The Protective Effect of Garlic Extract against Acetaminophen-Induced Loss of Mitochondrial Membrane Potential in Freshly Isolated Rat Hepatocytes

1. Introduction

       Garlic (Allium sativum) has been used as a medicinal plant, since before the time of the Sumerian civilization (2600-2100 BC), by when it was already widely cultivated in India and China [1]. The intact bulbs of garlic contain an odorless, sulfur-containing amino acid derivative known as alliin [(+)-S-allyl-L-cysteine sulfoxide]. When garlic bulbs are crushed, alliinase rapidly lyses the cytosolic alliin to form allicin (diallyl thiosulfinate) which is the dominant sulfur compound in crushed garlic [2]. Allicin is highly odoriferous, unstable, and undergoes a number of transformations depending on pH, temperature, and solvent conditions [3]. Its half-life in tissues is too short to ensure appreciable pathogen damage [4]. Systemic levels of the metabolic products of allicin are reported to be relatively high [5] and researchers have now turned their interest to these constituents due to their considerable antimicrobial and antioxidant properties. Allicin also has been shown to transform to diallyl disulfide and allyl mercaptan in the isolated perfused rat liver, with the former being rapidly metabolized to allyl mercaptan [6]. Acetaminophen or paracetamol, as it is known in Europe (N-acetyl-para-aminophenol; APAP) is a widely used analgesic and antipyretic drug throughout the world [7]. It has been long known that an overdose of APAP can cause severe damages to the liver or even death of the experimental animals and individuals who have ingested large quantities of APAP accidentally or in an attempt to commit suicide [8]. At pharmaco-logical doses, APAP is mainly metabolized by sulfation and glucuronidation [9]. A small proportion is metabolized through cytochrome CYP2E1 and to a lesser extent CYP1A2 and CYP3A4, which produce a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI) [10, 11]. Following therapeutic doses NAPQI is efficiently detoxified by conjugation with glutathione [12]. However,in overdoses a large amount of APAP is metabolized through the P450s, leading to GSH depletion by NAPQI conjugation followed by covalent binding of NAPQI to proteins [13]. Inhibition of mitochondrial respiration has been investigated as an important mechanism in acetaminophen toxicity [14]. Although the precise mechanism of APAP hepatotoxicity is not well understood, a number of studies have suggested that NAPQI exerts a cytotoxic effect through its covalent binding to cytosolic or microsomal proteins and membrane components, inhibition of mitochondrial respiration, depletion of ATP etc. [7]. Studies on APAP hepatotoxicity remain a very active area since much is still uncertain despite continuous efforts worldwide. On the other hand, considering repugnance results about cytoprotective effects of garlic and allicin, in the following, for the first time, the protetctive effect of garlic extract, allicin, and NAC towards APAP toxicity on freshly isolated rat hepatocytes have been compared. Also, the role of MMP has been studied with regards to the acetaminophen toxicity and the protective effects of agents mentioned above.

2. Materials and methods

2.1. Chemicals and plant materials

        Bovine serum albumin, collagenase A from Clostridium histolyticum and HEPES were obtained from Roche Diagnostics (Indianapolis, IN), acetaminophen (APAP; 4-acetamidophenol) from Sigma-Aldrich (St. Louis, MO), N-acetyl-cysteine (NAC) from Acros Pharmaceuticals, Rhodamine 123 from Fluka, b-naphto-flavon (BNF), heparin sodium salt grade 1-A, trypan blue, methanol, MgSO₄ and other buffer salts, were obtained from Merck (Germany). All other chemicals were of the highest grade commercially available.

2.2. Animals

      Male Sprague-Dawley rats (200-250 g) were obtained from the Laboratory of Animals Research Center of Tabriz University of Medical Sciences. The rats were housed in an air-conditioned room, under controlled temperature of 23±1 °C, relative humidity of 36±6% and 12 h light/12 h dark conditions for 1 week before starting the experiments. They were allowed to feed with standard laboratory chaw and tap water ad libitum. Procedures involving animals and their care were conducted in conformity with the NIH guidelines for the care and use of laboratory animals.

2.3. Preparation of garlic extract

       Fresh garlic (Allium sativum L.) was purchased from a retail food store (Tabriz, Iran) and identified by botanists in the herbarium of Tabriz University. On the day of experiments the garlic bulbs were peeled, weighed and ground to obtain a fine juice. It was then homogenized in either methanol or deionized water. The homogenized mixture was filtered through cheesecloth. Garlic extracts of lower concentrations were prepared by dilution of this solution with media used for cell suspensions [15].

2.4. Determination and preparation of allicin from garlic extract

       In order to determine and isolate the allicin content in garlic a modified method of Vargas et al. [16] was applied. Briefly, aqueous extract was assayed using analytical HPLC. Separation of allicin from extract was performed using a Spherisorb ODS2 column (4.6×250mm, 5μm, Waters, Ireland) and methanol (75%) and water (phosphate buffer pH=3), as a mobile phase, with a flow rate of 1 ml/min., detecting allicin at the wavelength of 254 nm. Retention time for allicin was 5.7 min. Pure allicin was obtained on the basis of analytical HPLC from a further preparative HPLC. The achieved allicin solution was dried with freeze drier and the resulting allicin powder was used for all experiments. Allicin content of garlic bulbs was quantified with analytical HPLC mentioned above as 80 mg%.

2.5. Preparation of allicin stock solution

       Allicin (32 mg) acquired from preparative HPLC was dissolved in 20 ml distilled water, so that the concentration reaches to 10 mM.

2.6. Preparation of hepatocytes

        Hepatocytes were isolated from male Sprague-Dawley rats by a two-step collagenase perfusion, as described previously [17, 18]. The first step involves the perfusion of a calcium-free buffer. The second step is circulation of a calcium-supplemented buffer containing collagenase. The initial perfusion facilitates desmosomal cleavage and further dispersion of liver cells. The addition of Ca²⁺ to the enzyme solution ensures adequate collagenase activity. After isolation, the cells were suspended (10⁶ cells/ml) in Krebs-Henseleit buffer containing 12.5 mM HEPES and incubated under a stream of 95% O₂ and 5% CO₂ in continuously rotating round-bottomed 50 ml flasks at 37 °C. Cell viability was measured by Trypan blue exclusion method. The hepatocytes used in this study were at least 85-90% viable immediately after isolation.

2.7. Measurement of mitochondrial membrane potential

       Mitochondrial membrane potential of cells was assessed by monitoring the uptake of cationic dye Rhodamine 123 [19]. Isolated cells were extracted, and then resuspended in original media containing 1 mM Rhodamine 123. After 10 min. of incubation, the cells were centrifuged and the supernatant was measured with a Shimadzu RF-5000U spec-trofluorimeter at the excitation wavelength 501 nm and the emission wavelength 530 nm. The amount of dye remaining in the supernatant was inversely proportional to the membrane potential of the cells [20].

2.8. Experimental protocols

       The animals of test group received 3 i.p. injections of BNF (80 mg/kg) during 72 h. before starting the experiments; while the animals of positive control and negative control groups received corn oil and no chemicals, respectively [21]. On the day of experiments, after induction of anesthesia using sodium pentobarbital, the liver cells were isolated via a two step model. The cells were allowed to get adapted with the incubation conditions for 20 min. before the addition of compounds to the incubation mixture. The cells were then exposed to different concentrations of allicin, garlic extract and/or NAC 30 min. before, at the same time with APAP and 30 min. after APAP addition. Aliquots of the cells were taken at different time points (0, 60, 120 and 180 min.) for determination of cell viability and MMP.

2.9. Statistical analysis

       Statistical comparisons were carried out using a one-way analysis of variance (ANOVA) followed by the Tukey post-hoc test for multiple comparisons in order to determine statistical significance (p< 0.05 or less) between treatments and control groups. Results represent the mean±SD of at least three independent samples.

3. Results

3.1. Protective effect of GE and/or NAC against APAP cytotoxicity

        As it has been shown in Table 1, APAP (500 μM) was not toxic in normal hepatocytes (the cells without any pretreatments) under controlled conditions. In contrast, it was extremely toxic towards the cells isolated from rats pretreated with BNF. On the other hand, neither the treatments, nor the solvents or carriers, showed significant cytotoxicity in the absence of APAP even in BNF pretreated cells. This means BNF itself could not be cytotoxic although it induces the microsomal metabolism at the doses administered. Among all control groups, only the groups receiving allicin demonstrated cytotoxicity after 180 min. There were no signs of toxicity in normal cells with treatments and chemicals even with APAP addition. Therefore, the results of normal cells have not been entered in Table 2. In order to investigate if the timing of the addition of APAP and the treatments is effective in the responses, three different protocols composed of 30 min. of time intervals were employed and the samples of control were prepared to avoid any possible experimental errors. As it can be seen in Table 2, GE, and NAC were effective against APAP hepatotoxicity in BNF treated hepatocytes, however, allicin had no protective effects. There was not a significant difference between protective effects of GE and NAC.

Figure 1. The effects of allicin, GE, and NAC on the APAP-induced MMP loss when added 30 min. before administration of APAP. Rhodamine was added to the aliquots of cell suspensions and the fluorescence amounts of the samples were measured with a Shimadzu RF-5000U spectrofluorimeter. The amount of dye remaining in the supernatant was inversely proportional to the membrane potential of the cells. All results were shown in mean±SD and are at least from 3 independent experiments.*Shows significant difference (p < 0.05) with APAP group. **Shows significant difference (p < 0.01) with APAP group. Shows significant difference (p

3.2. Protection of mitochondrial membrane potential against APAP toxicity by GE and/or NAC

       Figures 1 to 3 illustrate the relative percentages of mitochondrial membrane potential (MMP) in test groups and its comparison with the control group, where the 100 percent of Rhodamine 123 trapping occurred in the intact mitochondria. In order to separate the main protocols from each other, the results have been illustrated in three figures of 30 min. before APAP addition (Figure 1), at the same time with APAP (Figure 2) and 30 min. after APAP addition (Figure 3).

      Figure 1 illustrates that allicin was not only unable to maintain the mitochondrial membrane potential but also demonstrated APAP like effects; when NAC and/or GE showed significant differences with APAP treatment in MMP. On the other hand, there was no significant difference between NAC as the standard treatment and GE.

      It can be deducted from Figure 2 that both NAC and/or GE were effective in preventing the loss of MMP induced by APAP, but not allicin.

      In Figure 3, there is no significant protective effect by GE and/or allicin. However, NAC significantly protected MMP towards APAP toxicity. There were no significant differences between groups of GE and NAC in Figures 1 and 2, but a significant difference was observed between GE and NAC specially after 180 min. of monitoring (p < 0.05) .These findings indicate the decrease in efficacy of GE when administered after APAP addition which does not happen with NAC. Meanwhile the most non protective effect was shown with allicin 30 min. after APAP addition (Figure 3).

 Figure 2.The effects of allicin, GE, and NAC on the APAP-induced MMP loss when added at the same time with APAP.Rhodamine was added to the aliquots of cell suspensions and the fluorescence amounts of the samples were measured with a Shimadzu RF-5000U spectrofluorimeter. The amount of dye remaining in the supernatant was inversely proportional to the membrane potential of the cells. All results were shown in mean±SD and are at least from 3 independent experiments.*Shows significant difference (p < 0.05) with APAP group. ** Shows significant difference (p < 0.01) with APAP group. ***Shows significant difference (p < 0.001) with control group (BNF treated cells).

4. Discussion