Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium

Figures & data

Table 1. Parameters of oxidative status in the liver of male Wistar rats exposed solely to Cd or DSF.

Groups	Exposure (days)	OS parameters
		MDA (nmol/mg proteins)	O2^·− (nmol red NBT/mg proteins)	SOD (U SOD/mg proteins)	CAT (U CAT/mg proteins)
		Control	0	63.6 ± 7.16	67.3 ± 6.70	2.34 ± 0.174	279.2 ± 44.10
Cd	1	101.6 ± 17.62***	116.4 ± 1.58***	2.34 ± 0.325	351.7 ± 21.79**
	3	123.8 ± 17.77***	127.9 ± 25.19***	2.18 ± 0.416	327.9 ± 47.27
	10	123.1 ± 8.95***	134.4 ± 11.39***	1.89 ± 0.264	300.0 ± 50.73
	21	134.2 ± 5.68***	139.5 ± 21.73***	1.71 ± 0.145***	209.8 ± 11.11**
	42	138.1 ± 2.45***	179.2 ± 12.04***	1.44 ± 0.286***	168.21 ± 2.17***
DSF	1	65.5 ± 11.51	131.8 ± 25.78***	1.42 ± 0.135***	196.7 ± 30.59***
	3	77.8 ± 11.44	146.1 ± 26.64***	1.39 ± 0.133***	336 ± 23.92**
	10	77.3 ± 3.31	311.3 ± 10.63***	1.19 ± 0.018***	434.7 ± 21.24***
	21	107.9 ± 10.03***	672.6 ± 14.83***	0.9 ± 0.0.072***	515.7 ± 49.45***

Label of statistical significance refers to comparison between the experimental groups and the control group (intact animals); Level of significance is labeled as follows: *(p < 0.01), **(p < 0.001), ***(p < 0.0001); Values are expressed as mean ± standard deviation of six separate analyses (n = 6).

Table 2. Parameters of glutathione cycle in the liver of male Wistar rats exposed solely to Cd or DSF.

Groups	Exposure(days)	OS parameters
		GSH (nmol/mg proteins)	GSSG (nmol/mg proteins)	GSSG/GSH (ratio)	GR (U GR/mg proteins)	GST (U GST/mg proteins)	GPx (U GPx/mg proteins
		Control	0	32.8 ± 3.40	1.8 ± 0.08	0.05 ± 0.004	0.0024 ± 0.0001	0.0439 ± 0.0031	1.139 ± 0.0514
Cd	1	25.6 ± 2.57**	2.1 ± 0.13*	0.08 ± 0.011	0.0019 ± 0.0002	0.0430 ± 0.0072	1.282 ± 0.1310
	3	23.5 ± 2.42***	3.7 ± 0.31***	0.16 ± 0.011	0.0019 ± 0.0001*	0.0373 ± 0.0062	1.314 ± 0.1897
	10	22.2 ± 3.99***	4.7 ± 0.38***	0.21 ± 0.050	0.0018 ± 0.0003*	0.0173 ± 0.0025***	1.620 ± 0.1783***
	21	18.3 ± 2.00***	2.1 ± 0.13*	0.11 ± 0.018	0.0017 ± 0.0002**	0.0144 ± 0.0026***	1.954 ± 0.1055***
	42	12.5 ± 2.56***	1.7 ± 0.23	0.14 ± 0.012	0.0015 ± 0.0001***	0.012 ± 0.0017***	2.067 ± 0.0777***
DSF	1	32.5 ± 5.46	4.2 ± 0.77***	0.13 ± 0.028	0.0015 ± 0.0002***	0.0450 ± 0.0057	1.228 ± 0.1469
	3	21.7 ± 3.88**	0.6 ± 0.06***	0.03 ± 0.003	0.002 ± 0.0002**	0.0578 ± 0.0037*	1.344 ± 0.1530
	10	38.0 ± 3.85	4.5 ± 0.34***	0.12 ± 0.018	0.0029 ± 0.0001*	0.0594 ± 0.0013*	1.814 ± 0.0058***
	21	31.8 ± 4.89	0.7 ± 7.10***	0.02 ± 0.007	0.0021 ± 0.0002	0.1096 ± 0.016***	2.060 ± 0.2587***

Label of statistical significance refers to comparison between the experimental groups and the control group (intact animals); Level of significance is labeled as follows: *(p < 0.01), **(p < 0.001), ***(p < 0.0001); Values are expressed as mean ± standard deviation of six separate analyses (n = 6).

Table 3. Parameters of oxidative status in the liver of male Wistar rats co-exposed to Cd and DSF.

OS parameters	Control	Group
		Cd + DSF
		0 day	1 day	3 days	10 days	21 days
	MDA	63.6 ± 7.16	132.4 ± 7.10***	112.8 ± 10.51***	103.1 ± 9.98***	99.1 ± 11.64***
O2^·−	67.3 ± 6.70	140.9 ± 17.39***	127.2 ± 12.4***	127.1 ± 12.49***	106.7 ± 10.97***
SOD	2.34 ± 0.174	1.51 ± 0.277***	2.13 ± 0.261	2.16 ± 0.081	2.27 ± 0.284
CAT	279.2 ± 44.10	245.4 ± 30.29	388.6 ± 57.69***	221.7 ± 12.95	216.4 ± 3.35*
GSH	32.8 ± 3.40	10.8 ± 1.94***	26.5 ± 0.42**	28.1 ± 4.24	16.3 ± 2.35***
GSSG	1.8 ± 0.08	1.9 ± 0.22	1.9 ± 0.27	1.3 ± 0.01***	1.1 ± 0.09***
GSSG/GSH	0.05 ± 0.004	0.19 ± 0.049	0.07 ± 0.01	0.05 ± 0.007	0.07 ± 0.014
GR	0.0024 ± 0.1	0.0014 ± 0.01***	0.0028 ± 0.4	0.0029 ± 0.3*	0.0026 ± 0.5
GST	0.0439 ± 0.00306	0.087 ± 0.00502***	0.087 ± 0.01097***	0.0876 ± 0.01679***	0.0893 ± 0.00115***
GPx	1.139 ± 0.0514	2.656 ± 0.001***	1.852 ± 0.0704***	1.837 ± 0.0025***	1.157 ± 0.0037

Label of statistical significance refers to comparison between the experimental groups and the control group (intact animals); Level of significance is labeled as follows: *(p < 0.01), **(p < 0.001), ***(p < 0.0001); Values are expressed as mean ± standard deviation of six separate analyses (n = 6); 21-day period refers to administration of DSF for 21 days in the combined treatment; Units of OS parameters are expressed as: MDA (nmol/mg proteins), O₂^·− (nmol red NBT/mg proteins), SOD (U SOD/mg proteins), CAT (U CAT/mg proteins), GSH (nmol GSH/mg proteins), GSSG (nmol GSSG/mg proteins), GSSG/GSH ratio, GR (U GR/mg proteins), GST (U GST/mg proteins), GPx (U GPx/mg proteins).

Table 4. Metal content in the liver of male Wistar rats.

Groups	Exposure (days)	Metal
		Cd	Cu	Fe	Zn	Mg	Se
		Control	0	/	3.3 ± 0.28	66.0 ± 12.12	37.5 ± 4.44	240.2 ± 17.35	15.2 ± 0.39
Cd	1	55.8 ± 5.48	5.6 ± 0.93**	69.4 ± 9.53	54.4 ± 8.68*	240.5 ± 22.62	41.9 ± 1.95***
	3	54.22 ± 9.43	5.3 ± 0.43**	58.5 ± 7.32	58.1 ± 5.47**	240.0 ± 11.21	32.2 ± 4.97***
	10	57.5 ± 8.68	6.3 ± 0.38***	77.3 ± 10.21	78.1 ± 6.66***	261.9 ± 48.31	28.4 ± 1.13***
	21	107.3 ± 5.93 μ***	8.4 ± 1.64***	87.0 ± 7.03***	96.7 ± 12.59***	239.7 ± 31.11	22.6 ± 2.44*
	42	166.5 ± 26.3 μ***	8.7 ± 1.19***	138.5 ± 11.95***	96.3 ± 16.94***	242.0 ± 29.96	/
DSF	1	/	3.9 ± 0.62	64.2 ± 11.65	42.5 ± 3.53	242.8 ± 19.45	18.5 ± 0.52
	3	/	5.6 ± 0.97**	57.5 ± 9.50	67.9 ± 11.44***	244.4 ± 24.41	34.9 ± 6.44***
	10	/	5.7 ± 1.18**	62.1 ± 6.87	69.3 ± 12.38***	233.8 ± 34.33	43.6 ± 7.78***
	21	/	9.0 ± 1.74***	100.1 ± 14.31***	65.6 ± 6.39**	219.7 ± 16.80	44.8 ± 8.13***
Cd + DSF	1	133.0 ± 19.46 μ***	5.7 ± 0.73**	94.7 ± 13.34***	108.6 ± 7.31***	241.6 ± 16.61	22.0 ± 0.61*
	3	106.6 ± 13.8 μ***	13.9 ± 1.61***	94.4 ± 6.23***	114.3 ± 11.23***	230.6 ± 7.72	22.3 ± 3.14*
	10	152.2 ± 29.48 μ***	9.4 ± 1.48***	102.4 ± 10.68***	145.5 ± 9.87***	241.2 ± 14.37	23.3 ± 2.03*
	21	235.3 ± 11.46 μ***	5.6 ± 0.29**	197.6 ± 11.50***	156.1 ± 14.21***	242.5 ± 31.55	24.2 ± 0.99**

Significances between experimental groups in essential metal content determination refer to comparison between the experimental groups and the control group (intact animals); Significances between experimental groups in Cd content determination refer to comparison between the experimental groups and Cd1 subgroup (μ); Level of significance is labeled as follows: *(p < 0.01), **(p < 0.001), ***(p < 0.0001); Values are expressed as mean ± standard deviation of six separate analyses (n = 6); 21-day period refers to administration of DSF for 21 days in the combined treatment; Units of metals are expressed as μg/g tissue for Cd, Cu, Fe, Zn, Mg and ng/g tissue for Se.

Figure 1. Representative graphs of oxidative status in liver of Wistar rats intact and administered Cd, DSF and a combination of both substances. *See the experimental conditions presented in the section Experimental design. Values are presented as means ± S.D. Labels of statistical significance: compared with the control group (*) and compared with Cd42 subgroup (°). Statistical significance was considered at: ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001 and °°°p < 0.0001. (A) Superoxide anion radical content (O₂^·−): presented parameter of OS (expressed as nmol red NBT/mg proteins). (B) Lipid peroxidation: parameter of OS presented as MDA level (expressed as nmol MDA/mg proteins). (C) SOD activity: presented parameter of antioxidative defence (expressed as U SOD/mg proteins). (D) CAT activity: presented parameter of antioxidative defence (expressed as U CAT/mg proteins).

Figure 2. Representative graphs of glutathione cycle in liver of Wistar rats intact and administered Cd, DSF and a combination of both substances. *See the experimental conditions presented in the section Experimental design. Values are presented as means ± S.D. Labels of statistical significance: compared with the control group (*) and compared with Cd42 subgroup (°). Statistical significance was considered at: ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001 and °°°p < 0.0001. (A, B) Reduced and oxidized glutathione content: presented parameters of antioxidative defence (obtained values, expressed in μM, were recalculated in regard to protein status and expressed as nmol GSH or GSSG/mg proteins for the appropriate discussion of the obtained results). (C) Ratio of oxidized and reduced glutathione: presented parameter of glutathione homeostasis (values are presented as means of GSSG/GSH ratio through chosen time points). (D) GR activity: presented parameter of antioxidative defence (expressed as U GR/mg proteins). (E) GST activity: presented parameter of antioxidative defence (expressed as U GST/mg proteins). (F) GPx activity: presented parameter of antioxidative defence (expressed as U GPx/mg proteins).

Figure 3. Representative graphs of metal content in liver of Wistar rats intact and administered Cd, DSF and a combination of both substances. *See the experimental conditions presented in the section Experimental design. Values are presented as means ± S.D. Labels of statistical significance for toxic metal Cd: compared with the Cd1 group (*) and compared with Cd42 subgroup (°). Labels of statistical significance for essential metals (Cu, Fe, Zn, Mg, Se): compared with the control group (*) and compared with Cd42 subgroup (°). Statistical significance was considered at: ∗p < 0.01, ∗∗p < 0.001, ∗∗∗p < 0.0001, °°p < 0.001 and °°°p < 0.0001. (A) Cd content (expressed as μg Cd/g liver tissue). (B) Cu content (expressed as μg Cu/g liver tissue). (C) Fe content (expressed as μg Fe/g liver tissue). (D) Zn content (expressed as μg Zn/g liver tissue). (E) Mg content (expressed as μg Mg/g liver tissue). (F) Se content (expressed as ng Se/g liver tissue).

Figure 4. Representative photomicrographs of liver sections from Wistar rats exposed i.p. to 1 mg CdCl₂/kg BW/day (for 21 and 42 days) and orally to 178.5 mg DSF/kg BW/day (for 21 days), individually and/or in combination: (A) Control/intact group, showing normal appearance; (B) Cd21: showing infiltration of neutrophils (N) and focal necrosis (F). (C) Cd42: showing infiltration of neutrophils (N) and neutrophil infiltration of sinusoids (NS). (D) DSF21: showing normal appearance; and (E) Cd42 + DSF21: group showing infiltration of neutrophils (N), neutrophil infiltration of sinusoids (NS) and focal necrosis (F).

Table 5. Serum aminotransferases analysis in male Wistar rats.

Parameter	Groups
	Control	Cd	Cd	DSF	Cd + DSF
	0 day	21 days	42 days	21 days	21 days
	AST (U/L)	148 ± 15.8	184 ± 8.2¥*	230 ± 31.7¥**	136 ± 16.2	302 ± 2.5¥**;£**
ALT (U/L)	27 ± 0.6	42 ± 3.3¥**	46 ± 3.2¥**	28 ± 3.0	37 ± 2.6¥**;£**

Significances between experimental groups are labeled as follows: compared with controls (¥); compared with Cd42 subgroup (£); Level of significance is labeled as follows: *(p < 0.001), **(p < 0.0001); Values are expressed as mean ± standard deviation of six separate analyses (n = 6); 21-day period refers to administration of DSF for 21 days in the combined treatment.

Table 6. Spearman correlation between OS parameters and/or relevant metals in the liver of the Wistar rats subacutely exposed to Cd and DSF, subchronically exposed to Cd and subchronically co-exposed to Cd and DSF.

Groups	Correlated OS parameters and metals
Control	MDA	CAT	GSSG/GSH	Fe
	Se	GPx	GR	Mg
Spearman r	0.9429	0.9429	0.971	−0.8857
p Value	0.0167	0.0167	0.0028	0.0333
Cd21	SOD	GPx
	GPx	MDA
Spearman r	0.9276	−0.2899
p Value	0.0167	0.0167
Cd42	MDA	SAR	SOD	GST
	Cd	GSSG	GSSG/GSH	Mg
Spearman r	−0.9429	−0.9429	0.8533	−0.8827
p Value	0.0167	0.0167	0.0333	0.0333
DSF21	SAR			GSSG	GSSG/GSH	GPx
	GSSG	GPx	Zn	Zn	GST	MDA	Zn
Spearman r	−0.8697	0.9429	1	−0.8697	0.8452	−0.9429	0.9429
p Value	0.0333	0.0167	0.0028	0.0333	0.0333	0.0167	0.0167
Cd42 + DSF21	MDA		SAR	SOD		CAT	GSH		GSSG
	SOD	GR	Zn	GSH	GR	Fe	MDA	GR	MDA	GR
Spearman r	−0.8857	0.9856	0.8986	0.9429	−0.9276	0.8857	−0.9429	−0.9856	0.8986	0.8676
p Value	0.0333	0.0028	0.0333	0.0167	0.0167	0.0333	0.0167	0.0028	0.0333	0.0333

Correlation was calculated between all parameters across all chosen time points (Cd: 21 and 42 days; DSF: 21 days and Cd 42 days and DSF 21 days of co-exposure) in Wistar rats i.p. receiving 1 mg CdCl₂/kg/day and orally 178.5 mg DSF/kg BW/day. Spearman correlation coefficient (r) > ± 0.70 was the criteria for the segregation of the results. The units of the presented parameters are as follows: malondialdehyde (MDA): nmol MDA/mg proteins; superoxide anion radical (SAR): nmol red NBT/min/mg proteins; superoxide dismutase (SOD): units of SOD/mg proteins; catalase (CAT): units of CAT/mg proteins; glutathione – reduced and oxidized (GSH and GSSG): nmol of GSH and GSSG/mg proteins; glutathione reductase (GR): units of GR/mg proteins; Glutathione-S-transferase (GST): units of GST/mg proteins; glutathione peroxidase (GPx): units of GPx/mg proteins; and concentration of metals – iron (Fe): μg of Fe/g of wet tissue and selenium (Se): ng of Se/g of wet tissue. Differences were considered statistically significant for p < 0.05.