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Antioxidant effect of hydroxytyrosol (DPE) and Mn2+ in liver of cadmium-intoxicated rats
Comparative Biochemistry and Physiology Part C 133 (2002) 625–632
Antioxidant effect of hydroxytyrosol (DPE) and Mn2q in liver of cadmium-intoxicated rats Elisabetta Casalino, Giovanna Calzaretti, Cesare Sblano, Vito Landriscina, Mario Felice Tecce, Clemente Landriscina* Department of Pharmaco-Biology, Laboratory of Veterinary Biochemistry, University of Bari, Str. Prov. Per Casamassima, Km 3, 70010 Valenzano (Ba), Italy Received 19 July 2002; received in revised form 19 July 2002; accepted 27 August 2002
Abstract Liver TBARS formation in cadmium-intoxicated rats was completely reduced by administering a low amount of MnCl2 (2 mgykg b.w.) 1 h before intoxication. A similar antioxidant effect was first shown by hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, given twice to rats (9 mgykg b.w.) after cadmium administration. The antioxidant properties shown in vivo by both Mn2q and DPE were also active in vitro when rat liver microsomes were subjected to lipid peroxidation by cadmium or other prooxidant systems. The increase in liver glutathione concentrations occurring in cadmium-intoxicated rats, was also found, for the first time, 24 h after MnCl2 administration. Unlike cadmium intoxication, which caused a higher formation of both glutathione and TBARS, Mn2q induced glutathione synthesis without any TBARS formation. The same situation was also observed when cadmium plus Mn2q or cadmium plus DPE was given to rats. Our data show that: (a) both DPE and low Mn2q concentrations may have an antioxidant effect in the livers of cadmium-intoxicated rats and (b) Mn2q, like cadmium, induces liver glutathione synthesis and this effect is probably independent of TBARS formation. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cadmium intoxication; Glutathione; Hydroxytyrosol; Lipid peroxidation; Manganese antioxidant; Food antioxidants
1. Introduction Environmental pollutants, including a variety of industrial and domestic chemicals, pesticides, fertilizers, heavy metals and ionizing radiation are the major factors responsible for oxidative stress (Bolton et al., 2000; Hu, 2000; Banerjee et al., 2001; Li, 2001; Tsukamoto et al., 2002). Intoxication by heavy metals, particularly lead, cadmium, arsenic and mercury constitute serious threats to human health (Wenneberg, 1994; Hu, 2000). *Corresponding author. Tel.: q39-80-5443864; fax: q3980-5443863. E-mail address: [email protected] (C. Landriscina).
Among these metals, cadmium is of particular concern because it accumulates in many tissues with a half-life exceeding 10 years. Although this metal does not participate in the Fenton reaction and has no redox properties, it induces serious peroxidation in membrane structures (Stohs et al., 2000). Due to these negative effects, it has been associated with several diseases including osteoporosis, nephrotoxicity, pulmonary emphysema, liver dysfunction, and others (Piscator, 1986; Driscoll et al., 1992; Berglund et al., 2000). In addition, cadmium is weakly mutagenic (Rossmann, 1995) and has been shown to have carcinogenic activity (Waalkes et al., 1992). To preserve the integrity of biological membranes from detrimental oxida-
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tive processes caused by free radicals, both enzymatic and molecular defence mechanisms are present in the cell (McCord, 1993; Papas, 1996). The components of this defence system can be divided into two main groups: antioxidant enzymes (Fridovich, 1978; Powers and Lennon, 1999; Mates, 2000) and small endogenous antioxidant molecules such as glutathione (Deneke and Fanburg, 1989), coenzyme Q (Fernandez-Ayala et al., 2000; Crane, 2001) and urate (Sevanian et al., 1991). Other exogenous antioxidants, such as tocopherols, ascorbate, vitamin A and carotenoids and some metals, essential for the function of antioxidant enzymes, are of dietary origin (Papas, 1996). Constituents of some dietary plant products, such as flavonoids and phenolics, are powerful antioxidants and may play a protective role in several human pathologies (Rice-Evans, 1995; Croft, 1998). Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, DPE), one of the phenolic compounds present in extra virgin olive oil, has been suggested to be a potent antioxidant, thus contributing to the beneficial properties of olive oil (Deiana et al., 1999). Administration of DPE has been shown to reduce the consequences of passive smoking-induced oxidative stress (Visioli et al., 2000), prevents LDL oxidation (Wiseman et al., 1996) and platelet aggregation (Petroni et al., 1995) and inhibits leukocyte 5-lipoxygenases (Koyama et al., 1997; de la Puerta et al., 1999). As for the essential metals, dietary manganese deficiency has been reported to cause numerous pathological conditions such as growth retardation, sterility, skeletal deformities, alteration in lipoprotein metabolism and in glucose levels in experimental animals (Keen et al., 1999). Manganese is an important cofactor of the mitochondrial superoxide dismutase, an antioxidant enzyme which scavenges oxygen free radicals (Zidenberg-Cherr et al., 1983; Macmillan-Crow and Cruthirds, 2001). Furthermore, it has been shown that manganese inhibits microsomal lipid peroxidation (Coassin et al., 1992) and has antioxidant properties in an in vitro system generating oxygen free radicals (Hussain and Ali, 1999). For the first time, this study reports the antioxidant activity of DPE and Mn2q, singly administered to rats along with cadmium, on the peroxidation process induced by cadmium in liver. The antioxidant effectiveness of both DPE and manganese was further tested in in vitro experi-
ments by incubating rat liver microsomes with cadmium or with other prooxidant systems. Moreover, the results have been obtained indicate that the increase in liver glutathione concentration, observed following cadmium intoxication, also occurs 24 h after MnCl2 administration to rats. 2. Materials and methods 2.1. Reagents All chemicals used were of the highest quality and purchased from BDH Chemicals, Fisher Scientific Supply or Sigma Chemical Co. Chelex 100 ion exchange resin (Bio-Rad Laboratories) was used to remove contaminating metals from all reagents. Biochemicals were obtained from Boehringer, Mannheim. Organic solvents of analytical grade were used. All solutions were prepared in double-distilled water. 2.2. Animal treatment Male Wistar rats weighing 180–200 g were maintained on a standard diet. The animals were injected i.p. with CdCl2 (2.5 mgykg b.w.), DPE (9.0 mgykg b.w.) or MnCl2 (2.0 mgykg b.w.), in 0.1 ml saline and sacrificed 24 h after injection. When administered together, CdCl2 i.p. administration at 0 time was followed by two DPE i.p. administrations (the first after 1 h and the second after 8 h). When both CdCl2 and MnCl2 were given i.p. to rats, MnCl2 was injected at 0 time and CdCl2 after 1 h. Control animals received the equivalent volume of saline (0.1 mlykg body weight). In each experiment, a pair of animals (one control and one Cd-treated rat) was used. The animals were killed by decapitation under ether anesthesia. The livers were quickly excised, rinsed in ice-cold saline to clear them of blood, weighed, finely minced in the same solution, and homogenized (f10% wyv) in a Potter Elvehjem homogenizer with a Teflon pestle. Liver homogenate from both control and treated rats was used for TBARS and GSH determination. Liver microsomes from untreated rats, obtained by differential centrifugation (Landriscina et al., 1976), were used for the in vitro experiments.
E. Casalino et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 625–632 Table 1 Liver protective effect of 2-(3,4-dihydroxyphenyl)ethanol (DPE) and Mn2q ions in cadmium-intoxicated rats Treatment (ns4)
TBARS (nmolyg tissue)
% Variation
Control CdCl2 DPE MnCl2 CdCl2 and DPE CdCl2 and MnCl2
27.4"2.6 42.6"3.8* 28.3"3.1** 26.9"2.7** 29.8"3.1** 28.8"2.7**
– q55.5 q3.3 y1.8 q8.8 q5.1
Data are means"S.D. P-0.001 (vs. control). ** PsN.S. (vs. control). *
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2.5. GSH determination Tissue glutathione level was measured by a kinetic assay, using a dithionitrobenzoic acid recycling method (Anderson, 1985). 2.6. Protein concentration The protein content was estimated by the biuret method (Gornall et al., 1949) using bovine serum albumin as standard. 2.7. Statistics
2.3. In vitro experiments The incubation of liver microsomes (microsomal protein, 1 mg) was performed at 37 8C for 20 min in a mixture containing 0.175 M KCl, 25 mM Tris–HCl at pH 7.4. Final volume 1 ml; ascorbate, CdCl2, Fe2q, NADPH, DPE and MnCl2, were added at concentrations indicated in the tables. The reaction was stopped on ice. Aliquots of the suspension were used to determine lipid peroxidation. 2.4. Lipid peroxidation Lipid peroxidation was measured by determining the TBARS concentration in the presence of butylated hydroxytoluene (0.03%, final concentration) under continuous nitrogen flow to avoid any artifactual oxidation due to heating (Buege and Aust, 1987). In in vivo experiments, TBARS concentrations were measured in homogenate in the same way.
Statistical evaluation of the data was performed using the Student’s t-test. Differences from controls were considered significant at P-0.05. 3. Results The results in Table 1 show the effect of DPE and MnCl2 administration to cadmium-intoxicated rats. In this case, as expected, 24 h after i.p. cadmium administration, a remarkable liver TBARS increase was found compared to control. Both DPE and Mn2q exhibited good antioxidant properties. Two successive i.p. DPE administrations to intoxicated rats resulted in a beneficial effect, since no significant liver TBARS increase was observed 24 h after intoxication. Similar antioxidant effects were exerted by a low concentration of Mn2q administered i.p. 1 h before cadmium intoxication. The in vivo antioxidant property of manganese was also tested in vitro. Table 2 reports the effect of 25 mM Mn2q in the presence of an actively promoted lipid peroxidation by different prooxi-
Table 2 Antioxidant effect of Mn2q on differently-induced lipid peroxidation in rat liver microsomes Additions
Control Ascorbate Ascorbate and Fe2q Cd2q NADPH NADPH and Fe2q
TBARS (nmolymg protein) Mn2q absent (ns4)
Mn2q present (ns4)
0.72"0.06 6.32"0.71 25.28"2.73 2.25"0.26 5.82"0.55 28.72"3.16
0.61"0.05* 0.67"0.06** 0.75"0.08** 0.63"0.06** 3.61"0.32** 3.21"0.03**
The concentrations of the above compounds were: 5 mM FeCl2 , 100 mM ascorbate, 75 mM CdCl2, 0.3 mM NADPH, 25 mM MnCl2. Data are means"S.D. * P-0.05 (vs. Mn2q absent). ** P-0.001 (vs. Mn2q absent).
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dant systems in rat liver microsomes. Here, it is possible to see that TBARS production induced by ascorbate, both with and without iron, was completely removed when Mn2q was present. The same complete protective effect was exerted by Mn2q in the case of microsomal lipid peroxidation induced by cadmium. A slightly different situation occurred when this metal was added to liver microsomes in the course of their peroxidation by NADPH or NADPHyFe2q. In the latter conditions, the peroxidative process was about five times higher than that seen with NADPH alone (TBARS formed 28.72 vs. 5.82 nmol) and manganese was still antioxidant. However, in both cases, TBARS levels were not normalized, unlike the results achieved in the previous conditions. Table 3 shows that, as in Table 2, the TBARS quantities promoted by different prooxidants in rat liver microsomes were normalized in the presence of hydroxytyrosol (DPE). Particularly, in the case of cadmium-induced lipid peroxidation, DPE showed high antioxidant efficiency. It should be noted that with DPE, as had occurred before with Mn2q, it was not possible to completely remove TBARS produced by either NADPH or NADPHy Fe2q systems. Antioxidant stresses and heavy metals have been reported to induce the synthesis of glutathione (Ogino et al., 1989; Woods and Ellis, 1995; Shaikh et al., 1999). In the present study, we verified the effect on glutathione of Mn2q, compared to that of Cd2q, and that of Cd2q plus Mn2q. Table 4 shows that 24 h after cadmium intoxication, liver glutathione levels increased by 30.4%. A similar glutathione increase occurred 24 h after i.p. administration of a low concentration of Mn2q to rats.
Table 4 Total liver glutathione levels following cadmium andyor antioxidant administration to rats Treatment (ns3)
Glutathione (mmolyg tissue)
% Variation
Control CdCl2 DPE MnCl2 CdCl2 and MnCl2 CdCl2 and DPE
7.9"0.9 10.3"1.1* 7.7"0.8** 10.5"1.2* 10.4"0.9* 10.2"0.9*
– q30.4 y2.5 q32.9 q31.6 q29.1
Experimental conditions as in Table 3. Data are means"S.D. * P-0.05 (vs. control). ** PsN.S. (vs. control).
When Cd2q and Mn2q were administered together, a similar liver glutathione increase was found. It is important to note that, in cadmium-intoxicated rats, the glutathione level increase took place concomitantly with high TBARS formation, as previously shown in Table 1. In contrast, when Mn2q was administered alone or with Cd2q, only glutathione synthesis was stimulated but no TBARS increase occurred (cf. Table 1). The same situation was observed when the antioxidant DPE was administered to cadmium-intoxicated rats. In this case, only the liver glutathione increase was seen with no significant TBARS formation (cf. Table 1). 4. Discussion Cadmium toxicity is associated with increased lipid peroxidation, enzyme inactivation and several specific cell membrane lesions (Casalino et al., 2000; Stohs et al., 2000). The field of cadmium
Table 3 Antioxidant effect of 2-(3,4-dihydroxphenyl) ethanol (DPE) on differently-induced lipid peroxidation in rat liver microsomes Additions
Control Ascorbate Ascorbate and Fe2q Cd2q NADPH NADPH and Fe2q
TBARS (nmolymg protein) DPE absent (ns3)
DPE present (ns3)
0.66"0.07 5.64"0.61 30.78"2.95 2.83"0.31 5.31"0.49 26.74"2.73
0.51"0.06* 0.72"0.08*** 1.15"0.12*** 0.41"0.04*** 3.45"0.35** 6.21"0.59***
The concentrations of the above compounds were: 5 mM FeCl2 , 100 mM ascorbate, 75 mM CdCl2 , 0.3 mM NADPH, 50 mM 2-(3,4dihydroxyphenyl)ethanol (DPE). Data are means"S.D. * P-0.05 (vs. DPE absent). ** P-0.01 (vs. DPE absent). *** P-0.001 (vs. DPE absent).
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intoxication therapy has recently been looking at different chemical compounds isolated from plants with hepatoprotective and antitoxic effects (Shaikh et al., 1999; Sugihara et al., 1999; Sunitha et al., 2001). The finding that, in addition to containing vitamin E, olive oil has relatively high amounts of polyphenolic compounds with several beneficial effects has given rise to great interest from many laboratories. Among these polyphenols, DPE is the major phenolic compound, and its antioxidant properties have been well-assessed (Stupans et al., 2000; Gordon et al., 2001). This feature was clearly shown in rat liver microsomes subjected to lipid peroxidation by ascorbateyFe2q (Table 3), thus confirming previous evidence from other laboratories (Gutierrez et al., 2001). Antioxidant effect of DPE was also seen in liver microsomal lipid peroxidation induced by the NADPH and NADPHyFe2q enzymatic systems or by cadmium (Table 3). Data on in vivo DPE metabolism relative to its absorption, bioavailability and elimination have been previously reported (D’Angelo et al., 2001; Tuck et al., 2001). Now we present the first evidence showing the antioxidant capacity of DPE in rat liver following acute cadmium intoxication (Table 1). The mechanism by which DPE scavenges free radicals requires further investigation; however, many proposals have been advanced, according to which DPE, like other simple phenols, probably acts via proton donation to radical species (Owen et al., 2000) or by binding iron ions (de la Puerta et al., 1999). Manganese is an essential nutritional element which activates glycosyltransferases and is present in two mitochondrial metalloenzymes, pyruvate carboxylase and superoxide dismutase. At higher doses, this metal causes neurotoxicity in experimental animals and man (Zheng et al., 1998), with the neurochemical changes being attributed to oxidative stress. However, the prooxidant properties of manganese are controversial, because antioxidant effects have also been associated with this metal. Indeed, in vivo studies have shown that this dual behaviour depends on the quantity of manganese administered, since high doses cause oxidative injury while low doses have an antioxidant effect (Sziraki et al., 1995). The in vitro antioxidant action of Mn2q in differently mediated lipid peroxidation conditions (Coassin et al., 1992; Tampo and Yonaha, 1992) and its ability to scavenge oxygen free radicals have been described (Hussain
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and Ali, 1999; Anand and Kanwar, 2001). The results in Table 2 confirm the in vitro antioxidant properties of Mn2q, since in rat liver microsomes this ion greatly reduced TBARS formation in NADPH and principally in NADPHyFe2q-mediated lipid peroxidation (Tampo and Yonaha, 1992). Analogously, Mn2q was able to completely decrease ascorbate and ascorbateyFe2q-induced lipid peroxidation in rat liver microsomes, a result similar to that reported by Anand and Kanwar (2001) in placental membranes. In any event, the novelty in Table 2 is the antioxidant effect shown by Mn2q in microsomal cadmium-induced lipid peroxidation. With regard to the role of manganese on in vivo cadmium toxicity, few investigations have so far been reported. Goering and Klaassen (1985) found that, in acute cadmium intoxication, Mn2q pretreatment greatly decreased hepatotoxicity in rats, and this positive effect was attributed to a different liver cadmium subcellular distribution with more Cd2q binding to induced metallothioneins. Other studies suggested that the beneficial effect of Mn2q administration to cadmium-intoxicated rats was probably due to a decreased rate of gastrointestinal absorption of this detrimental metal (Sarhan et al., 1986). In this paper, we also show that a low amount of Mn2q administered to rats 1 h before intoxication with cadmium, strongly reduced liver TBARS concentrations (Table 1). As regards the antioxidant effect mechanism by Mn2q on cadmium-induced lipid peroxidation, in some laboratories it has been found that non-toxic doses of manganese may behave as quenchers of superoxide anions and hydroxyl radicals as well as chain-breaking compounds (Hussain and Ali, 1999; Anand and Kanwar, 2001). Mn2q could also have superoxide dismutase-like activity (Singh et al., 1992). It must be said, however, that the antioxidant properties of Mn2q and DPE shown here in cadmium-intoxicated rats have no effect on the inhibition by this metal on antioxidant enzymes. Indeed, in our laboratory, it has been found that Cd2q plus Mn2q or Cd2q plus DPE administration totally fails to restore the liver cadmium-reduced activities of CuZnSOD, MnSOD and catalase (unpublished results). Also, in vitro the removal of TBARS by vitamin E does not prevent cadmium inhibition on the above enzymes. The only positive effect observed in vitro is the capacity of Mn2q to remove the inhibition of this metal on MnSOD activity (Casalino et al., 2002).
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Heavy metal intoxication or peroxide-dependent tissue glutathione level changes are currently a controversial problem. The liver glutathione concentration increase seen following Mn2q or Mn2q plus Cd2q administration to rats does not seem to depend on the antioxidant properties of manganese, considering the absence of TBARS formation in these conditions (Table 1). Similarly, when cadmium intoxication was followed by two DPE i.p. injections, the higher glutathione level was not accompanied by a TBARS increase. However, it must be noted that cadmium alone caused both TBARS and glutathione increases (Tables 1 and 4, respectively). Hence, it seems that the higher liver concentration of this tripeptide caused by these metals probably does not depend on the presence of TBARS, in contrast with other studies indicating that the addition of peroxidation products to alveolar epithelial cells stimulated glutathione synthesis (Liu et al., 2001). Other authors have reported that exposure to cadmium, in addition to causing lipid peroxidation, stimulated endogenous defences such as increases in antioxidant enzyme activities and in liver glutathione concentration (Gupta et al., 1991; Shaikh et al., 1999). Also, a single exposure to cadmium aerosol caused glutathione elevations in lung (Shukla et al., 2000). By contrast, other studies have suggested that cadmium intoxication reduced the glutathione level, thus causing the production of hydroxyl radicals and other species responsible for increased lipid peroxidation (Stohs et al., 2000). As for other heavy metals, oxidative stress by mercury (Woods and Ellis, 1995) and iron loading (Ogino et al., 1989) stimulated glutathione synthesis in kidney and liver, respectively. In any case, the stimulatory mechanism by these metals was the same, in that they induced the expression of g-glutamylcysteine synthetase, the rate-limiting enzyme in de novo glutathione synthesis. The present paper indicates now that manganese too stimulated liver glutathione synthesis. As regards this metal, it has been recently suggested that it may play an important role in ATP binding to the active site of g-glutamylcysteine synthetase (Kelly et al., 2002). In this way, Mn2q helps the phosphoryl transfer to L-glutamate, thus improving the rate of the first reaction step catalysed by this enzyme. In conclusion, the positive effect shown by manganese here and in other laboratories (Goering and Klaassen, 1985; Sarhan et al., 1986) makes a
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Tampo, Y., Yonaha, M., 1992. Antioxidant mechanism of Mn(II) in phospholipid peroxidation. Free Radic. Biol. Med. 13, 115–120. Tsukamoto, M., Tampo, Y., Sawada, M., Yonaha, M., 2002. Paraquat-induced oxidative stress and dysfunction of the glutathione redox cycle in pulmonary microvascular endothelial cells. Toxicol. Appl. Pharmacol. 178, 62–92. Tuck, K.L., Freeman, M.P., Hayball, P., Stretch, G., Stupans, I., 2001. The in vivo fate of hydroxytyrosol and tyrosol, antioxidant phenolic constituents of olive oil, after intravenous and oral dosing of labelled compounds to rats. J. Nutr. 131, 1993–1996. Visioli, F., Galli, C., Plasmati, E., et al., 2000. Olive oil hydroxytyrosol prevents passive smoking-induced oxidative stress. Circulation 102, 2169–2171. Waalkes, M.P., Coogan, T.P., Barter, R.A., 1992. Toxicological principle of metal carcinogenesis with special emphasis on cadmium. Crit. Rev. Toxicol. 22, 175–201.
Wenneberg, A., 1994. Neurotoxic effects of selected metals. Scand. J. Work Environ. Heath 20, 65–71. Wiseman, S.A., Mathot, J.N., de Fouw, N.J., Tijburg, L.B., 1996. Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the resistance of low density lipoproteins to oxidation in rabbits. Atherosclerosis 120, 15–23. Woods, J.S., Ellis, M.E., 1995. Up-regulation of glutathione synthesis in rat kidney by methyl mercury. Relationship to mercury-induced oxidative stress. Biochem. Pharmacol. 50, 1719–1724. Zheng, W., Ren, S., Graziano, J.H., 1998. Manganese inhibits mitochondrial aconitase: a mechanism of manganese neurotoxicity. Brain Res. 799, 334–342. Zidenberg-Cherr, S., Keen, C.L., Lonnerdal, B., Hurley, L.S., 1983. Superoxide dismutase activity and lipid peroxidation in the rat: developmental correlations affected by manganese deficiency. J. Nutr. 113, 2498–2504.
Antioxidant effect of hydroxytyrosol (DPE) and Mn2q in liver of cadmium-intoxicated rats Elisabetta Casalino, Giovanna Calzaretti, Cesare Sblano, Vito Landriscina, Mario Felice Tecce, Clemente Landriscina* Department of Pharmaco-Biology, Laboratory of Veterinary Biochemistry, University of Bari, Str. Prov. Per Casamassima, Km 3, 70010 Valenzano (Ba), Italy Received 19 July 2002; received in revised form 19 July 2002; accepted 27 August 2002
Abstract Liver TBARS formation in cadmium-intoxicated rats was completely reduced by administering a low amount of MnCl2 (2 mgykg b.w.) 1 h before intoxication. A similar antioxidant effect was first shown by hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, given twice to rats (9 mgykg b.w.) after cadmium administration. The antioxidant properties shown in vivo by both Mn2q and DPE were also active in vitro when rat liver microsomes were subjected to lipid peroxidation by cadmium or other prooxidant systems. The increase in liver glutathione concentrations occurring in cadmium-intoxicated rats, was also found, for the first time, 24 h after MnCl2 administration. Unlike cadmium intoxication, which caused a higher formation of both glutathione and TBARS, Mn2q induced glutathione synthesis without any TBARS formation. The same situation was also observed when cadmium plus Mn2q or cadmium plus DPE was given to rats. Our data show that: (a) both DPE and low Mn2q concentrations may have an antioxidant effect in the livers of cadmium-intoxicated rats and (b) Mn2q, like cadmium, induces liver glutathione synthesis and this effect is probably independent of TBARS formation. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Cadmium intoxication; Glutathione; Hydroxytyrosol; Lipid peroxidation; Manganese antioxidant; Food antioxidants
1. Introduction Environmental pollutants, including a variety of industrial and domestic chemicals, pesticides, fertilizers, heavy metals and ionizing radiation are the major factors responsible for oxidative stress (Bolton et al., 2000; Hu, 2000; Banerjee et al., 2001; Li, 2001; Tsukamoto et al., 2002). Intoxication by heavy metals, particularly lead, cadmium, arsenic and mercury constitute serious threats to human health (Wenneberg, 1994; Hu, 2000). *Corresponding author. Tel.: q39-80-5443864; fax: q3980-5443863. E-mail address: [email protected] (C. Landriscina).
Among these metals, cadmium is of particular concern because it accumulates in many tissues with a half-life exceeding 10 years. Although this metal does not participate in the Fenton reaction and has no redox properties, it induces serious peroxidation in membrane structures (Stohs et al., 2000). Due to these negative effects, it has been associated with several diseases including osteoporosis, nephrotoxicity, pulmonary emphysema, liver dysfunction, and others (Piscator, 1986; Driscoll et al., 1992; Berglund et al., 2000). In addition, cadmium is weakly mutagenic (Rossmann, 1995) and has been shown to have carcinogenic activity (Waalkes et al., 1992). To preserve the integrity of biological membranes from detrimental oxida-
1532-0456/02/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 2 . 0 0 1 8 0 - 1
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tive processes caused by free radicals, both enzymatic and molecular defence mechanisms are present in the cell (McCord, 1993; Papas, 1996). The components of this defence system can be divided into two main groups: antioxidant enzymes (Fridovich, 1978; Powers and Lennon, 1999; Mates, 2000) and small endogenous antioxidant molecules such as glutathione (Deneke and Fanburg, 1989), coenzyme Q (Fernandez-Ayala et al., 2000; Crane, 2001) and urate (Sevanian et al., 1991). Other exogenous antioxidants, such as tocopherols, ascorbate, vitamin A and carotenoids and some metals, essential for the function of antioxidant enzymes, are of dietary origin (Papas, 1996). Constituents of some dietary plant products, such as flavonoids and phenolics, are powerful antioxidants and may play a protective role in several human pathologies (Rice-Evans, 1995; Croft, 1998). Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol, DPE), one of the phenolic compounds present in extra virgin olive oil, has been suggested to be a potent antioxidant, thus contributing to the beneficial properties of olive oil (Deiana et al., 1999). Administration of DPE has been shown to reduce the consequences of passive smoking-induced oxidative stress (Visioli et al., 2000), prevents LDL oxidation (Wiseman et al., 1996) and platelet aggregation (Petroni et al., 1995) and inhibits leukocyte 5-lipoxygenases (Koyama et al., 1997; de la Puerta et al., 1999). As for the essential metals, dietary manganese deficiency has been reported to cause numerous pathological conditions such as growth retardation, sterility, skeletal deformities, alteration in lipoprotein metabolism and in glucose levels in experimental animals (Keen et al., 1999). Manganese is an important cofactor of the mitochondrial superoxide dismutase, an antioxidant enzyme which scavenges oxygen free radicals (Zidenberg-Cherr et al., 1983; Macmillan-Crow and Cruthirds, 2001). Furthermore, it has been shown that manganese inhibits microsomal lipid peroxidation (Coassin et al., 1992) and has antioxidant properties in an in vitro system generating oxygen free radicals (Hussain and Ali, 1999). For the first time, this study reports the antioxidant activity of DPE and Mn2q, singly administered to rats along with cadmium, on the peroxidation process induced by cadmium in liver. The antioxidant effectiveness of both DPE and manganese was further tested in in vitro experi-
ments by incubating rat liver microsomes with cadmium or with other prooxidant systems. Moreover, the results have been obtained indicate that the increase in liver glutathione concentration, observed following cadmium intoxication, also occurs 24 h after MnCl2 administration to rats. 2. Materials and methods 2.1. Reagents All chemicals used were of the highest quality and purchased from BDH Chemicals, Fisher Scientific Supply or Sigma Chemical Co. Chelex 100 ion exchange resin (Bio-Rad Laboratories) was used to remove contaminating metals from all reagents. Biochemicals were obtained from Boehringer, Mannheim. Organic solvents of analytical grade were used. All solutions were prepared in double-distilled water. 2.2. Animal treatment Male Wistar rats weighing 180–200 g were maintained on a standard diet. The animals were injected i.p. with CdCl2 (2.5 mgykg b.w.), DPE (9.0 mgykg b.w.) or MnCl2 (2.0 mgykg b.w.), in 0.1 ml saline and sacrificed 24 h after injection. When administered together, CdCl2 i.p. administration at 0 time was followed by two DPE i.p. administrations (the first after 1 h and the second after 8 h). When both CdCl2 and MnCl2 were given i.p. to rats, MnCl2 was injected at 0 time and CdCl2 after 1 h. Control animals received the equivalent volume of saline (0.1 mlykg body weight). In each experiment, a pair of animals (one control and one Cd-treated rat) was used. The animals were killed by decapitation under ether anesthesia. The livers were quickly excised, rinsed in ice-cold saline to clear them of blood, weighed, finely minced in the same solution, and homogenized (f10% wyv) in a Potter Elvehjem homogenizer with a Teflon pestle. Liver homogenate from both control and treated rats was used for TBARS and GSH determination. Liver microsomes from untreated rats, obtained by differential centrifugation (Landriscina et al., 1976), were used for the in vitro experiments.
E. Casalino et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 625–632 Table 1 Liver protective effect of 2-(3,4-dihydroxyphenyl)ethanol (DPE) and Mn2q ions in cadmium-intoxicated rats Treatment (ns4)
TBARS (nmolyg tissue)
% Variation
Control CdCl2 DPE MnCl2 CdCl2 and DPE CdCl2 and MnCl2
27.4"2.6 42.6"3.8* 28.3"3.1** 26.9"2.7** 29.8"3.1** 28.8"2.7**
– q55.5 q3.3 y1.8 q8.8 q5.1
Data are means"S.D. P-0.001 (vs. control). ** PsN.S. (vs. control). *
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2.5. GSH determination Tissue glutathione level was measured by a kinetic assay, using a dithionitrobenzoic acid recycling method (Anderson, 1985). 2.6. Protein concentration The protein content was estimated by the biuret method (Gornall et al., 1949) using bovine serum albumin as standard. 2.7. Statistics
2.3. In vitro experiments The incubation of liver microsomes (microsomal protein, 1 mg) was performed at 37 8C for 20 min in a mixture containing 0.175 M KCl, 25 mM Tris–HCl at pH 7.4. Final volume 1 ml; ascorbate, CdCl2, Fe2q, NADPH, DPE and MnCl2, were added at concentrations indicated in the tables. The reaction was stopped on ice. Aliquots of the suspension were used to determine lipid peroxidation. 2.4. Lipid peroxidation Lipid peroxidation was measured by determining the TBARS concentration in the presence of butylated hydroxytoluene (0.03%, final concentration) under continuous nitrogen flow to avoid any artifactual oxidation due to heating (Buege and Aust, 1987). In in vivo experiments, TBARS concentrations were measured in homogenate in the same way.
Statistical evaluation of the data was performed using the Student’s t-test. Differences from controls were considered significant at P-0.05. 3. Results The results in Table 1 show the effect of DPE and MnCl2 administration to cadmium-intoxicated rats. In this case, as expected, 24 h after i.p. cadmium administration, a remarkable liver TBARS increase was found compared to control. Both DPE and Mn2q exhibited good antioxidant properties. Two successive i.p. DPE administrations to intoxicated rats resulted in a beneficial effect, since no significant liver TBARS increase was observed 24 h after intoxication. Similar antioxidant effects were exerted by a low concentration of Mn2q administered i.p. 1 h before cadmium intoxication. The in vivo antioxidant property of manganese was also tested in vitro. Table 2 reports the effect of 25 mM Mn2q in the presence of an actively promoted lipid peroxidation by different prooxi-
Table 2 Antioxidant effect of Mn2q on differently-induced lipid peroxidation in rat liver microsomes Additions
Control Ascorbate Ascorbate and Fe2q Cd2q NADPH NADPH and Fe2q
TBARS (nmolymg protein) Mn2q absent (ns4)
Mn2q present (ns4)
0.72"0.06 6.32"0.71 25.28"2.73 2.25"0.26 5.82"0.55 28.72"3.16
0.61"0.05* 0.67"0.06** 0.75"0.08** 0.63"0.06** 3.61"0.32** 3.21"0.03**
The concentrations of the above compounds were: 5 mM FeCl2 , 100 mM ascorbate, 75 mM CdCl2, 0.3 mM NADPH, 25 mM MnCl2. Data are means"S.D. * P-0.05 (vs. Mn2q absent). ** P-0.001 (vs. Mn2q absent).
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dant systems in rat liver microsomes. Here, it is possible to see that TBARS production induced by ascorbate, both with and without iron, was completely removed when Mn2q was present. The same complete protective effect was exerted by Mn2q in the case of microsomal lipid peroxidation induced by cadmium. A slightly different situation occurred when this metal was added to liver microsomes in the course of their peroxidation by NADPH or NADPHyFe2q. In the latter conditions, the peroxidative process was about five times higher than that seen with NADPH alone (TBARS formed 28.72 vs. 5.82 nmol) and manganese was still antioxidant. However, in both cases, TBARS levels were not normalized, unlike the results achieved in the previous conditions. Table 3 shows that, as in Table 2, the TBARS quantities promoted by different prooxidants in rat liver microsomes were normalized in the presence of hydroxytyrosol (DPE). Particularly, in the case of cadmium-induced lipid peroxidation, DPE showed high antioxidant efficiency. It should be noted that with DPE, as had occurred before with Mn2q, it was not possible to completely remove TBARS produced by either NADPH or NADPHy Fe2q systems. Antioxidant stresses and heavy metals have been reported to induce the synthesis of glutathione (Ogino et al., 1989; Woods and Ellis, 1995; Shaikh et al., 1999). In the present study, we verified the effect on glutathione of Mn2q, compared to that of Cd2q, and that of Cd2q plus Mn2q. Table 4 shows that 24 h after cadmium intoxication, liver glutathione levels increased by 30.4%. A similar glutathione increase occurred 24 h after i.p. administration of a low concentration of Mn2q to rats.
Table 4 Total liver glutathione levels following cadmium andyor antioxidant administration to rats Treatment (ns3)
Glutathione (mmolyg tissue)
% Variation
Control CdCl2 DPE MnCl2 CdCl2 and MnCl2 CdCl2 and DPE
7.9"0.9 10.3"1.1* 7.7"0.8** 10.5"1.2* 10.4"0.9* 10.2"0.9*
– q30.4 y2.5 q32.9 q31.6 q29.1
Experimental conditions as in Table 3. Data are means"S.D. * P-0.05 (vs. control). ** PsN.S. (vs. control).
When Cd2q and Mn2q were administered together, a similar liver glutathione increase was found. It is important to note that, in cadmium-intoxicated rats, the glutathione level increase took place concomitantly with high TBARS formation, as previously shown in Table 1. In contrast, when Mn2q was administered alone or with Cd2q, only glutathione synthesis was stimulated but no TBARS increase occurred (cf. Table 1). The same situation was observed when the antioxidant DPE was administered to cadmium-intoxicated rats. In this case, only the liver glutathione increase was seen with no significant TBARS formation (cf. Table 1). 4. Discussion Cadmium toxicity is associated with increased lipid peroxidation, enzyme inactivation and several specific cell membrane lesions (Casalino et al., 2000; Stohs et al., 2000). The field of cadmium
Table 3 Antioxidant effect of 2-(3,4-dihydroxphenyl) ethanol (DPE) on differently-induced lipid peroxidation in rat liver microsomes Additions
Control Ascorbate Ascorbate and Fe2q Cd2q NADPH NADPH and Fe2q
TBARS (nmolymg protein) DPE absent (ns3)
DPE present (ns3)
0.66"0.07 5.64"0.61 30.78"2.95 2.83"0.31 5.31"0.49 26.74"2.73
0.51"0.06* 0.72"0.08*** 1.15"0.12*** 0.41"0.04*** 3.45"0.35** 6.21"0.59***
The concentrations of the above compounds were: 5 mM FeCl2 , 100 mM ascorbate, 75 mM CdCl2 , 0.3 mM NADPH, 50 mM 2-(3,4dihydroxyphenyl)ethanol (DPE). Data are means"S.D. * P-0.05 (vs. DPE absent). ** P-0.01 (vs. DPE absent). *** P-0.001 (vs. DPE absent).
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intoxication therapy has recently been looking at different chemical compounds isolated from plants with hepatoprotective and antitoxic effects (Shaikh et al., 1999; Sugihara et al., 1999; Sunitha et al., 2001). The finding that, in addition to containing vitamin E, olive oil has relatively high amounts of polyphenolic compounds with several beneficial effects has given rise to great interest from many laboratories. Among these polyphenols, DPE is the major phenolic compound, and its antioxidant properties have been well-assessed (Stupans et al., 2000; Gordon et al., 2001). This feature was clearly shown in rat liver microsomes subjected to lipid peroxidation by ascorbateyFe2q (Table 3), thus confirming previous evidence from other laboratories (Gutierrez et al., 2001). Antioxidant effect of DPE was also seen in liver microsomal lipid peroxidation induced by the NADPH and NADPHyFe2q enzymatic systems or by cadmium (Table 3). Data on in vivo DPE metabolism relative to its absorption, bioavailability and elimination have been previously reported (D’Angelo et al., 2001; Tuck et al., 2001). Now we present the first evidence showing the antioxidant capacity of DPE in rat liver following acute cadmium intoxication (Table 1). The mechanism by which DPE scavenges free radicals requires further investigation; however, many proposals have been advanced, according to which DPE, like other simple phenols, probably acts via proton donation to radical species (Owen et al., 2000) or by binding iron ions (de la Puerta et al., 1999). Manganese is an essential nutritional element which activates glycosyltransferases and is present in two mitochondrial metalloenzymes, pyruvate carboxylase and superoxide dismutase. At higher doses, this metal causes neurotoxicity in experimental animals and man (Zheng et al., 1998), with the neurochemical changes being attributed to oxidative stress. However, the prooxidant properties of manganese are controversial, because antioxidant effects have also been associated with this metal. Indeed, in vivo studies have shown that this dual behaviour depends on the quantity of manganese administered, since high doses cause oxidative injury while low doses have an antioxidant effect (Sziraki et al., 1995). The in vitro antioxidant action of Mn2q in differently mediated lipid peroxidation conditions (Coassin et al., 1992; Tampo and Yonaha, 1992) and its ability to scavenge oxygen free radicals have been described (Hussain
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and Ali, 1999; Anand and Kanwar, 2001). The results in Table 2 confirm the in vitro antioxidant properties of Mn2q, since in rat liver microsomes this ion greatly reduced TBARS formation in NADPH and principally in NADPHyFe2q-mediated lipid peroxidation (Tampo and Yonaha, 1992). Analogously, Mn2q was able to completely decrease ascorbate and ascorbateyFe2q-induced lipid peroxidation in rat liver microsomes, a result similar to that reported by Anand and Kanwar (2001) in placental membranes. In any event, the novelty in Table 2 is the antioxidant effect shown by Mn2q in microsomal cadmium-induced lipid peroxidation. With regard to the role of manganese on in vivo cadmium toxicity, few investigations have so far been reported. Goering and Klaassen (1985) found that, in acute cadmium intoxication, Mn2q pretreatment greatly decreased hepatotoxicity in rats, and this positive effect was attributed to a different liver cadmium subcellular distribution with more Cd2q binding to induced metallothioneins. Other studies suggested that the beneficial effect of Mn2q administration to cadmium-intoxicated rats was probably due to a decreased rate of gastrointestinal absorption of this detrimental metal (Sarhan et al., 1986). In this paper, we also show that a low amount of Mn2q administered to rats 1 h before intoxication with cadmium, strongly reduced liver TBARS concentrations (Table 1). As regards the antioxidant effect mechanism by Mn2q on cadmium-induced lipid peroxidation, in some laboratories it has been found that non-toxic doses of manganese may behave as quenchers of superoxide anions and hydroxyl radicals as well as chain-breaking compounds (Hussain and Ali, 1999; Anand and Kanwar, 2001). Mn2q could also have superoxide dismutase-like activity (Singh et al., 1992). It must be said, however, that the antioxidant properties of Mn2q and DPE shown here in cadmium-intoxicated rats have no effect on the inhibition by this metal on antioxidant enzymes. Indeed, in our laboratory, it has been found that Cd2q plus Mn2q or Cd2q plus DPE administration totally fails to restore the liver cadmium-reduced activities of CuZnSOD, MnSOD and catalase (unpublished results). Also, in vitro the removal of TBARS by vitamin E does not prevent cadmium inhibition on the above enzymes. The only positive effect observed in vitro is the capacity of Mn2q to remove the inhibition of this metal on MnSOD activity (Casalino et al., 2002).
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Heavy metal intoxication or peroxide-dependent tissue glutathione level changes are currently a controversial problem. The liver glutathione concentration increase seen following Mn2q or Mn2q plus Cd2q administration to rats does not seem to depend on the antioxidant properties of manganese, considering the absence of TBARS formation in these conditions (Table 1). Similarly, when cadmium intoxication was followed by two DPE i.p. injections, the higher glutathione level was not accompanied by a TBARS increase. However, it must be noted that cadmium alone caused both TBARS and glutathione increases (Tables 1 and 4, respectively). Hence, it seems that the higher liver concentration of this tripeptide caused by these metals probably does not depend on the presence of TBARS, in contrast with other studies indicating that the addition of peroxidation products to alveolar epithelial cells stimulated glutathione synthesis (Liu et al., 2001). Other authors have reported that exposure to cadmium, in addition to causing lipid peroxidation, stimulated endogenous defences such as increases in antioxidant enzyme activities and in liver glutathione concentration (Gupta et al., 1991; Shaikh et al., 1999). Also, a single exposure to cadmium aerosol caused glutathione elevations in lung (Shukla et al., 2000). By contrast, other studies have suggested that cadmium intoxication reduced the glutathione level, thus causing the production of hydroxyl radicals and other species responsible for increased lipid peroxidation (Stohs et al., 2000). As for other heavy metals, oxidative stress by mercury (Woods and Ellis, 1995) and iron loading (Ogino et al., 1989) stimulated glutathione synthesis in kidney and liver, respectively. In any case, the stimulatory mechanism by these metals was the same, in that they induced the expression of g-glutamylcysteine synthetase, the rate-limiting enzyme in de novo glutathione synthesis. The present paper indicates now that manganese too stimulated liver glutathione synthesis. As regards this metal, it has been recently suggested that it may play an important role in ATP binding to the active site of g-glutamylcysteine synthetase (Kelly et al., 2002). In this way, Mn2q helps the phosphoryl transfer to L-glutamate, thus improving the rate of the first reaction step catalysed by this enzyme. In conclusion, the positive effect shown by manganese here and in other laboratories (Goering and Klaassen, 1985; Sarhan et al., 1986) makes a
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