Investigation into the Hepatoprotective effect of Heparvit in mice with Acetaminophen Induced Hepatotoxicity

Dr.mr.sci. Marija Renic, Prof. Dr.sci. Filip Culo, Department of Physiology and Immunology, School of Medicine, University of Zagreb, Šalata 3, 10 000 Zagreb, Croatia.

• Tel: +385 1 4566-777 Fax: +385 1 272-050
• faculty web-site: www.mef.hr/homepage/html/fizio.htm
• email: Marija [email protected]

Introduction

Acetaminophen: Acetaminophen (Paracetamol; Tylenol, N-acetyl-para-aminophenol; AAP) is an active metabolite of the other para-aminophenol compounds, such as phenacetin and acetanilide. Acetaminophen has analgesic, antipyretic and only a weak anti-inflammatory action when taken in therapeutic doses.

Metabolism of acetaminophen: Acetaminophen is rapidly and almost completely absorbed from the gastrointestinal tract. The concentrations in plasma reaches a peak within 30 to 60 minutes, and the half-life in plasma varies from 1 to 3 hours after therapeutic dose in normal subjects. Acetaminophen is relatively uniformly distributed throughout most body fluids. Binding of the drug to serum proteins is variable but does not exceed 50 per cent of the total drug concentrations in the serum. After therapeutic dose, acetaminophen is predominantly metabolised by conjugation with glucuronic acid (60%), sulfuric acid (35%) and cystein (3%) and excreted in the urine. A small proportion of acetaminophen is hydroxylated by cytochrome P-450-dependent mixed function oxidises to form highly reactive metabolite N-acetyl-p-benzoquinoneimine. This metabolite is normally detoxified by conjugation with hepatic gluthatione and subsequently excreted in the urine as conjugates with cysteine and mercapturic acid. Only a few per cent of a dose of acetaminophen are eliminated unchanged by the kidneys (ref.1).

Toxic action of acetaminophen: Acetaminophen is rarely toxic when used in a therapeutic dose. Hypersensitivity to acetaminophen occurs rarely and is usually manifested by erythematous or urticarial skin rashes. Therapeutic doses of acetaminophen do not produce gastric irritation, erosion or bleeding, do not interfere with platelet function. When overdoses of acetaminophen are taken, abnormally large amounts of the toxic arylating metabolite N-acetyl-p-benzoquinoneimine are formed, which covalently bind to sulfuric group of hepatic glutathione. When hepatic glutathione is exhausted, N-acetyl-p-benzoquinoneimine then bind covalently to essential hepatocellular macromolecules and thereby causes hepatic necrosis. Hepatic necrosis caused by acetaminophen overdose is centrilobular, with sparing of the periportal areas. The severity of the hepatic injury by acetaminophen is increased with consumption of alcohol, phenobarbitone and some other drugs that induce hepatic drug-metabolising enzymes. The liver may be injured by a single dose of 10 g, and doses of 20 to 25 g or more are potentially fatal. Initial symptoms of acetaminophen toxicity are nausea, vomiting, diarrhoea and abdominal pain. Hepatic injury becomes apparent 24 to 48 hours after ingestion by increases in serum activities of aminotransferases and lactic dehydrogenase, in prothrombin time and in serum bilirubin concentrations. In severely intoxicated patients hepatic failure may progress to encephalopathy, coma and death within a few days (ref. 2). Treatment of acetaminophen overdose include gastric lavage and subsequent administration of activated charcoal or administration of glutathione precursors (ref. 3).

		Acetaminophen
				Hepatic microsomal P-450
Inactivated by glutathione		Toxic metabolite N-acetyl-p-benzoquinoneimine
		Binding liver cell macromolecules
		Necrosis

Figure 1. The mechanism of acetaminophen liver injury

Materials and Methods

Animals: CBA/H Zgr inbred mice were raised in an animal colony unit at the Department of Physiology. Mice of both sexes aged 3 to 4 months were used in the experiments. Mice were maintained under standard laboratory conditions, fed with commercially available murine food pallets (K-l, Faculty of Biotechnology, Domzale, Slovenia) and allowed water ad libitum.

Chemical substances:

Acetaminophen - [Paracetamol/Tylenol]. Pure acetaminophen substance was kindly provided by the Belupo pharmaceutical company (Koprivnica, Croatia).

Heparvit - Pure standardised heparvit [95.2% Fraxinus Ornus (Manna Ash) extr.sic., 2.4% Matricaria Camomile (Camomile) pulv., Bee Pollen] was kindly provided by the herbal/dietary supplement company Bio-Monde Preparations (Douglas, Isle of Man, UK), LD50 in CBA/H Zgr mice was 0.5 g/kg i.p. Heparvit was dissolved in phosphate-buffered saline (PBS) to which several drops of Tween 20 were added (50µl/ml) (Serva, Heidelberg, Germany). The resulting white milk suspension administered to mice intraperitoneally.

Induction of hepatotoxicity with acetaminophen: The procedure of Guarner was followed (ref. 4). To induce hepatic drug-metabolising enzymes, mice were given phenobarbitone-sodium (Kemika, Zagreb, Croatia) in drinking water during 7 days (0.3 g/L). Thereafter, mice were fasted overnight. Acetaminophen, dissolved in heated PBS, was given intragastrically, by stomach tube, in a volume of 0.5 ml. Animals were allowed food 4 hours later. Mortality of mice was followed for 48 hours, as previous results have shown that control mice (given saline before acetaminophen) after that time either die within this period or fully recover and survive indefinitely. Heparvit was given to mice intraperitoneally in volume of 0.5 ml 30 minutes before acetaminophen. At the same time, control animals were given 0.5 ml of pyrogen-free saline to which several drops of Tween 20 (50µl/ml) were added.

Measurement of plasma aminotransferase concentrations: Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 24 hours after the administration of acetaminophen, because it has been observed that the levels of AST and ALT are at their peak values at this time. Mice were given 250 units intraperitoneal heparin 15 minutes before bleeding. Blood was obtained by puncture of medial orbital angle using heparinised glass capillary tubes. Plasma was stored at -20 C until aminotransferase determination, which was carried out using standard laboratory techniques (ref: 5, 6).

Statistical analysis: Differences in survival between groups of mice were compared by X2 - test. Plasma AST and ALT concentrations are expressed as means ± SEM and differences between groups were compared by Student s t-test.

Results

The effect of different doses of heparvit on survival of mice with AAP-induced hepatotoxicity: Three different doses of heparvit (10, 50, or 250 mg/kg) were given to mice intraperitoneally 30 minutes before intragastric administration of acetaminophen (300 mg/kg). Table 1 shows the mortality of mice 48 hours after acetaminophen administration. In comparison with control group of mice given saline, pre-treatment of mice with 250 mg/kg of heparvit significantly reduced the mortality of mice (P < 0.01). The mortality of mice given 10 or 50 mg/kg of heparvit was similar to that observed in control mice given saline. Tables 2 and 3 show the mortality of mice of two further experiments. As shown, pre-treatment of mice with heparvit (250 mg/kg) statistically significantly reduced the mortality of mice in comparison with control mice given saline (P < 0.001).

The effect of heparvit on plasma aminotransferase levels in mice with AAP-induced hepatotoxicity: Heparvit (250 mg/kg) or saline were given to mice 30 minutes before administration of acetaminophen (220 mg/kg). Plasma AST and ALT were measured 24 hours after acetaminophen administration. Table 4 shows plasma aminotransferase levels in normal and in mice treated with heparvit or saline. As shown, in comparison with normal mice, the administration of acetaminophen increased AST and ALT by approximately 19 and 100 times, respectively. Pre-treatment of mice with heparvit significantly reduced the increase of AST and ALT in comparison with control mice pre-treated with saline (AST five times and ALT three times; P < 0.005).

Table 1. The effect of different doses of heparvit on survival of mice with AAP-induced hepatotoxicity

{a} Mice were given 300 mg/kg AAP intragastrically in volume of 0.5 ml. Saline or different doses of heparvit were given intraperitoneally 30 minutes before AAP administration

{b} Mortality was recorded 48 hours after AAP administration

{c} Significantly different in relation to the control group of mice treated with saline (P < 0.01)

---

Table 2. The effect of different doses of heparvit on survival of mice with AAP-induced hepatotoxicity

{a} Mice were given 250 mg/kg AA.P intragastrically in volume of 0.5 ml. Saline or heparvit were given intraperitoneally 30 minutes before AAP administration

{b} Mortality was recorded 48 hours after AAP administration

{c} Significantly different in relation to the control group of mice treated with saline (P < 0.01)

Table 3. The effect of heparvit on survival of mice with AAP-induced hepatotoxicity

{a} Mice were given 250 mg/kg AA.P intragastrically in volume of 0.5 ml. Saline or heparvit (250 mg/kg) were given intraperitoneally 30 minutes before AAP administration

{b} Mortality was recorded 48 hours after AAP administration

{c} Significantly different in relation to the control group of mice treated with saline (P < 0.01)

Table 4. Plasma aminotransferase levels in mice with AAP-induced hepatotoxicity pre-treated with saline or heparvit

Treatment{a}	AST (U/L){b}	ALT (U/L){b}
Normal mice	75 ± 4 (n = 19)	23 ± 1 (n = 19)
Heparvit + AAP	1417 ± 355 (n = 16)	2300 ± 376 (n = 16)
Heparvit + AAP	257 ± 56{c} (n = 16)	776 ± 166{c} (n = 16)

{a} Saline or heparvit (250 mg/kg) were given 30 minutes before AAP administration (220 mg/kg)

{b} Determined 24 hours after AAP administration; mean ± SEM

{c} Significantly different in relation to the control group of mice given saline before AAP administration (P < 0.05)

Conclusion

The results of the investigation have shown that heparvit has hepatoprotective effect in mice with acetaminophen-induced acute hepatotoxicity. Heparvit in a dose of 250 mg/kg given intraperitoneally to mice 30 minutes before administration of acetaminophen, significantly reduced the mortality of mice and reduced plasma aminotransferase levels in comparison with control mice given saline before administration of acetaminophen.

References

1. Koch-Weser J. Drug therapy. Acetaminophen. N Engl J Med 1976; 295:1297-300.

2. Black M. Acetaminophen hepatotoxicity. Ann Rev Med 1984; 35:577-93.

3. Prescott L F, Newton R W, Swainson C P. Successful treatment of severe paracetamol overdosage with cisteamine. Lancet 1974; 1:588-92.

4. Guarner F, Boughton-Smith N K, Blackwell G J, Moncada S. Reduction by prostacyclin of acetaminophen-induced liver toxicity in the mouse. Hepatology 1988; 8:248-53.

5. Expert Panel on Enzymes, Committee on Standards (IFCC). Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes part 2. IFCC methods for aspartate aminotransferase. J Clin Chem Biochem 1977; 15:39-51.

6. Expert Panel on Enzymes (IFCC). Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. IFCC methods for alanine aminotransferase. Clin Chem Acta 1980; 105:147F-54F.