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Treatment with aged garlic extract protects against bromobenzene toxicity to precision cut rat liver slices
Toxicology 132 (1999) 215 – 225
Treatment with aged garlic extract protects against bromobenzene toxicity to precision cut rat liver slices Bo Han Wang, Katherine A. Zuzel, Khalid Rahman, David Billington * School of Biomolecular Sciences, Li6erpool John Moores Uni6ersity, Byrom Street, Li6erpool L3 3AF, UK Received 10 September 1998; accepted 23 December 1998
Abstract Precision-cut liver slices from phenobarbital-induced rats were incubated for 6 h with the model hepatotoxin bromobenzene (BB) at a final concentration of 1 mM. Severe toxicity was indicated by a decreased K + , adenosine triphosphate and glutathione (GSH) content of the slices, increased release of alanine aminotransferase and lactate dehydrogenase into the medium, and increased formation of thiobarbituric acid reacting substances. Pretreatment of animals for 7 days with aged garlic extract (AGE) (Kyolic®) at doses of 2 and 10 ml/kg/day dramatically reduced the toxicity of BB in a dose-dependent manner. The GSH content of liver slices from rats treated with AGE at 2 or 10 ml/kg/day increased by 50 and 80%, respectively. The BB-induced decrease in GSH content was less in slices derived from AGE-treated rats compared with slices from control rats. Pretreatment with AGE did not affect cytochrome P450 when assayed as 7-ethoxycoumarin O-deethylase and 7-pentoxyresorufin O-depentylase activities in hepatic microsomes. Thus, the mechanism by which pretreatment with AGE protects against BB hepatotoxicity involves both an elevation of hepatic GSH content, and a GSH sparing effect, possibly due to conjugation of organosulphur compounds in AGE with toxic BB metabolites. Only this GSH sparing effect was seen in our earlier study on the in vitro hepatoprotective effect of AGE [Wang et al., 1998. Toxicology 126, 213 – 222]. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bromobenzene; Garlic; Hepatotoxicity; Glutathione; Liver slices
Abbre6iations: AGE, Aged garlic extract; ALT, Alanine aminotransferase; BB, Bromobenzene; DMSO, Dimethylsulphoxide; DTNB, 5,5%-dithiobis-(2-nitrobenzoic acid); ECOD, 7-ethoxycoumarin O-de-ethylase; GSH, Total glutathione; LDH, Lactate dehydrogenase; NPSH, Non-protein sulphydryl; PROD, 7-pentoxyresorufin O-depentylase; TBARS, Thiobarbituric acid reacting substances. * Corresponding author. Tel: +44-151-2312077; fax: + 44-151-2982821; e-mail: [email protected]. 0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 0 4 - 9
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1. Introduction Garlic is reputed to possess a wide range of therapeutic properties including anti-tumourigenic (Milner, 1996), anti-thrombotic (Lawson et al., 1992), and hypocholesterolaemic actions (Steiner et al., 1996). It has also been reported that garlic exerts protective effects against the action of certain toxins. For example, garlic and its constituents diallyl sulphide, S-allyl cysteine and S-allyl mercaptocysteine have been shown to protect mice against paracetamol hepatotoxicity (Nakagawa et al., 1989; Hu et al., 1996a; Wang et al., 1996). It has been suggested that this hepatoprotection is due, at least in part, to inhibition of the formation of the toxic metabolite N-acetyl-pbenzoquinone by cytochromes P450 2E1 and P450 1A2, since fresh garlic homogenate and diallyl sulphide have been shown to inhibit the 2E1 isoform of cytochrome P450 (Hu et al., 1996a; Wang et al., 1996). We have demonstrated previously that inclusion of an aged garlic extract (AGE) in the incubation medium of precision-cut rat liver slices protects against the toxic effects of the industrial solvent bromobenzene (BB) (Wang et al., 1998). BB is metabolised primarily to the corresponding 3,4-oxide by cytochromes P450 2B1 and P450 2B2; thus, its toxicity is potentiated by preferential induction of these isoforms by sodium phenobarbital. At low doses the 3,4-oxide is detoxified by conjugation with glutathione, but at doses sufficient to cause glutathione depletion, the accumulating 3,4-oxide causes lipid peroxidation and conjugates to proteins (Jollow et al., 1974). While the precise mode of toxicity is not clear, it appears that cell damage is due to the 3,4-oxide as the other metabolites are not hepatotoxic (Lau and Monks, 1988). BB has also been shown to form secondary quinone metabolites (Buben et al., 1988; Narasimhan et al., 1988), and the formation of hydrogen peroxide in HepG2 cells exposed to BB has been reported (Wu et al., 1997). In our previous study, AGE was added to the incubation medium of rat liver slices (Wang et al., 1998). In order to mimic more closely the in vivo situation where garlic would be ingested orally on a regular basis, we have now investigated the
effects of feeding rats with AGE for 7 days on BB toxicity to subsequently isolated precision-cut liver slices. Slice K + , adenosine triphosphate (ATP), non-protein sulphydryl (NPSH) and glutathione (GSH) content, lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) release, and the formation of thiobarbituric acid reacting substances (TBARS) have been used as indicators of toxicity. In addition, the effects of feeding AGE on the hepatic content of GSH and on some cytochrome P450 activities were also investigated.
2. Materials and methods
2.1. Chemicals AGE (Kyolic®) was kindly provided by Wakunaga of America (Mission Viejo, CA). It is prepared by soaking sliced raw garlic (Allium sati6um) in 15–20% aqueous ethanol for at least 10 months at room temperature. The extract is then filtered and concentrated under reduced pressure at low temperature. The content of water-soluble compounds is relatively high while that of oil-soluble compounds is low. The AGE used in these experiments contained 30.5% extracted solids (305 mg/ml), and S-allyl cysteine, the most abundant water-soluble compound in AGE, was present at 1.47 mg/ml. NADH, NADPH and diagnostic kits for assaying ALT were purchased from Boehringer Mannheim UK (Lewes, East Sussex, UK). All other chemicals were of reagent grade and were obtained from Sigma (Poole, Dorset, UK).
2.2. Animals Adult male Wistar rats (200–300 g) were used throughout. These were bred within the Life Sciences Support Unit of Liverpool John Moores University and maintained in a relative humidity of 50% and in a 12-h light/12-h dark cycle at 22oC. All dosings were performed between 10.00 and 12.00 h and all animals were fasted for 2 h before and 2 h after gavage. AGE was administered
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orally by stomach tube for 7 days at 10 ml/kg/ day; some animals were administered the same volume of a 20% (v/v) dilution of AGE in water (equivalent to a dose of 2 ml/kg/day). Control animals received distilled water only. In order to potentiate BB toxicity, mixed function oxygenases were induced by the intraperitoneal injection of sodium phenobarbital at 100 mg/kg/day for the last 4 days of treatment of all rats.
2.3. Preparation of li6er slices and microsomes Precision-cut rat liver slices (8 mm diameter, 250920 mm thick) and microsomes (5 – 10 mg of protein/ml) were prepared as described previously (Wang et al., 1998).
2.4. Incubation of li6er slices Slices were incubated essentially as described by Connors et al. (1990) in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 1.2 ml of 100X MEM non-essential amino acids, 1.2 ml of 200 mM L-glutamine, 22.8 ml of 10 mg/ml gentamicin solution, 25.2 ml of 33.7 mg/ml hydrocortisone solution, 4.5 ml of 130.8 mg/ml insulin solution and 1 ml of 5000 IU/ml +5000 mg/ml penicillin/streptomycin solution per 100 ml. The medium was gassed with O2:CO2 (19:1 v/v) for 10 min prior to use. Slices were placed in individual wells of a 24-well plastic tissue culture plate containing 0.5 ml of supplemented DMEM per well. Plates were incubated on an orbital shaker oscillating at 60 rpm and contained within an incubator at 37°C with saturated humidity and an atmosphere of 95% air/5% CO2. All slices were pre-incubated for 30 min before any experiments were undertaken. Stock BB solutions were prepared in dimethyl sulphoxide (DMSO) and added to the culture medium such that final BB and DMSO concentrations were 1 mM and 1% (v/v),respectively. Control incubations contained 1% (v/v) DMSO only. Bromobenzene has been reported previously to produce concentration-dependent toxicity in rat liver slices (Smith et al., 1987), and at concentra-
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tions of 1 mM, BB produces substantial toxicity in liver slices from phenobarbital-induced rats, but not in slices from control rats (Smith et al., 1985).
2.5. Biochemical assays The following were determined as described previously (Wang et al., 1998): protein by a Coomassie blue binding method; slice K + content by flame photometry; slice ATP content by the firefly luciferase assay; LDH and ALT leakage from slices by NAD + -linked reactions; slice NPSH content by reaction with 5,5%-dithiobis-(2nitrobenzoic acid) (DTNB); slice total GSH using the DTNB–glutathione disulphide reductase recycling method; lipid peroxidation by formation of TBARS, and, microsomal 7-ethoxycoumarin Odeethylase (ECOD) and 7-pentoxyresorufin O-depentylase (PROD) activities by fluorimetric assays.
2.6. Statistical analyses Values are presented as means 9S.D. from at least four experiments. The data were analysed with an unpaired two-tailed Student’s t-test and differences were considered significant at PB 0.05.
3. Results
3.1. Slice K + and ATP content Slices from control animals maintained their K + content at close to that in freshly isolated slices for the duration of the 6-h incubation period (Fig. 1A). The K + content of slices was not affected by the pre-treatment of animals with AGE at either dose used. BB (1 mM) produced a marked loss of intracellular K + from slices from control animals such that after 6 h in culture, it was reduced by approximately 75%. This BB-induced loss of K + was partially prevented in a dose-dependent manner by pretreatment of animals with AGE (Fig. 1A).
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Fig. 1. Effects of pretreatment with AGE on the BB-induced loss of (A) K + and (B) ATP from rat liver slices. Animals were dosed orally for 7 days at 10 ml/kg/day with either water (control), AGE or a 20% (v/v) dilution of AGE (AGE20). All animals were administered sodium phenobarbital (100 mg/kg/day) by intraperitoneal injection for the last 4 days of oral dosing. Subsequently isolated slices were incubated in the presence or absence of 1 mM BB as described in Section 2. Values are means 9 S.D. from four animals. + , P B 0.05 compared with control; *, PB 0.05 compared with control+1 mM BB.
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Fig. 2. Effects of pretreatment with AGE on the BB-induced release of (A) ALT and (B) LDH from rat liver slices. See Fig. 1 for symbols and abbreviations.
Essentially similar results were obtained for slice ATP content (Fig. 1B). The ATP content of slices from control animals was unchanged from that of freshly isolated slices after 6 h in culture.
BB produced a dramatic decrease in slice ATP content, which was partially prevented in a dosedependent manner by pretreatment with AGE (Fig. 1B).
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3.2. ALT and LDH leakage from slices
3.4. TBARS formation
Release of intracellular enzymes from control slices into the medium after 6 h in culture was B 15% of total slice activity for ALT (Fig. 2A) and B 30% of total slice activity for LDH (Fig. 2B). ALT and LDH leakage was increased by approximately threefold when slices from control animals were incubated with 1 mM BB. AGE exhibited a marked dose-dependent inhibition of BB-induced ALT and LDH release (Fig. 2).
BB caused a time-dependent increase in TBARS formation by slices from control animals such that after 6 h in culture, TBARS were increased by approximately fivefold (Fig. 4). This was completely inhibited by pre-treatment of animals with AGE. Indeed, a dose dependency was not observed in that pretreatment with the 20% (v/v) dilution of AGE restored TBARS formation to values similar to those for slices from control animals (Fig. 4).
3.3. Slice NPSH and GSH content Since measurement of NPSH by the DTNB method detects all sulphydryl-containing compounds, the reductant status of slices was monitored by measuring NPSH together with GSH by a specific enzymic method. Pretreatment of rats with AGE for 7 days increased the content of NPSH and GSH by approximately 60 and 80%, respectively in subsequently isolated liver slices (Fig. 3). Pretreatment with low-dose AGE produced more modest, but significant, increases in slice NPSH and GSH content. The NPSH and GSH content of slices from both control and AGE-treated rats remained constant during 6 h in culture (Fig. 3). Inclusion of 1 mM BB in the culture medium of slices from control animals resulted in a rapid and extensive depletion of both NPSH and GSH; indeed, almost total depletion of GSH was observed after 6 h. The NPSH and GSH content of slices from AGE-treated rats still decreased in the presence of 1 mM BB. However, this decrease was less marked than that in slices from control animals. Thus, because of the elevated NPSH and GSH contents at time zero, even after 6 h in culture with BB, slices from AGE-treated rats still had NPSH and GSH contents above those of slices from control animals incubated in the absence of BB. Furthermore, after 6 h in culture with BB, slices from rats pretreated with low-dose AGE had NPSH and GSH contents similar to those of slices from control animals.
3.5. Time course of the increase in slice NPSH and GSH content The NPSH and GSH contents of slices from control animals are given in Table 1; GSH accounted for approximately 70% of slice NPSH. Treatment with AGE caused a time-dependent increase in both NPSH and GSH which was maximal after 5 days. The non-GSH NPSH content of slices was calculated by difference and can be seen to be approximately constant throughout 7 days of AGE administration (Table 1).
3.6. Effects of AGE on ECOD and PROD acti6ities ECOD and PROD activities in microsomes from control livers were 10.49 0.6 nmol/min/mg protein and 62.89 4.7 pmol/min/mg protein respectively (means9 S.D., n= 5). Pretreatment of animals for 7 days with both doses of AGE did not affect either activity in subsequently isolated microsomes (results not shown).
4. Discussion Garlic contains a number of organosulphur compounds which are generally believed to be the active constituents responsible for its biological actions (Agarwal, 1996). These can be divided into water-soluble compounds such as S-allyl cysteine and oil-soluble compounds such as diallyl sulphide. The aged garlic extract that we have used (Kyolic®) is prepared by slicing fresh garlic
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Fig. 3. Effects of pretreatment with AGE on the BB-induced depletion of (A) NPSH and (B) GSH in rat liver slices. See Fig. 1 for symbols and abbreviations.
and soaking it in dilute ethanol for at least 10 months. Thus, it is relatively enriched in the water-soluble constituents and contains only low levels of oil-soluble constituents; indeed, by far the most abundant organosulphur compound in AGE
is S-allyl cysteine. The doses of AGE used in this study (2 and 10 ml/kg/day) are comparable with those used by other workers. For example, Efendy et al. (1997) administered 0.8 ml/kg/day to rabbits when investigating the effect of AGE on experi-
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Fig. 4. Effects of pretreatment with AGE on the BB-induced TBARS formation by rat liver slices. See Fig. 1 for symbols and abbreviations.
mental atherosclerosis, while Kyo et al. (1997) administered 10 ml/kg when assessing the anti-allergic effects of AGE in rats and Steiner et al. (1996) gave 7.2 g of AGE powder to hypercholesterolaemic men. It is clear that slices prepared from livers of rats which have been treated with AGE for 7 days are protected against BB toxicity. All the indicators of toxicity studied showed that liver slices from AGE-treated rats are resistant to BB toxicity, and this is more pronounced at the higher of the two doses administered. Thus, the BB-induced leakage of K + , loss of ATP (Fig. 1) and leakage of the two intracellular enzymes LDH and ALT (Fig. 2) were all reduced by pretreatment with AGE. Similarly, lipid peroxidation, as judged by the formation of TBARS, was reduced to control levels even at the lower dose of AGE (Fig. 4). A possible explanation for the protective effect of AGE is that the toxic P450-derived metabolites of BB are not formed due to inhibition of some cytochrome P450 isoforms. This cannot be the case here since both ECOD and PROD activities were similar in microsomes isolated from livers of
control and AGE-treated rats. ECOD activity estimates total cytochrome P450, while PROD is relatively specific for those isoforms responsible for BB metabolism, namely 2B1 and 2B2 (Gonzalez, 1990). Although garlic and some organosulphur compounds inhibit some isoforms of P450, such effects are limited to the oil-soluble organosulphur compounds (Reicks and Crankshaw, 1996). Indeed, diallyl sulphide has been shown to inhibit cytochrome 2E1 (Hu et al., 1996a). Of more interest is the substantial increase in the hepatic content of NPSH and GSH by AGE (Fig. 3). This effect occurred over several days and was specific to glutathione in that the hepatic content of other non-GSH NPSH (presumably mainly free cysteine) was not increased by pretreatment with AGE (Table 1). Similar increases in GSH content have been shown in both bovine pulmonary artery endothelial cells after 24 h in culture with AGE (Geng and Lau, 1997) and in human prostate carcinoma cells after 3 h in culture with S-allyl cysteine and S-allyl mercaptocysteine (Pinto et al., 1997) However, it must be
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Table 1 Effect of pretreatment with AGE on the NPSH and GSH content of rat liver slicesa Treatment
NPSH content (nmol/mg protein)
GSH content (nmol/mg protein)
Non-GSH NPSH content (nmol/mg protein)
Control AGE, 1 AGE, 3 AGE, 5 AGE, 7
52.39 8.6 50.0, 65.6 64.4, 79.1 82.6, 84.9 85.29 10.5*
36.79 4.2 36.2, 47.0 45.6, 59.1 64.4, 61.3 64.4 910.2*
16.4 98.5 13.8, 18.6 18.8, 20.0 18.2, 23.6 17.89 4.5
day days days days
a
Rats were administered AGE via a stomach tube at 10 ml/kg/day for the number of days shown; control animals received similar volumes of water for 7 days. Individual values are given for days 1, 3 and 5; values for controls and day 7 are means9 S.D. (n =4). Values for non-GSH NPSH content were calculated by difference. * Significant differences from corresponding controls (PB0.05).
emphasised that in our previous study, the GSH content of rat liver slices was not increased after 6 h in culture with up to 5% (v/v) AGE (Wang et al., 1998). Since the primary mode of detoxification of the 3,4-oxide metabolite of BB is by conjugation with GSH, an increased hepatic content of GSH would be expected to confer a higher capacity for detoxification. The mechanism by which treatment of rats with AGE increases the hepatic content of GSH must involve either increased synthesis and/or decreased utilisation. It is doubtful that decreased glutathione S-transferase activity could account for decreased utilisation; indeed, the garlic constituents diallyl sulphide, diallyl trisulphide (Sparnins et al., 1988; Hu et al., 1996b) and S-allyl cysteine (Hatono et al., 1996) have been shown to induce some isoforms of glutathione S-transferase in liver. Alternatively, an increase in the bioavailability of cysteine might increase GSH synthesis since this is usually the limiting substrate. Such a mechanism has been proposed for N-acetyl cysteine, a hepatoprotective agent used widely to treat paracetamol poisoning (Rafeiro et al., 1994). Thus, hepatic concentrations of cysteine increase dramatically 10 min after intraperitoneal injection of N-acetyl cysteine into rats (Yao et al., 1994). In addition, it has been reported that the hepatoprotective effect of N-acetyl cysteine is blocked by the GSH synthesis inhibitor buthionine sulphoxide (Miners et al., 1984; Rafeiro et al., 1994), and it is not reproduced by the non-physiological stereoisomer N-acetyl-D-
cysteine (Corcoran and Wong, 1986). This suggests that N-acetyl cysteine is deacetylated relatively easily. If the organosulphur compounds in AGE are acting in a similar manner here, they must also be metabolised to yield free cysteine. However, the observations that the NPSH content of rat liver slices does not increase following incubation with AGE (Wang et al., 1998), nor do hepatic concentrations of non-GSH NPSH increase after treatment of rats with AGE (Table 1), do not support this hypothesis. It is interesting to note that in slices from AGE-treated rats, not only was the initial hepatic content of GSH elevated, but the GSH content fell less on exposure to BB. Thus, slices from control rats lost approximately 35 nmol GSH/mg protein over 6 h, while slices from rats treated with both high-dose and low-dose AGE lost only approximately 20 nmol GSH/mg protein (Fig. 3B). It is well established that in cases where xenobiotic metabolism generates electrophilic metabolites leading to depletion of GSH, administration of some thiol-containing compounds may decrease toxicity, either by facilitating GSH synthesis or by sparing GSH as a result of direct formation of conjugates with the electrophilic species (Thor et al., 1979). Although garlic and commercial garlic preparations contain many organosulphur compounds which are nucleophilic, none of those identified carry free sulphydryl groups. The possibility that these are generated by metabolism cannot be eliminated, but is unlikely here since treatment of rats with
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AGE did not increase the hepatic content of non-GSH NPSH (Table 1). It is more likely that any GSH sparing effect exerted by the organosulphur compounds in AGE is due to their nuleophilic nature allowing them to react directly with electrophilic species such as BB-3,4-oxide. Such a situation must occur in vitro since the hepatic content of GSH was not increased when liver slices were cultured for 6 h in the presence of 5% (v/v) AGE, although protection against BBinduced toxicity and GSH depletion was still observed (Wang et al., 1998). In conclusion, it would appear that the mechanism of the protective effect of AGE against BB hepatotoxicity involves two possibly inter-related components. The first, which is seen both in liver slices from AGE-treated rats and when AGE is added to liver slices in vitro, represents a GSH sparing effect, presumably due to conjugation of organosulphur compounds in AGE with the 3,4oxide of BB. The second, which is seen only after pretreating animals with AGE, is a slow process occurring over several days and leads to a specific elevation in the GSH content of liver. The GSHsparing effect would be expected to decrease the utilisation of GSH and hence may explain the long-term elevation of hepatic GSH content. The origin of these effects may be multifactorial involving many of the organosulphur compounds in AGE and/or their metabolites. Clearly, a precise understanding of the mechanisms involved will only be possible after more experiments using defined components in AGE.
Acknowledgements BHW is in receipt of a Liverpool John Moores University research studentship. We thank Wakunaga of America for their generous support.
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Treatment with aged garlic extract protects against bromobenzene toxicity to precision cut rat liver slices Bo Han Wang, Katherine A. Zuzel, Khalid Rahman, David Billington * School of Biomolecular Sciences, Li6erpool John Moores Uni6ersity, Byrom Street, Li6erpool L3 3AF, UK Received 10 September 1998; accepted 23 December 1998
Abstract Precision-cut liver slices from phenobarbital-induced rats were incubated for 6 h with the model hepatotoxin bromobenzene (BB) at a final concentration of 1 mM. Severe toxicity was indicated by a decreased K + , adenosine triphosphate and glutathione (GSH) content of the slices, increased release of alanine aminotransferase and lactate dehydrogenase into the medium, and increased formation of thiobarbituric acid reacting substances. Pretreatment of animals for 7 days with aged garlic extract (AGE) (Kyolic®) at doses of 2 and 10 ml/kg/day dramatically reduced the toxicity of BB in a dose-dependent manner. The GSH content of liver slices from rats treated with AGE at 2 or 10 ml/kg/day increased by 50 and 80%, respectively. The BB-induced decrease in GSH content was less in slices derived from AGE-treated rats compared with slices from control rats. Pretreatment with AGE did not affect cytochrome P450 when assayed as 7-ethoxycoumarin O-deethylase and 7-pentoxyresorufin O-depentylase activities in hepatic microsomes. Thus, the mechanism by which pretreatment with AGE protects against BB hepatotoxicity involves both an elevation of hepatic GSH content, and a GSH sparing effect, possibly due to conjugation of organosulphur compounds in AGE with toxic BB metabolites. Only this GSH sparing effect was seen in our earlier study on the in vitro hepatoprotective effect of AGE [Wang et al., 1998. Toxicology 126, 213 – 222]. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Bromobenzene; Garlic; Hepatotoxicity; Glutathione; Liver slices
Abbre6iations: AGE, Aged garlic extract; ALT, Alanine aminotransferase; BB, Bromobenzene; DMSO, Dimethylsulphoxide; DTNB, 5,5%-dithiobis-(2-nitrobenzoic acid); ECOD, 7-ethoxycoumarin O-de-ethylase; GSH, Total glutathione; LDH, Lactate dehydrogenase; NPSH, Non-protein sulphydryl; PROD, 7-pentoxyresorufin O-depentylase; TBARS, Thiobarbituric acid reacting substances. * Corresponding author. Tel: +44-151-2312077; fax: + 44-151-2982821; e-mail: [email protected]. 0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 9 ) 0 0 0 0 4 - 9
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1. Introduction Garlic is reputed to possess a wide range of therapeutic properties including anti-tumourigenic (Milner, 1996), anti-thrombotic (Lawson et al., 1992), and hypocholesterolaemic actions (Steiner et al., 1996). It has also been reported that garlic exerts protective effects against the action of certain toxins. For example, garlic and its constituents diallyl sulphide, S-allyl cysteine and S-allyl mercaptocysteine have been shown to protect mice against paracetamol hepatotoxicity (Nakagawa et al., 1989; Hu et al., 1996a; Wang et al., 1996). It has been suggested that this hepatoprotection is due, at least in part, to inhibition of the formation of the toxic metabolite N-acetyl-pbenzoquinone by cytochromes P450 2E1 and P450 1A2, since fresh garlic homogenate and diallyl sulphide have been shown to inhibit the 2E1 isoform of cytochrome P450 (Hu et al., 1996a; Wang et al., 1996). We have demonstrated previously that inclusion of an aged garlic extract (AGE) in the incubation medium of precision-cut rat liver slices protects against the toxic effects of the industrial solvent bromobenzene (BB) (Wang et al., 1998). BB is metabolised primarily to the corresponding 3,4-oxide by cytochromes P450 2B1 and P450 2B2; thus, its toxicity is potentiated by preferential induction of these isoforms by sodium phenobarbital. At low doses the 3,4-oxide is detoxified by conjugation with glutathione, but at doses sufficient to cause glutathione depletion, the accumulating 3,4-oxide causes lipid peroxidation and conjugates to proteins (Jollow et al., 1974). While the precise mode of toxicity is not clear, it appears that cell damage is due to the 3,4-oxide as the other metabolites are not hepatotoxic (Lau and Monks, 1988). BB has also been shown to form secondary quinone metabolites (Buben et al., 1988; Narasimhan et al., 1988), and the formation of hydrogen peroxide in HepG2 cells exposed to BB has been reported (Wu et al., 1997). In our previous study, AGE was added to the incubation medium of rat liver slices (Wang et al., 1998). In order to mimic more closely the in vivo situation where garlic would be ingested orally on a regular basis, we have now investigated the
effects of feeding rats with AGE for 7 days on BB toxicity to subsequently isolated precision-cut liver slices. Slice K + , adenosine triphosphate (ATP), non-protein sulphydryl (NPSH) and glutathione (GSH) content, lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) release, and the formation of thiobarbituric acid reacting substances (TBARS) have been used as indicators of toxicity. In addition, the effects of feeding AGE on the hepatic content of GSH and on some cytochrome P450 activities were also investigated.
2. Materials and methods
2.1. Chemicals AGE (Kyolic®) was kindly provided by Wakunaga of America (Mission Viejo, CA). It is prepared by soaking sliced raw garlic (Allium sati6um) in 15–20% aqueous ethanol for at least 10 months at room temperature. The extract is then filtered and concentrated under reduced pressure at low temperature. The content of water-soluble compounds is relatively high while that of oil-soluble compounds is low. The AGE used in these experiments contained 30.5% extracted solids (305 mg/ml), and S-allyl cysteine, the most abundant water-soluble compound in AGE, was present at 1.47 mg/ml. NADH, NADPH and diagnostic kits for assaying ALT were purchased from Boehringer Mannheim UK (Lewes, East Sussex, UK). All other chemicals were of reagent grade and were obtained from Sigma (Poole, Dorset, UK).
2.2. Animals Adult male Wistar rats (200–300 g) were used throughout. These were bred within the Life Sciences Support Unit of Liverpool John Moores University and maintained in a relative humidity of 50% and in a 12-h light/12-h dark cycle at 22oC. All dosings were performed between 10.00 and 12.00 h and all animals were fasted for 2 h before and 2 h after gavage. AGE was administered
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orally by stomach tube for 7 days at 10 ml/kg/ day; some animals were administered the same volume of a 20% (v/v) dilution of AGE in water (equivalent to a dose of 2 ml/kg/day). Control animals received distilled water only. In order to potentiate BB toxicity, mixed function oxygenases were induced by the intraperitoneal injection of sodium phenobarbital at 100 mg/kg/day for the last 4 days of treatment of all rats.
2.3. Preparation of li6er slices and microsomes Precision-cut rat liver slices (8 mm diameter, 250920 mm thick) and microsomes (5 – 10 mg of protein/ml) were prepared as described previously (Wang et al., 1998).
2.4. Incubation of li6er slices Slices were incubated essentially as described by Connors et al. (1990) in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 1.2 ml of 100X MEM non-essential amino acids, 1.2 ml of 200 mM L-glutamine, 22.8 ml of 10 mg/ml gentamicin solution, 25.2 ml of 33.7 mg/ml hydrocortisone solution, 4.5 ml of 130.8 mg/ml insulin solution and 1 ml of 5000 IU/ml +5000 mg/ml penicillin/streptomycin solution per 100 ml. The medium was gassed with O2:CO2 (19:1 v/v) for 10 min prior to use. Slices were placed in individual wells of a 24-well plastic tissue culture plate containing 0.5 ml of supplemented DMEM per well. Plates were incubated on an orbital shaker oscillating at 60 rpm and contained within an incubator at 37°C with saturated humidity and an atmosphere of 95% air/5% CO2. All slices were pre-incubated for 30 min before any experiments were undertaken. Stock BB solutions were prepared in dimethyl sulphoxide (DMSO) and added to the culture medium such that final BB and DMSO concentrations were 1 mM and 1% (v/v),respectively. Control incubations contained 1% (v/v) DMSO only. Bromobenzene has been reported previously to produce concentration-dependent toxicity in rat liver slices (Smith et al., 1987), and at concentra-
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tions of 1 mM, BB produces substantial toxicity in liver slices from phenobarbital-induced rats, but not in slices from control rats (Smith et al., 1985).
2.5. Biochemical assays The following were determined as described previously (Wang et al., 1998): protein by a Coomassie blue binding method; slice K + content by flame photometry; slice ATP content by the firefly luciferase assay; LDH and ALT leakage from slices by NAD + -linked reactions; slice NPSH content by reaction with 5,5%-dithiobis-(2nitrobenzoic acid) (DTNB); slice total GSH using the DTNB–glutathione disulphide reductase recycling method; lipid peroxidation by formation of TBARS, and, microsomal 7-ethoxycoumarin Odeethylase (ECOD) and 7-pentoxyresorufin O-depentylase (PROD) activities by fluorimetric assays.
2.6. Statistical analyses Values are presented as means 9S.D. from at least four experiments. The data were analysed with an unpaired two-tailed Student’s t-test and differences were considered significant at PB 0.05.
3. Results
3.1. Slice K + and ATP content Slices from control animals maintained their K + content at close to that in freshly isolated slices for the duration of the 6-h incubation period (Fig. 1A). The K + content of slices was not affected by the pre-treatment of animals with AGE at either dose used. BB (1 mM) produced a marked loss of intracellular K + from slices from control animals such that after 6 h in culture, it was reduced by approximately 75%. This BB-induced loss of K + was partially prevented in a dose-dependent manner by pretreatment of animals with AGE (Fig. 1A).
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Fig. 1. Effects of pretreatment with AGE on the BB-induced loss of (A) K + and (B) ATP from rat liver slices. Animals were dosed orally for 7 days at 10 ml/kg/day with either water (control), AGE or a 20% (v/v) dilution of AGE (AGE20). All animals were administered sodium phenobarbital (100 mg/kg/day) by intraperitoneal injection for the last 4 days of oral dosing. Subsequently isolated slices were incubated in the presence or absence of 1 mM BB as described in Section 2. Values are means 9 S.D. from four animals. + , P B 0.05 compared with control; *, PB 0.05 compared with control+1 mM BB.
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Fig. 2. Effects of pretreatment with AGE on the BB-induced release of (A) ALT and (B) LDH from rat liver slices. See Fig. 1 for symbols and abbreviations.
Essentially similar results were obtained for slice ATP content (Fig. 1B). The ATP content of slices from control animals was unchanged from that of freshly isolated slices after 6 h in culture.
BB produced a dramatic decrease in slice ATP content, which was partially prevented in a dosedependent manner by pretreatment with AGE (Fig. 1B).
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3.2. ALT and LDH leakage from slices
3.4. TBARS formation
Release of intracellular enzymes from control slices into the medium after 6 h in culture was B 15% of total slice activity for ALT (Fig. 2A) and B 30% of total slice activity for LDH (Fig. 2B). ALT and LDH leakage was increased by approximately threefold when slices from control animals were incubated with 1 mM BB. AGE exhibited a marked dose-dependent inhibition of BB-induced ALT and LDH release (Fig. 2).
BB caused a time-dependent increase in TBARS formation by slices from control animals such that after 6 h in culture, TBARS were increased by approximately fivefold (Fig. 4). This was completely inhibited by pre-treatment of animals with AGE. Indeed, a dose dependency was not observed in that pretreatment with the 20% (v/v) dilution of AGE restored TBARS formation to values similar to those for slices from control animals (Fig. 4).
3.3. Slice NPSH and GSH content Since measurement of NPSH by the DTNB method detects all sulphydryl-containing compounds, the reductant status of slices was monitored by measuring NPSH together with GSH by a specific enzymic method. Pretreatment of rats with AGE for 7 days increased the content of NPSH and GSH by approximately 60 and 80%, respectively in subsequently isolated liver slices (Fig. 3). Pretreatment with low-dose AGE produced more modest, but significant, increases in slice NPSH and GSH content. The NPSH and GSH content of slices from both control and AGE-treated rats remained constant during 6 h in culture (Fig. 3). Inclusion of 1 mM BB in the culture medium of slices from control animals resulted in a rapid and extensive depletion of both NPSH and GSH; indeed, almost total depletion of GSH was observed after 6 h. The NPSH and GSH content of slices from AGE-treated rats still decreased in the presence of 1 mM BB. However, this decrease was less marked than that in slices from control animals. Thus, because of the elevated NPSH and GSH contents at time zero, even after 6 h in culture with BB, slices from AGE-treated rats still had NPSH and GSH contents above those of slices from control animals incubated in the absence of BB. Furthermore, after 6 h in culture with BB, slices from rats pretreated with low-dose AGE had NPSH and GSH contents similar to those of slices from control animals.
3.5. Time course of the increase in slice NPSH and GSH content The NPSH and GSH contents of slices from control animals are given in Table 1; GSH accounted for approximately 70% of slice NPSH. Treatment with AGE caused a time-dependent increase in both NPSH and GSH which was maximal after 5 days. The non-GSH NPSH content of slices was calculated by difference and can be seen to be approximately constant throughout 7 days of AGE administration (Table 1).
3.6. Effects of AGE on ECOD and PROD acti6ities ECOD and PROD activities in microsomes from control livers were 10.49 0.6 nmol/min/mg protein and 62.89 4.7 pmol/min/mg protein respectively (means9 S.D., n= 5). Pretreatment of animals for 7 days with both doses of AGE did not affect either activity in subsequently isolated microsomes (results not shown).
4. Discussion Garlic contains a number of organosulphur compounds which are generally believed to be the active constituents responsible for its biological actions (Agarwal, 1996). These can be divided into water-soluble compounds such as S-allyl cysteine and oil-soluble compounds such as diallyl sulphide. The aged garlic extract that we have used (Kyolic®) is prepared by slicing fresh garlic
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Fig. 3. Effects of pretreatment with AGE on the BB-induced depletion of (A) NPSH and (B) GSH in rat liver slices. See Fig. 1 for symbols and abbreviations.
and soaking it in dilute ethanol for at least 10 months. Thus, it is relatively enriched in the water-soluble constituents and contains only low levels of oil-soluble constituents; indeed, by far the most abundant organosulphur compound in AGE
is S-allyl cysteine. The doses of AGE used in this study (2 and 10 ml/kg/day) are comparable with those used by other workers. For example, Efendy et al. (1997) administered 0.8 ml/kg/day to rabbits when investigating the effect of AGE on experi-
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Fig. 4. Effects of pretreatment with AGE on the BB-induced TBARS formation by rat liver slices. See Fig. 1 for symbols and abbreviations.
mental atherosclerosis, while Kyo et al. (1997) administered 10 ml/kg when assessing the anti-allergic effects of AGE in rats and Steiner et al. (1996) gave 7.2 g of AGE powder to hypercholesterolaemic men. It is clear that slices prepared from livers of rats which have been treated with AGE for 7 days are protected against BB toxicity. All the indicators of toxicity studied showed that liver slices from AGE-treated rats are resistant to BB toxicity, and this is more pronounced at the higher of the two doses administered. Thus, the BB-induced leakage of K + , loss of ATP (Fig. 1) and leakage of the two intracellular enzymes LDH and ALT (Fig. 2) were all reduced by pretreatment with AGE. Similarly, lipid peroxidation, as judged by the formation of TBARS, was reduced to control levels even at the lower dose of AGE (Fig. 4). A possible explanation for the protective effect of AGE is that the toxic P450-derived metabolites of BB are not formed due to inhibition of some cytochrome P450 isoforms. This cannot be the case here since both ECOD and PROD activities were similar in microsomes isolated from livers of
control and AGE-treated rats. ECOD activity estimates total cytochrome P450, while PROD is relatively specific for those isoforms responsible for BB metabolism, namely 2B1 and 2B2 (Gonzalez, 1990). Although garlic and some organosulphur compounds inhibit some isoforms of P450, such effects are limited to the oil-soluble organosulphur compounds (Reicks and Crankshaw, 1996). Indeed, diallyl sulphide has been shown to inhibit cytochrome 2E1 (Hu et al., 1996a). Of more interest is the substantial increase in the hepatic content of NPSH and GSH by AGE (Fig. 3). This effect occurred over several days and was specific to glutathione in that the hepatic content of other non-GSH NPSH (presumably mainly free cysteine) was not increased by pretreatment with AGE (Table 1). Similar increases in GSH content have been shown in both bovine pulmonary artery endothelial cells after 24 h in culture with AGE (Geng and Lau, 1997) and in human prostate carcinoma cells after 3 h in culture with S-allyl cysteine and S-allyl mercaptocysteine (Pinto et al., 1997) However, it must be
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Table 1 Effect of pretreatment with AGE on the NPSH and GSH content of rat liver slicesa Treatment
NPSH content (nmol/mg protein)
GSH content (nmol/mg protein)
Non-GSH NPSH content (nmol/mg protein)
Control AGE, 1 AGE, 3 AGE, 5 AGE, 7
52.39 8.6 50.0, 65.6 64.4, 79.1 82.6, 84.9 85.29 10.5*
36.79 4.2 36.2, 47.0 45.6, 59.1 64.4, 61.3 64.4 910.2*
16.4 98.5 13.8, 18.6 18.8, 20.0 18.2, 23.6 17.89 4.5
day days days days
a
Rats were administered AGE via a stomach tube at 10 ml/kg/day for the number of days shown; control animals received similar volumes of water for 7 days. Individual values are given for days 1, 3 and 5; values for controls and day 7 are means9 S.D. (n =4). Values for non-GSH NPSH content were calculated by difference. * Significant differences from corresponding controls (PB0.05).
emphasised that in our previous study, the GSH content of rat liver slices was not increased after 6 h in culture with up to 5% (v/v) AGE (Wang et al., 1998). Since the primary mode of detoxification of the 3,4-oxide metabolite of BB is by conjugation with GSH, an increased hepatic content of GSH would be expected to confer a higher capacity for detoxification. The mechanism by which treatment of rats with AGE increases the hepatic content of GSH must involve either increased synthesis and/or decreased utilisation. It is doubtful that decreased glutathione S-transferase activity could account for decreased utilisation; indeed, the garlic constituents diallyl sulphide, diallyl trisulphide (Sparnins et al., 1988; Hu et al., 1996b) and S-allyl cysteine (Hatono et al., 1996) have been shown to induce some isoforms of glutathione S-transferase in liver. Alternatively, an increase in the bioavailability of cysteine might increase GSH synthesis since this is usually the limiting substrate. Such a mechanism has been proposed for N-acetyl cysteine, a hepatoprotective agent used widely to treat paracetamol poisoning (Rafeiro et al., 1994). Thus, hepatic concentrations of cysteine increase dramatically 10 min after intraperitoneal injection of N-acetyl cysteine into rats (Yao et al., 1994). In addition, it has been reported that the hepatoprotective effect of N-acetyl cysteine is blocked by the GSH synthesis inhibitor buthionine sulphoxide (Miners et al., 1984; Rafeiro et al., 1994), and it is not reproduced by the non-physiological stereoisomer N-acetyl-D-
cysteine (Corcoran and Wong, 1986). This suggests that N-acetyl cysteine is deacetylated relatively easily. If the organosulphur compounds in AGE are acting in a similar manner here, they must also be metabolised to yield free cysteine. However, the observations that the NPSH content of rat liver slices does not increase following incubation with AGE (Wang et al., 1998), nor do hepatic concentrations of non-GSH NPSH increase after treatment of rats with AGE (Table 1), do not support this hypothesis. It is interesting to note that in slices from AGE-treated rats, not only was the initial hepatic content of GSH elevated, but the GSH content fell less on exposure to BB. Thus, slices from control rats lost approximately 35 nmol GSH/mg protein over 6 h, while slices from rats treated with both high-dose and low-dose AGE lost only approximately 20 nmol GSH/mg protein (Fig. 3B). It is well established that in cases where xenobiotic metabolism generates electrophilic metabolites leading to depletion of GSH, administration of some thiol-containing compounds may decrease toxicity, either by facilitating GSH synthesis or by sparing GSH as a result of direct formation of conjugates with the electrophilic species (Thor et al., 1979). Although garlic and commercial garlic preparations contain many organosulphur compounds which are nucleophilic, none of those identified carry free sulphydryl groups. The possibility that these are generated by metabolism cannot be eliminated, but is unlikely here since treatment of rats with
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AGE did not increase the hepatic content of non-GSH NPSH (Table 1). It is more likely that any GSH sparing effect exerted by the organosulphur compounds in AGE is due to their nuleophilic nature allowing them to react directly with electrophilic species such as BB-3,4-oxide. Such a situation must occur in vitro since the hepatic content of GSH was not increased when liver slices were cultured for 6 h in the presence of 5% (v/v) AGE, although protection against BBinduced toxicity and GSH depletion was still observed (Wang et al., 1998). In conclusion, it would appear that the mechanism of the protective effect of AGE against BB hepatotoxicity involves two possibly inter-related components. The first, which is seen both in liver slices from AGE-treated rats and when AGE is added to liver slices in vitro, represents a GSH sparing effect, presumably due to conjugation of organosulphur compounds in AGE with the 3,4oxide of BB. The second, which is seen only after pretreating animals with AGE, is a slow process occurring over several days and leads to a specific elevation in the GSH content of liver. The GSHsparing effect would be expected to decrease the utilisation of GSH and hence may explain the long-term elevation of hepatic GSH content. The origin of these effects may be multifactorial involving many of the organosulphur compounds in AGE and/or their metabolites. Clearly, a precise understanding of the mechanisms involved will only be possible after more experiments using defined components in AGE.
Acknowledgements BHW is in receipt of a Liverpool John Moores University research studentship. We thank Wakunaga of America for their generous support.
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