Acetaminophen-induced Hepato- and Nephrotoxicity and Amelioration by Silymarin and Terminalia chebula in Rats.

Experimental study was conducted to evaluate the hepato- and renoprotective effect of silymarin and Terminalia chebula against experimentally-induced acetaminophen (APAP) toxicity in rats. Oral administration of APAP @ 500 mg/kg for 1 to 3 days to all the four groups (six rats in each) resulted in significant elevation of serum triglycerides, total cholesterol, blood urea nitrogen, serum creatinine, and aspartate transaminase activity. Post-treatment with silymarin @ 25 mg/kg and T. chebula 125 mg/kg in groups 2 and 3 and their combination to group 4 from day 4 to 14 has significantly reversed the alterations of above said markers and offered better protection. The results of the study enunciated that silymarin and T. chebula exhibit good hepato- and nephro-protection against APAP toxicity.

INTRODUCTION

Liver diseases remain one of the serious health problems. Hepatic dysfunction due to ingestion or inhalation of hepatotoxin is increasing worldwide.[1] Acetaminophen (APAP; paracetamol-[PCM]) is commonly prescribed analgesic and antipyretic drug producing dose-dependent hepato- and nephrotoxicity. Herbal alternatives are one of the best ways to minimize liver damage and were used prophylactically and also as antidotes. Silymarin, a flavonolignan obtained from Silybum marianum, comprises of silybin as the most active component that counteracts the leakage of alanine transaminase (ALT) and gamma glutamyl transferase and prevent lipid peroxidation (LPO).[2] The fruits of Terminalia chebula were used as common household remedy,[3] and its phenolics prevent nickel chloride-induced oxidative stress by decreasing LPO, restoring the activity of glutathione-S-transferase, glutathione reductase, and glutathione peroxidase.[4] The present investigation was carried out to study the hepato- and renoprotective effects of silymarin and T. chebula.

MATERIALS AND METHODS

A total of 24 male Wistar kyoto rats (200 – 250 g) were obtained from National Institute of Nutrition, Hyderabad. The animals were maintained and used in accordance with guideline of the Control and Prevention Supervision Committee Experimental on Animals, and the protocols were accepted by Instituitional animal ethics committee (IAEC). The rats were randomly divided into four groups of six rats in each; APAP @ 500 mg/kg was administered to all the groups from 1 to 3 days. Group 1 served as toxic control and was maintained with distilled water from 4 to 14 days. Groups 2 and 3 were administered orally with silymarin (25 mg/kg) and T. chebula (125 mg/kg) from 4 to 14 days. Group 4 was administered with a combination of silymarin + T. chebula at the doses mentioned in groups 2 and 3, respectively. The experiment was carried out for 14 days.

The sera samples were separated from blood on day 0, 4, and 14 for the estimation of triglycerides, total cholesterol, blood urea nitrogen (BUN), serum creatinine, and aspartate transaminase (AST) activity by using commercially available diagnostic kits (Qualigens Pvt. Ltd, Mumbai, India). All data were expressed as means ± SE. The means were analyzed by One Way Analysis of Variance (ANOVA) by using SPSS software package. Values of P<0.05 were considered as significant.

RESULTS AND DISCUSSION

The activity of AST (U/L) [Table 1] and concentration of triglycerides (mg/dl) and total cholesterol (mg/dl) [Table 2] were significantly (P<0.05) elevated on day 4 in groups 1, 2, 3, and 4 as compared with the mean values of day 0, and these observations coincided with earlier studies where oral administration of PCM at 750 mg/kg/day for 7 days increased the level of liver marker enzymes viz., AST, ALT, Alkaline phosphatase (ALP), creatinine, and BUN.[5] Following administration with silymarin and T. chebula in groups 2, 3, and 4, the levels were significantly (P<0.05) reduced on day 14 as compared with that of hepatotoxic control.

Table 1

Aspartate transaminase activity (U/l) in different groups of rats

Group	Day
	0	4	14
Acetaminophen (APAP) (1 – 3 days)	71.00 ± 3.07aA	127.65 ± 8.90aBC	119.78 ± 4.23bB
APAP (1 – 3 days) + silymarin (4 – 14 days)	67.71 ± 3.40aA	124.65 ± 8.62aBC	81.57 ± 3.33aA
APAP (1 – 3 days) + Terminalia chebula (4 – 14 days)	69.94 ± 6.21aA	140.94 ± 5.78aBC	74.01 ± 8.85aA
APAP (1 – 3 days) + silymarin + Terminalia chebula (4 – 14 days)	74.20 ± 5.16aA	130.56 ± 8.31aBC	83.42 ± 4.76aA

Values are mean ± SE of six observations; Means with different alphabets as superscripts differ significantly (P<0.05) ANOVA; Capital alphabets (horizontal comparison), small alphabets (vertical comparison); SE - Standard error

Table 2

Serum triglycerides and total cholesterol concentration in different groups of rats

Group	Serum triglycerides concentration (mg/dl)			Serum total cholesterol concentration (mg/dl)
	Day 0	Day 4	Day 14	Day 0	Day 4	Day 14
Acetaminophen (APAP) (1 – 3 days)	26.34 ± 2.32aAB	58.09 ± 4.53aC	53.97 ± 2.12bC	54.95 ± 2.97aA	88.83 ± 5.80aC	84.86 ± 2.26bC
APAP (1 – 3 days) + silymarin (4–14 days)	24.44 ± 2.58aAB	62.86 ± 2.70aC	30.16 ± 3.53aAB	56.58 ± 3.73aA	84.50 ± 3.99aC	69.55 ± 2.48aB
APAP (1 – 3 days) +Terminalia chebula (4 – 14 days)	30.47 ± 2.25aAB	56.83 ± 3.46aC	32.38 ± 2.95aB	55.86 ± 2.11aA	84.86 ± 4.95aC	67.93 ± 2.68aB
APAP (1 – 3 days) + silymarin + Terminalia chebula (4 – 14 days)	21.58 ± 3.86aA	53.65 ± 4.73aC	24.76 ± 2.30aAB	62.70 ± 2.33aAB	90.09 ± 4.98aC	71.53 ± 2.09aB

Values are mean ± SE of six observations; Means with different alphabets as superscripts differ significantly (P<0.05) ANOVA; Capital alphabets (horizontal comparison), small alphabets (vertical comparison)

In the current investigation, renal biomarkers like BUN (mg/dl) and serum creatinine (mg/dl) were significantly (P<0.05) elevated on day 4 in groups 1, 2, 3, and 4 [Table 3] as compared with the mean values of day 0. The N-acetyl-p-benzoquinoneimine, a reactive metabolite of APAP that causes oxidative damage to tissues, might be the reason for its renotoxic effects.[6] At the end of treatment on day 14, the BUN level was significantly (P<0.05) decreased in groups 2, 3, and 4. However, groups treated with silymarin (group 2) and silymarin + T. chebula (group 4) showed a significant (P<0.05) decrease in BUN level when compared with T. chebula alone treated group (group 3) at 14th day [Table 3]. Nonprotein nitrogenous substances such as BUN and serum creatinine are increased only when renal function is below 30% of its original capacity. An increase in BUN reflects an accelerated rate of protein catabolism.[7] Cytochrome P450-mediated toxic metabolite of acetaminophen binds with cell macromolecules, which might be the reason for its nephrotoxic effects.[6] The beneficial effects of the drugs in test may be attributed to free radical scavenging activity of T. chebula[8] and renoprotective effects of T. chebula.[9] Silymarin has been reported to possess protective action on kidney cells against PCM-induced toxicity.[10]

Table 3

Blood urea nitrogen and serum creatinine concentration in different groups of rats

Group	Blood urea nitrogen (BUN) (mg/dl)			Serum creatinine concentration (mg/dl)
	Day 0	Day 4	Day 14	Day 0	Day 4	Day 14
Acetaminophen (APAP) (1 – 3 days)	23.14 ± 1.85aABC	38.49 ± 2.65aEF	36.10 ± 2.57cE	0.77 ± 0.03aAB	1.42 ± 0.14aE	1.09 ± 0.05bCD
APAP (1 – 3 days) + silymarin (4 – 14 days)	18.24 ± 2.05aA	38.62 ± 0.81aEF	29.18 ± 0.77abcD	0.70 ± 0.06aAB	1.18 ± 0.10aD	0.88 ± 0.08aABC
APAP (1 – 3 days) +Terminalia chebula (4 – 14 days)	20.63 ± 2.97aAB	40.50 ± 2.45aEF	33.98 ± 0.73bcDE	0.66 ± 0.04aA	1.16 ± 0.06aD	0.90 ± 0.07abBC
APAP (1 – 3 days) + silymarin +Terminalia chebula (4 – 14 days)	20.88 ± 2.04aAB	44.65 ± 2.66aF	26.92 ± 1.87aBC	0.77 ± 0.03aAB	1.18 ± 0.06aD	0.85 ± 0.02aAB

Values are mean ± SE of six observations; Means with different alphabets as superscripts differ significantly (P<0.05) ANOVA; Capital alphabets (horizontal comparison), small alphabets (vertical comparison)