing 3	−2.03	1.28	3.42*
Birc5	Baculoviral IAP repeat-containing 5	1.12	2.35*	−1.52
Casp1	Caspase 1	−1.13	−1.22	−3.71*
Casp7	Caspase 7	1.31	1.88*	6.51*
Casp9	Caspase 9, apoptosis-related cysteine peptidase	−1.94	−1.26	2.67*
Cidea	Cell death-inducing DFFA-like effector a	1.99	1.90*	5.85*
Dapk1	Death associated protein kinase 1	−3.46*	−3.99	2.68
Dffa	DNA fragmentation factor, alpha subunit	−2.13*	1.16	1.37
Fadd	Fas (TNFRSF6)-associated via death domain	1.03	1.09	2.62*
Ltbr	Lymphotoxin beta receptor (TNFR superfamily, member 3)	−1.06	1.12	5.55*
Nfkb1	Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1	−2.17	−1.44	2.21*
Nol3	Nucleolar protein 3 (apoptosis repressor with CARD domain)	7.72*	11.61*	7.18*
Prlr	Prolactin receptor	−3.81*	1.91	3.15
Prok2	Prokineticin 2	1.11	3.36*	−1.33
Tnfrsf1a	Tumor necrosis factor receptor superfamily, member 1a	1.40	1.49	2.89*
Cd40	CD40 molecule, TNF receptor superfamily member 5	−1.13	1.08	−2.02*
Fas	Fas (TNF receptor superfamily, member 6)	−1.73	2.32*	−1.07
Tnfsf10	Tumor necrosis factor (ligand) superfamily, member 10	−3.09*	−1.12	1.10
Tnfsf12	Tumor necrosis factor ligand superfamily member 12	−1.58	−1.37	4.50*
Tradd	TNFRSF1A-associated via death domain	−1.29	−1.28	2.61*

Values are expressed as fold-change in AMG-treated compared to control rats (n = 4).

*p < 0.05 compared to control.

Table 2. Mitochondrial gene expression changes in the livers of AMG-treated rats.

Gene symbol	Gene name	7 days	14 days
Aifm2	Apoptosis-inducing factor, mitochondrion-associated 2	1.52	2.54*
Bbc3	Bcl-2 binding component 3	2.67*	2.80*
Bid	BH3 interacting domain death agonist	2.37*	2.00
LOC691853	Similar to COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase	1.53	−2.26*
Mfn1	Mitofusin 1	−1.63	2.21*
Mipep	Mitochondrial intermediate peptidase	1.62*	2.05*
Msto1	Misato homolog 1 (Drosophila)	1.35	3.30*
Ppargc1a	Peroxisome proliferator-activated receptor gamma, coactivator 1 alpha	−3.97*	2.51
Rnf135	Ring finger protein 135	1.17	2.38*
Sfn	Stratifin	2.98*	−1.44
Slc25a12	Solute carrier family 25 (mitochondrial carrier, Aralar), member 12	−1.26	2.64*
Slc25a13	Solute carrier family 25, member 13 (citrin)	−1.23	2.20*
Slc25a19	Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19	−1.13	3.61*
Slc25a23	Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 23	1.13	2.91*
Slc25a25	Solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 25	−2.23*	2.08*
Slc25a30	Solute carrier family 25, member 30	−3.79*	−1.29
Slc25a5	Solute carrier family 25 (mitochondrial carrier: adenine nucleotide translocator) member 5	−1.13	2.00*
Tomm34	Translocase of outer mitochondrial membrane 34	1.33	2.06*
Tomm40	Translocase of outer mitochondrial membrane 40 homolog (yeast)	−1.02	2.69*
Tspo	Translocator protein	27.30	4.95*
Timm17b	Translocase of inner mitochondrial membrane 17 homolog B (yeast)	−1.31	2.43*

Values are expressed as fold-change in AMG-treated compared to control rats (n = 4).

*p < 0.05 compared to control.

Table 3. AMG-induced hepatic gene changes in chemokines and receptors.

Gene symbol	Gene name	Day 1	Day 7	Day 14
C3	Complement component 3	−1.08	1.90*	2.16*
Ccbp2	Chemokine binding protein 2	−3.54*	−2.03	1.62
Ccl19	Chemokine (C-C motif) ligand 19	1.64*	2.24*	1.30
Ccl3	Chemokine (C-C motif) ligand 3	−4.53*	1.09	1.25
Ccr5	Chemokine (C-C motif) receptor 5	−5.08*	−1.13	−2.03
Cxcr7	Chemokine (C-X-C motif) receptor 7	−1.75	−4.66*	3.46*
Cmtm3	CKLF-like MARVEL transmembrane domain containing 3	−2.47*	−1.40	1.03
Cx3cl1	Chemokine (C-X3-C motif) ligand 1	1.19	2.06*	−1.42
Ppbp	Pro-platelet basic protein (chemokine (C-X-C motif) ligand 7)	−2.11*	1.49	2.26*
Tymp	Thymidine phosphorylase	−3.87*	−2.80*	1.64
Cxcl13	Chemokine (C-X-C motif) ligand 13	−5.05*	−2.21	−5.61*
Mmp2	Matrix metallopeptidase 2	−2.11*	−1.67	−1.99
Mmp7	Matrix metallopeptidase 7	1.41	2.32*	−1.80
Nfkb1	Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1	−2.21*	1.11	2.79*
Tlr2	Toll-like receptor 2	−3.76*	−1.43	1.30
Tnf	Tumor necrosis factor (TNF superfamily, member 2)	−2.41*	−1.05	1.08
Tnfrsf1a	Tumor necrosis factor receptor superfamily, member 1a	−1.16	1.11	2.57*
Trem1	Triggering receptor expressed on myeloid cells 1	1.32	2.42*	−1.26

Values are expressed as fold-change in AMG-treated compared to control rats (n = 4).

*p < 0.05 compared to control.

Table 4. Hepatic gene changes induced by AMG in various immune pathways after 14 days of treatment.

Gene symbol	Gene name	p Value	Fold-change
Common cytokines pathway
Bmp1	Bone morphogenetic protein 1	0.019	2.48
Il16	Interleukin 16	0.035	−2.89
Il1f5	Interleukin 1 family, member 5 (delta)	0.001	−2.38
Il1rn	Interleukin 1 receptor antagonist	0.004	2.49
Ltb	Lymphotoxin beta (TNF superfamily, member 3)	0.001	3.28
Tnfsf12	Tumor necrosis factor ligand superfamily member 12	0.019	2.83
Toll-like receptor signaling pathway
Cd14	CD14 molecule	0.005	6.48
Hmgb1	High mobility group box 1	0.032	−3.27
Irak1	Interleukin-1 receptor-associated kinase 1	0.008	3.23
Map2k3	Mitogen activated protein kinase kinase 3	0.047	2.86
Map4k4	Mitogen-activated protein kinase kinase kinase kinase 4	0.028	4.26
Nfkbil1	Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1	0.017	−1.75
Ppara	Peroxisome proliferator activated receptor alpha	0.006	3.55
Sarm1	Sterile alpha and TIR motif containing 1	0.032	5.43
Ticam2	Toll-like receptor adaptor molecule 2	0.022	4.14
Tlr2	Toll-like receptor 2	0.035	2.28
Tnfrsf1a	Tumor necrosis factor receptor superfamily, member 1a	0.006	3.27
Th17 response pathway
Cd4	Cd4 molecule	0.015	4.39
Icam1	Intercellular adhesion molecule 1	0.005	3.04
Il17rb	Interleukin 17 receptor B	0.009	5.57
Il18	Interleukin 18	0.008	−2.91
Nfatc2	Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2	0.004	3.41
Stat3	Signal transducer and activator of transcription 3	0.004	2.73
Stat5a	Signal transducer and activator of transcription 5A	0.004	2.40
Stat6	Signal transducer and activator of transcription 6	0.016	3.17

Values are expressed as fold-change in AMG-treated compared to control rats (n = 4).