1α, 25-dihydroxyvitamin D3 prevents DNA damage and restores antioxidant enzymes in rat hepatocarcinogenesis induced by diethylnitrosamine and promoted by phenobarbital

Table 1 Body and liver masses in each group of rats at end of the study (after 20 wk) (mean ± SE)

Group	No. of rats	Body mass (g)	Liver mass (g)	Relative liver mass (g) (Liver/100 g body mass)
A	6	282.5 ± 15.8a	14.6 ± 2.6	5.16 ± 0.47b
B	10	338.0 ± 18.3	10.8 ± 1.9	3.19 ± 0.21
C	8	334.3 ± 13.8c	12.3 ± 1.8	3.67 ± 0.36e
D	10	348.9 ± 21.6	11.9 ± 1.5	3.41 ± 0.29
E	8	308.1 ± 19.1	13.1 ± 2.2	4.25 ± 0.45
F	10	336.6 ± 22.2	12.1 ± 1.8	3.59 ± 0.42

Statistical level of significance by using Dunnett’s t-test.

^aP < 0.05,

^bP < 0.01, significantly different from normal control (group B),

^cP < 0.05,

^eP < 0.05, significantly different from DEN control (group A).

Table 2 Effect of 1α, 25(OH)₂D₃ on inhibition of GGT-positive foci in DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE)

Group	No. of rats	No. of GGT-positive foci/cm2	Decrease (%)
A	0 6	26.16±2.16
C	0 8	8.16±0.59b	68.8
E	0 8	14.32±1.23d	45.25

Statistical level of significance by using Dunnett’s t-test,

^bP < 0.01,

^dP < 0.01 compared with group A.

Table 3 Effect of 1α, 25(OH)₂D₃ on incidence, number and size of hepatocellular lessions during DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE)

Group	No. of rats with nodules	Nodule incidence (%)	Total No. of nodule	Average No. of nodules per nodule bearing liver	Relative size
					< 1 mm	> 1 mm, < 3 mm	> 3 mm
A	10/10	100	168	16.80±1.33	94 (55.95)	48 (28.57)	26 (15.47)
C	6/10	60b	22	3.66±0.88	11 (50.0)	6 (27.27)	05 (22.72)
E	8/10	80d	72	8.00±1.05	28 (38.88)	24 (33.33)	20 (27.77)

Statistical level of significance by using Dunnett’s t-test,

^bP < 0.01,

^dP < 0.01 compared with group A.

Table 4 Effect of 1α, 25dihydroxyvitamin D₃ on DNA damage [based on mean tailed cells (%) and mean length: width ratios ± SEM of DNA pattern] in hepatic cells of rats during DEN in-duced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE, n = 100)

Groups	Length and width ratio of DNA mass	Decrease (%)	Tailed cells (%)	Decrease (%)
A	2.44±0.0681		84±0.020
B	1.029±0.005		29±0.008
C	1.12±0.025b	54.09	39±0.014d	53.57
D	1.02±0.0005		29±0.008
E	1.63±0.026g	33.19	62±0.026f	26.19
F	1.02±0.005		29±0.008

Group A: DEN control; Group B: Normal control; Group C: Long term study; Groups D, F 1α, 25(OH)₂D₃ control; Group E: promotion. Statistical level of significance by using Dunnett’s t-test,

^bP < 0.01,

^gP < 0.01,

^dP < 0.01 and

^fP < 0.01 compared to group A.

Table 5 Changes in activities of superoxide dismutase (units/mg protein) in different groups of rats treated with 1α, 25(OH)₂D₃ during DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE, n = 5)

Groups	Nodules	Surrounding	Control
A	4.38 ± 0.56b	5.28 ± 0.57a	8.64 ± 0.87
C	6.84 ± 0.81c	7.60 ± 0.73	8.84 ± 0.80
E	4.88 ± 0.66	5.64 ± 0.53	8.52 ± 0.77

Statistical level of significance by using Dunnett’s t-test.

^bP < 0.01,

^aP < 0.05, compared with control.

^cP < 0.05 compared with group A.

Table 6 Changes in total hepatic lipid peroxidation (nmol MDA/100 mg protein) level in different groups of rats treated with 1α, 25(OH)₂D₃ during DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE, n = 5)

Groups	Nodules	Surrounding	Control
A	18.72 ± 1.59b	17.4 ± 1.34d	2.44 ± 0.84
C	8.80 ± 1.02a	7.6 ± 0.88	3.16 ± 0.94
E	13.16 ± 1.23	12.32 ± 1.03	2.8 ± 0.79

Statistical level of significance by using Dunnett’s t-test.

^bP < 0.01,

^dP < 0.01 compared with control;

^aP < 0.05 compared with group A.

Table 7 Changes in total hepatic reduced glutathione (GSH) (mg/100 g tissue) level in different groups of rats treated with 1α, 25(OH)₂D₃ during DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE, n = 5)

Groups	Nodules	Surrounding	Control
A	367.6 ± 25.5b	310.0 ± 24.9c	226.0 ± 22.5
C	268.0 ± 21.5a	254.0 ± 20.1	248.0 ± 21.7
E	303.2 ± 22.5	284.0 ± 21.6	256.0 ± 22.5

Statistical level of significance by using Dunnett’s t-test.

^bP < 0.01,

^cP < 0.05 compared with control.

^aP < 0.05 compared with group A.

Table 8 Changes in activity of 1-chloro-2,4-dinitro benzene (CDNB) conjugated (μmol CDNB conjugated/mg protein/mL) hepatic cytosolic glutathione S-transferase (GST) in different groups of rats treated with 1α, 25(OH)₂D₃ during DEN induced rat hepatocarcinogenesis promoted by phenobarbital (mean ± SE, n = 5)

Groups	Nodules	Surrounding	Control
A	2.52 ± 0.23b	1.54 ± 0.23c	0.9 ± 0.10
C	1.45 ± 0.27a	1.24 ± 0.11	1.12 ± 0.13
E	1.76 ± 0.29	1.48 ± 0.19	1.10 ± 0.11

Statistical level of significance by using Dunnett’s t-test.

^bP < 0.01,

^cP < 0.05 compared with control.

^aP < 0.05 compared with group A.