Ameliorative effects of pyrazinoic acid against oxidative and metabolic stress manifested in rats with dimethylhydrazine induced colonic carcinoma

Figures & data

Figure 1. Effects of PA after oral administration of 10 and 25 mg/kg doses for 15 d. Data represented as mean ± SD (n = 6). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (**p < 0.01, *p < 0.001)].

Table 1. Effects of PA on oxidative stress parameters in colon after 10 and 25 mg/kg doses for 15 d.

Groups	SOD (U/mg of Protein)	CT mM H2O2 decomposed/min/mg of protein	GSH (µM/mg of Protein)	TBARS (nM of MDA/mg of protein)	PC (µg/mg of protein)
Control	0.21 ± 0.02	14.48 ± 0.72	6.59 ± 0.24	0.12 ± 0.02	0.77 ± 0.46
Carcinogen (DMH)	0.08 ± 0.07	7.50 ± 0.44	3.17 ± 0.29	0.19 ± 0.01	1.40 ± 0.29
DMH^+5-FU	0.20 ± 0.01*	13.84 ± 0.58*	5.00 ± 0.20*	0.13 ± 0.01*	0.80 ± 0.19***
DMH^+ PA(10)	0.18 ± 0.03**	8.67 ± 0.49**	3.76 ± 0.18**	0.15 ± 0.02**	0.84 ± 0.16***
DMH^+ PA(25)	0.19 ± 0.04**	8.95 ± 0.23*	3.90 ± 0.14**	0.14 ± 0.01**	0.82 ± 0.26***

Data represented as mean ± SD (n = 6). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (***p < 0.05, **p < 0.01, *p < 0.001)].

Figure 2. Effect of PA on enzyme levels in plasma after 10 and 25 mg/kg doses for 15 d. Data represented as mean ± SD (n = 6). Statistically significant differences were observed between carcinogen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (*p < 0.001, **p < 0.01)].

Table 2. Effects of PA on (A) COX-2, IL-2 and IL-6 (B) caspase 3 in colon carcinogenic tissue after oral administration of 10 and 25 mg/kg for 15 d.

(A)
Groups	COX-2 (pg/mL)	IL-2 (pg/mL)	IL-6 (pg/mL)
Control	622.69 ± 63.11	340.84 ± 71.11	485.14 ± 75.58
Carcinogen (DMH)	1047.06 ± 142.24	866.42 ± 97.41	970.29 ± 94.05
DMH^+5-FU	646.15 ± 23.06*	538.63 ± 50.00*	542.99 ± 73.41*
DMH^+ PA(10)	848.74 ± 35.23**	701.90 ± 41.78**	802.35 ± 94.05***
DMH^+ PA(25)	786.89 ± 61.02*	700.04 ± 28.25**	779.96 ± 81.56**
(B)
Groups	Caspase 3 (pg/mL)
Control	75.5 ± 11.84
Carcinogen (DMH)	50.46 ± 7.80
DMH^+5-FU	71.86 ± 9.04**
DMH^+ PA(10)	66.76 ± 5.80*
DMH^+ PA(25)	69.93 ± 9.72*

Data represented as mean ± SD (n = 6). Statistically significant differences were observed between carcinigen control and test groups [one way-ANOVA followed by Bonferroni multiple comparison test (***p < 0.05, **p < 0.01, *p < 0.001)].

Figure 3. Gene expression levels of proinflammatory cytokines like (A) IL-6 and (B) COX-2 after PA administration in DMH treated rats. Data represented as mean ± SD (n = 6). Statistically significant differences were observed between carcinogen control and test groups [Paired T-test, (*p<0.001)].

Figure 4. The colonic pathological changes in DMH-induced CC rats (Scale bar 50 µm). Tumoral vacuoles were prominent in DMH group which was absent after 5-FU and PA administration. (Cr- Crypts, NE- Normal epithelium, CL⁻ Colon lumen, TA- Tubular adenoma, TD- Tumoral deposits, Ly- Lymphatics (lined by endothelium), TS- Tumor stroma).

Figure 5. (A-D) OPLS-DA score plots with their respective loading S-plots (A′-D′) derived from 1D 1H CPMG NMR spectra. (E-H) OPLS-DA score plots with their respective loading S-plots (E’-H’) derived from 1D DE ¹H NMR spectra. The notions used in the Figure represent respectively: Control (NC), DMH (TC), DMH⁺5-FU (PC), DMH⁺PA-10mg (DA) and DMH⁺PA-25 mg (DB) groups.

Table 3. Goodness-of-fit of the PLS-DA models obtained from 1D CPMG and 1D DE of rat serum samples.

Comparison	NMR spectra	R^2Y (cum)	Q^2 (cum)	Number of latent variables
Control vs DMH	1D CPMG	0.89	0.59	5
DMH vs DMH +5-FU	1D CPMG	0.72	0.26	5
DMH vs DMH +PA-10 mg	1D CPMG	0.99	0.76	5
DMH vs DMH +PA-25 mg	1D CPMG	0.99	0.79	5
Control vs DMH	1D DE	0.82	0.55	5
DMH vs DMH +5-FU	1D DE	0.99	0.40	5
DMH vs DMH +PA-10 mg	1D DE	0.95	0.81	5
DMH vs DMH +PA-25 mg	1D DE	0.97	0.84	5

Table 4. Key observed metabolic differences between the healthy control and DMH rat sera. chemical shift, variation, VIP score and p-values of the individual biomarkers are given. p-values less than 0.05 were considered as significant. The metabolic differences between DMH and DMH⁺5-FU, DMH and DMH⁺PA-10 mg, and DMH and DMH⁺PA-25 mg have also been presented.

					Variation with respect to DMH
Metabolites	Chemical shift	Variation in DMH with respect to control	VIP	p-value	5-FU	PA-10 mg	PA-25 mg
3-Hydroxy butyrate	0.88,1.55, 2.15	↓	1.03	0.04	—	—	—
Acetate	1.92	↑	1.17	0.12	—	↑	↑
Glutamine	2.35	↑	1.49	0.28	↑	↓	↑
O-acetyl-glycoprotein	2.13	↑	1.33	0.26	—	↑	↑
Succinate	2.39	↑	1.09	0.48	↓	—	↓
Citrate	2.52	↑	2.29	0.19	—	↓	↑
Dimethyl amine	2.72	↓	1.50	0.25	↑	↑	↑
Citrulline	2.28	↑	2.19	0.20	—	↓	↑
Choline	3.19	↑	1.51	0.20	—	↓	↓
O-acetyl choline	3.21	↑	1.23	0.17	↑	—	↑
Glucose	3.23–3.92, 4.63, 5.21	↓	3.08	0.08	↑	↑	↑
Maltose	3.25	↓	6.88	0.39	↑	↓	↓
Tryptophan	3.35, 7.18	↑	1.66	0.08	—	↓	—
Glycerol	3.55, 3.65, 3.78	↑	5.93	0.31	↑	↓	↑
Myoinositol	3.27,3.53, 3.61, 4.06	↓	8.82	0.26	↑	↓	↑
Creatinine	3.03	↑	1.63	0.15	↑	—	↑
Lactate	1.31, 4.11	↑	—	—	↓	↓	↓