- Email: [email protected]
Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
Chapter 136
Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity Vincenzo Zappia1, Patrizia Galletti1, Caterina Manna1, Stefania D’Angelo1,2, Daniela Napoli1, Maria Luigia De Bonis1 and Giovambattista Capasso3 1
Department of Biochemistry and Biophysics, School of Medicine, Second University of Naples, Italy Faculty of Motor Sciences, Parthenope University, Naples, Italy 3 Chair of Nephrology, Department of Internal Medicine, Second University of Naples, Italy 2
136.1 Introduction Olive oil represents the typical lipidic source of the Mediterranean diet that has been associated with a low incidence of several pathologies, including cardiovascular diseases and neurological disorders (Bendini et al., 2007; Trichopoulou and Dilis, 2007). The beneficial properties of olive oil have been mainly attributed to its high content of monounsaturated oleic acid, however, in recent years converging evidence indicates that the non-glyceride fraction of olive oil, rich in vitamin and non-vitamin antioxidants including polyphenols, significantly contributes to its benefits on human health (Visioli and Galli, 2002). Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET) is the main ortho-diphenolic compound of olive oil and is mainly responsible for the antioxidant properties of this nutrient. Indeed, it has been shown to function as an efficient scavenger of peroxyl radicals in several biological systems and contributes to increase the shelf-life of the oil, preventing its auto-oxidation (Visioli and Galli, 2002).
136.2 Biological effects of hydroxytyrosol The biological activities of DOPET have been explored by several groups and are summarized in Table 136.1 (for a comprehensive review see Bendini et al., 2007). Even though the majority of them can be directly ascribed to its antioxidant activity (Manna et al., 1999; Visioli and Galli 2002), emerging evidence (Della Ragione et al., 2002) supports the view that some effects of this molecule are independent from its scavenging properties. DOPET inhibits in vitro low-density lipoprotein oxidation and modulates the Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
oxidative/antioxidative balance in plasma (Covas et al., 2006). Experiments from our group demonstrated that DOPET, which effectively permeates cell membranes via passive diffusion (Manna et al., 2000), counteracts the cytotoxic effects of reactive oxygen species (ROS) in various human systems. Preincubation of intestinal Caco-2 cells with DOPET prevents the typical damages of oxidative stress (Manna et al., 1999). Similarly, this polyphenol exerts a protective effect against the H2O2-induced oxidative hemolysis and lipid peroxidation in red blood cells (Manna et al., 1999). Moreover, it has been demonstrated that the molecule counteracts the up-rise of specific markers of oxidative stress in UVA-irradiated melanoma cells. In fact it prevents the formation of abnormal L-isoaspartyl residues in proteins and reduces lipid peroxidation in irradiated M14 cells (D’Angelo et al., 2005). Furthermore, pretreatment of human hepatocarcinoma HepG2 cells with micromolar concentrations of DOPET completely prevents the decrease of reduced glutathione and the rise of malondialdehyde induced by tert-butyl hydroperoxide in this cell line (Goya et al., 2007). As already mentioned, it has been proposed that the antioxidant effect of DOPET probably contributes to the prevention of some degenerative diseases and supports brain cell survival after oxidative injuries (Bendini et al., 2007; Trichopoulou and Dilis, 2007). The effects of DOPET on inflammation/atherogenesis have also been thoroughly investigated. It has been demonstrated that this antioxidant inhibits the expression of adhesion molecules in a human endothelial cell line (HUVEC) exposed to proinflammatory cytokines (Carluccio et al., 2003). Moreover, DOPET administration in hyperlipidemic rabbits is able to reduce the size of atherosclerotic lesions of the aortic arch (González-Santiago et al., 2006). The ability of DOPET to counteract inflammation is also
1245
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
1246
Section | III Tyrosol and Hydroxytyrosol
Table 136.1 Biological effects of hydroxytyrosol. Affected biological systems
Experimental models
References
LDL oxidation in vitro and in vivo*
Human
Covas et al., 2006
Cytokine-induced endothelial activation and macrophage activation*
Human umbilical vein endothelial cells (HUVEC)
Carluccio et al., 2003
Bovine aortic endothelial cells (BAEC)
Maiuri et al., 2005
Murine macrophages (J774) Platelet and leukocytes arachidonate lipoxygenases*
Rat
de la Puerta et al., 1999
PMA-induced respiratory burst*
Human neutrophils
Visioli and Galli, 2002
Peroxynitrite-dependent tyrosine nitration*
Neuronal hybridoma cell line N-18-RE-105
Deiana et al., 1999
Platelet aggregation*
Human
Dell’Agli et al., 2008
Cell proliferation* and accumulation of cells in the G0/G1 phase**
Human myeloid leukemia cells (HL-60)
Fabiani et al., 2008
Apoptosis**
Human myeloid leukemia cells (HL-60)
Della Ragione et al., 2000
Human carcinoma colon cell line (HT-29)
Fabiani et al., 2008
Atherosclerotic lesion development (*)
Rabbit
González-Santiago et al., 2006
ROS-mediated citotoxicity (*)
Human carcinoma colon cell line (Caco-2)
Manna et al., 1999
Human erythrocytes
Manna et al., 1999
Human melanoma cell line (M14)
D’Angelo et al., 2005
Human hepatocarcinoma cell line (HepG2)
Goya et al., 2007
*
Inhibition. Induction.
**
supported by the evidence that extra virgin olive oil administration significantly decreases the levels of known markers of this process, such as thromboxane B2 (Bendini et al., 2007) and 5- and 12-lipoxygenases (de la Puerta et al., 1999). On the other hand, the anti-inflammatory properties of the phenolic fraction of olive oil are widely documented in the literature, DOPET being a powerful inhibitor of neutrophil respiratory burst (Visioli and Galli, 2002). The prevention of abnormal cell proliferation and the induction of apoptosis, both events involved in carcinogenesis, have been described as effects of DOPET not directly attributable to its antioxidant properties (Bendini et al., 2007). Indeed, it has been recently demonstrated that this compound, in a micromolar concentration range, inhibits the proliferation and induces apoptosis in different human cell lines (Della Ragione et al., 2000; Fabiani et al., 2008). In detail, the polyphenol alters HL60 cell cycle progression, inducing
an accumulation of cells in the G0/G1 phase (Manna et al., 1999; Fabiani et al., 2008). In the same cell line, it has been recently demonstrated that this effect is associated with an up-regulation of cyclin-dependent protein kinase inhibitors and with an induction of cell differentiation (Fabiani et al., 2008). In this respect, it has been proposed that DOPET may affect the expression of genes involved in the regulation of tumor cell proliferation and differentiation, such as p27Kip1 and p21WAF/Cip1 (Fabiani et al., 2008). It is worthwhile mentioning that tyrosol (4-hydroxyphenylethanol), the DOPET analogue which lacks the ortho-diphenolic moiety, does not show any antioxidant activity and fails to exert any inhibitory effect on cell growth, indicating that the two ortho-hydroxyl groups are critical for both antioxidant and antiproliferative effects (Manna et al., 1999). The prevention of DNA damage, responsible for mutagenesis and carcinogenesis, can be envisioned as another
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
mechanism associated with the DOPET anticancer effect. This phenol, indeed, exerts an inhibitory effect on peroxynitrite-dependent DNA base modifications as well as on tyrosine nitration (Deiana et al., 1999).
1247
Table 136.2 Key features of cyclosporine nephrotoxicity. 1. Cyclosporine A (CsA), is a cyclic undecapeptide produced by the fungus Tolypocladium inflatum Gams
136.3 Cyclosporine and ROS
2. CsA is an immunosuppressant drug primarily used in humans to prevent organ transplant rejection
Cyclosporine A (CsA), a cyclic undecapeptide of fungal origin, is the most widely used immunosuppressive drug in organ transplantation and in the treatment of autoimmune disorders (Table 136.2). However, its clinical use is often limited by the frequent and dose-dependent severe side effects, including nephrotoxicity, hypertension, hepatotoxicity, lymphoproliferative disorders, atherosclerosis of the graft and interstitial myocardial fibrosis (Rezzani, 2006). Nephrotoxicity is the main untoward consequence of CsA treatment; moderate to severe renal dysfunction has been, indeed, documented in 30% of CsA-treated patients. On the basis of the length of treatment, two forms of CsA nephrotoxicity have been described: acute CsA treatment induces reversible reduction of both glomerular filtration rate (GFR) and renal blood flow, related to the afferent arteriolar vasoconstriction. This latter effect is associated with an increase in vasoconstrictor factors such as endothelin, thromboxane A2, angiotensin II and/or a decrease in vasodilators, such as prostacyclin and nitric oxide. Thus, an imbalance in the release of vasoactive substances may account for the reported renal vasoconstriction. On the other hand, when chronically administered, CsA can lead to irreversible renal failure due to the synergical effect of several factors, such as renal vasoconstriction, tubulointerstitial fibrosis, tubular atrophy and glomerular sclerosis (Rezzani, 2006). Even though the mechanisms of CsA toxicity are not fully understood, in recent years several experimental evidences have been collected on the involvement of oxidative stress in the toxic effects of CsA treatment. It has been reported that this drug induces membrane lipid peroxidation in several in vitro and in vivo experimental models, as well as in transplanted patients. In vivo, CsA increases lipoperoxidation in the rat kidney and liver, depletes the hepatic and renal pool of glutathione and impairs antioxidant defenses. Moreover, it has been reported that mRNA and protein levels of heme oxygenase-1, an enzyme responsive to changes in the redox status, vary after treatment with CsA (Rezzani, 2006). Several hypotheses have been proposed in order to correlate CsA treatment and oxidative stress, such as up-regulation of the kidney cytochrome P450-dependent ROSproducing system, perturbation of the balance between vaso dilation–vasoconstriction, in turn responsible for tubular hypoxia–reoxygenation, increased formation of renal thromboxane A2, induction of nitric oxide production. In addition, a possible direct interference of CsA with the intracellular homeostasis of glutathione has been suggested (Rezzani, 2006).
3. CsA inhibits interleukin-2 gene transcription and the transition of T-lymphocytes from the G0 to G1 phase of the cell cycle 4. CsA, although extensively used in kidney transplantation, causes important renal adverse effects, including acute and chronic renal dysfunction, hemolytic-uremic syndrome, hypertension, electrolyte disturbances and defects in urinary concentrating ability 5. Prolonged CsA administration may lead to structural changes which are no longer dose-dependent and reversible and cause end-stage renal failure 6. Although the mechanisms of nephrotoxicity have not been well defined, some evidence suggests that reactive oxygen species play a causative role
The hypothesis that CsA toxicity is mediated by ROS led investigators to use antioxidant molecules such as taurine, lipoic acid, melatonin and N-acetylcysteine to prevent or ameliorate its adverse effects (Rezzani, 2006). Since many plant-derived ‘phytonutrients’ are becoming increasingly known for their antioxidant activity, the use of plant-derived antioxidants against nephrotoxicity induced by treatment with CsA has been thoroughly explored in animal models. The administration of shallot (Allium ascalonicum) extract along with CsA counteracts its deleterious effects on renal dysfunction, oxidative stress markers and morphological changes (Wongmekiat et al., 2008). Lycopene, the carotenoid pigment found in tomatoes and other red fruits, ameliorates the CsA-induced pathological alterations including: tubular necrosis, degeneration, thickened basement membranes and intertubular fibrosis (Atessahin et al., 2007). Resveratrol, a naturally occurring phenolic compound abundantly present in grapes and red wine, protects against CsA-induced nephrotoxicity through a nitric oxide-dependent mechanism (Rezzani, 2006). Comparable results have been obtained using curcumin, the principal component of the Indian curry spice turmeric (Rezzani, 2006). Similarly, provinol, a polyphenolic extract obtained from red wine, prevents the increase of systolic blood pressure and nephrotoxicity in rat, through a mechanism that involves reduction of oxidative stress and iNOS expression, via nuclear factor-B pathway (Rezzani, 2006).
1248
6
Fluorescence intensity/106 cells
Both dried black grape and aqueous garlic counteract CsA nephrotoxicity, reducing the malondialdehyde level in the kidney tissue, possibly by preventing oxidative reactions (Durak et al., 2007). Vitamin E protects renal function and structure when administered in vivo to CsA-treated rats (Rezzani, 2006). Moreover, it has been demonstrated that the combination of quercetin and vitamin E plays a protective role against the imbalance elicited by CsA between the production of free radicals and the antioxidant defense systems, suggesting that a combination of these two antioxidants may find clinical application where cellular damage is a consequence of ROS (Mustafi-Pour et al., 2008).
Section | III Tyrosol and Hydroxytyrosol
5 4 3
*
2 1 0
A
Control
DOPET
CsA
CsA + DOPET
Control
DOPET
CsA
CsA + DOPET
The aim of the in vitro study (Galletti et al., 2005) was to investigate the possible protective effect of DOPET on CsA-induced nephrotoxicity using immortalized renal proximal tubule cells (RPTc) from normotensive Wistar–Kyoto rats. This cell line has been frequently selected as a model system, since tubular cells represent in vivo the major target of CsA-induced nephrotoxicity both in humans and in animal models (Galletti et al., 2005 and references therein). In the RPT cellular model system, CsA toxicity is a relatively late event, since 95% of RPTc are still largely viable up to 8 h in the presence of 25 M CsA. Conversely, prolonged incubation time leads to a dramatic decrease in cell viability, with 30% cell death after 20 h exposure to CsA. When RPTc have been incubated with CsA in the presence of 10 M DOPET, ROS formation was measured using the dichlorofluorescein (DCF) assay. As shown in Figure 136.1A, CsA treatment results in a significant increase in ROS formation. Moreover, the CsA-induced fluorescence signal is totally quenched by DOPET treatment. However, this phenol appears completely ineffective in preventing the toxic effect of the drug and in restoring cell viability (Figure 136.1B). The effect of DOPET on CsA-induced membrane lipoperoxidation has also been evaluated (Figure 136.2). RPTc incubation in the presence of CsA significantly increases the lipoperoxidation products (TBARS) (30% above basal), confirming that CsA treatment exposes cells to an oxidative microenvironment. Again, DOPET effectively counteracts the increase in TBARS formation. The pivotal role played by glutathione (GSH) in cellular protection against free radical damage is well known. The effect of DOPET on CsA-induced imbalance of the GSH redox state is shown in Table 136.3. CsA treatment induces in RPTc a significant increase in oxidized gluta thione (GSSG), resulting in a 50% reduction in the [GSH]/ [GSSG] ratio. This result supports a direct interference of
100 80 60 40 20 0
B
Figure 136.1 Effect of hydroxytyrosol (DOPET) on cyclosporine A (CsA)-induced reactive oxygen species (ROS) production (A) and cytotoxicity (B) in renal proximal tubule cells (RPTc). RPTc were exposed for 20 h to 25 M CsA in the presence or absence of 10 M DOPET. ROS production was detected by means of the fluorescent indicator dichlorofluorescein, as reported by Galletti et al. (2005). Cell viability was evaluated by the trypan blue exclusion method. Data are expressed as means SD (n 3) *p 0.001 vs CsA. 3
TBARS nmol−1 µg protein
136.4 Effects of hydroxytyrosol on cyclosporine cytotoxicity in rat renal tubular cells
Cell viability (% of control)
120
2
* 1
0 Control
DOPET
CsA
CsA + DOPET
Figure 136.2 Effect of hydroxytyrosol (DOPET) on cyclosporine A (CsA)-induced lipid peroxidation in renal proximal tubule cells (RPTc). RPTc were incubated for 20 h with 25 M CsA in the presence or absence of 10 M DOPET. Data are expressed as means SD (n 3) *p 0.05 vs CsA.
the drug with the intracellular GSH homeostasis. However, DOPET fails to provide any appreciable protection (Table 136.3) against the CsA-induced alterations in gluta thione metabolism.
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
Table 136.3 Effect of hydroxytyrosol (DOPET) on glutathione redox state in CsA-treated RPTc. GSSG nmol mg1 protein
GSH nmol mg1 protein
GSH/GSSG molar ratio
Control
1.32 0.65
15.9 4.2
12 3.8
CsA 25 M
2.85 1.14*
17 3.8
6.0 3.4*
CsA 25 M DOPET 10 M
2.8 0.15*
18.3 0.54 6.5 0.15*
Effect of cyclosporine-A (CsA) on the glutathione redox state in RPTc. Control samples received CsA vehicle. The glutathione (GSH), the oxidized glutathione (GSSG) and the GSH/GSSG ratio values are reported as mean SD. Significance (t-test)/ANOVA: *p 0.01 vs control.
The reported data indicate that DOPET effectively counteracts both CsA-induced ROS production and membrane lipoperoxidation in RPTc; however, the protection against the CsA-induced oxidative stress is not paralleled by an equivalent decrease in CsA cytotoxicity. On the basis of these findings, ROS generation induced by CsA does not appear strictly related to its nephrotoxi city; therefore, the generalization that antioxidants might exert a protective effect against the adverse effects of CsA, proposed by several authors and shared by nephrologists, is called into question by the reported in vitro approach.
136.5 Cyclosporine nephrotoxicity in rats: effect of hydroxytyrosol in vivo To further elucidate whether oxidative stress is responsible for CsA toxicity, a recent study assayed the protective effect of DOPET on oxidative stress, renal histology and hemodynamic alterations induced in rats by chronic CsA treatment (Capasso et al., 2008). In order to evaluate CsA-induced superoxide production within cells of the abdominal aorta and renal artery, the changes in fluorescence resulting from the oxidation of dihydroethidium (DHE) were monitored. The red fluorescence generated by the binding of ethidium–DNA complex is considered an appropriate indicator of superoxide production within the cells. As reported in Figure 136.3, the abdominal aorta of CsA-treated animals presents a red fluorescent signal significantly brighter than controls. When the rats were treated with CsA plus DOPET the fluorescence intensity was similar to controls, indicating that the polyphenol is able to completely prevent the production of superoxide.
1249
This experimental approach indicates that also in vivo DOPET is able to quench CsA-induced ROS production both in aorta and renal artery. These results are fully in agreement with the data of the in vitro study previously reported. TBARS were measured in the rat kidney to evaluate the CsA-induced oxidative alteration of cellular components. A significant increase (p 0.01) of TBARS was observable after CsA treatment (Table 136.4); the simultaneous administration of DOPET and CsA restored TBARS to control level, thus demonstrating that, also when administered in vivo, the polyphenol completely prevents the effect of CsA on lipid peroxidation. Moreover, chronic CsA treatment significantly increased the GSSG level (p 0.01 versus control). Also in this case, the co-administration of DOPET completely reversed this effect. Accordingly, CsA treatment lowered the cellular [GSH]/[GSSG] ratio by 44%, while DOPET completely prevented this alteration. As already mentioned, clinical observations and experimental evidence (Capasso et al., 2008 and references therein) indicate that CsA administration is associated with major side effects, including hypertension and renal failure related with fibrosis and vasoconstriction. It has been proposed that ROS overproduction induced by CsA may lead to the inhibition of NO synthesis with the consequent appearance of hypertension. Consistently, as shown in Figure 136.4, starting from the second week of CsA treatment, an increase of about 15 mmHg in both systolic blood pressure (BP) and diastolic BP can be observed. According to literature data, CsA increased both systolic BP and diastolic BP by 16–17 mmHg at the end of the treatment period. However, the administration of DOPET did not yield any observable effect on CsA-induced hypertension. A severe impairment of renal hemodynamics is another side effect of chronic administration of CsA. Among the implicated mechanisms, there is general agreement that the major factor of the CsA effect on GFR is mediated by its action on afferent arteriolar resistance. In this study, GFR was measured by means of inulin clearance. The effect of DOPET on chronic CsA-induced renal failure is reported in Figure 136.5. DOPET per se did not exert any change on glomerular function (0.93 0.05 mL min1 100 g1 b.w.). CsA treatment significantly decreased GFR compared to control animals (0.51 0.03 versus 0.94 0.05 mL min1 100 g1 b.w., respectively) (p 0.01), while DOPET, in combination with CsA, did not exert any protection (0.46 0.02 mL min1 100 g1 b.w.). Therefore, in contrast with its ROS-quenching effect, DOPET is unable to prevent both the increase in BP and the decrease in GFR. It is worth underlining that DOPET alone had almost no detrimental effect, confirming the low toxicity of this antioxidant phenol. The lack of a protective effect of DOPET on BP and kidney hemodynamics fits very nicely with the histological data, indicating that the antioxidant was unable to act on the arteriolopathy.
1250
Section | III Tyrosol and Hydroxytyrosol
Figure 136.3 Effect of cyclosporine A (15 mg kg1 day1) and hydroxytyrosol (20 mg kg1 twice daily) on the production of superoxide in the abdominal aorta. Superoxide concentrations within cells of the abdominal aorta were monitored by measuring the changes in fluorescence resulting from the oxidation of dichlorofluorescein. The figure has been reproduced with permission from Capasso et al., (2008).
Table 136.4 Oxidative stress markers evaluation after cyclosporine A (CsA) treatment. Control
DOPET (20 mg kg1)
CsA (15 mg kg1)
CsA (15 mg kg1) DOPET (20 mg kg1)
TBARS (nmol/g tissue)
20.8 3.4
23.8 2.5
37.2 7.4a**
28.0 2.3b*
GSSG (nmol GSH/g tissue)
1.66 0.11
1.50 0.10
2.17 0.29a**
1.60 0.19b*
GSH/GSSG ratio
31.09 3.4
29.62 2.3
17.08 2.1a**
33.57 2.4b*
Lipid peroxidation was measured in kidney tissues as thiobarbituric acid reacting substances (TBARS); oxidized glutathione (GSSG) was assessed by enzymatic assay. Control group received CsA vehicle, CsA group received CsA 15 mg kg1 day1 and DOPET group received DOPET 20 mg kg1 twice daily. Significance (t-test)/ANOVA: a vs control; b vs CsA; *p 0.05; **p 0.01.
136.6 Conclusions All together these observations lead to the conclusion that CsA-induced kidney injury is only partially due to oxidative stress. Moreover, the in vivo results are in agreement with in vitro data indicating that DOPET is able to completely prevent CsA-induced oxidative stress in rat tubular cells, but is ineffective in ameliorating the associated reduction of cell viability. The most reasonable interpretation of the data obtained in vivo is that the process leading to BP increase and GFR decrease is not necessarily related to ROS-dependent mechanism(s). Thus, even when CsA-induced oxidative stress is completely reverted by DOPET, renal failure and hypertension cannot be prevented, probably because
of other underlying mechanisms, such as artheriolopathy. Such a hypothesis seems to be in contrast with literature data showing that the administration of ‘antioxidant drugs’ like vitamin E (Rezzani, 2006) and lycopene (Atessahin et al., 2007) is able to reduce oxidative stress and ameliorate renal function after CsA treatment. It is highly likely that these two effects are mutually independent. Indeed, it should be underlined that both compounds, beside their antioxidant activity, exert key functions such as modulation of enzymatic activities and alteration of gene expression, which could account for their protective effect against CsA treatment. In this respect, it has been demonstrated that the overexpression of superoxide dismutase 1 by gene delivery, 3 days prior to the in vivo CsA administration, can partially reduce CsA-induced pathological alterations and inhibition of renal function (Rezzani, 2006).
mmHg
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
1251
Systolic blood pressure
140 135 130 125 120 115 110 105 100 0
5
10
20
15
25
Time (days)
mmHg
A
CsA+DOPET
CsA
DOPET
Control
Diastolic blood pressure
125 120 115 110 105 100 95 90 85 0
5
10
15
20
25
Time (days) CsA+DOPET
CsA Control
DOPET
B
Figure 136.4 Effect of cyclosporine A (15 mg kg day1) and hydroxytyrosol (20 mg kg1 twice daily) on systolic and diastolic pressure in rats. The rats were treated as described in the text. Diastolic and systolic blood pressure were measured on conscious rats using the tail method. Panel A shows the systolic pressure trend during the treatment period. Panel B shows the diastolic pressure results. †p 0.05, ††p 0.01, †††p 0.001 versus day 0; *** p 0.001 versus control. The figure has been reproduced with permission from Capasso et al., (2008). 1
thus supporting the view that kidney injury by CsA is mainly related to pathogenetic mechanisms independent from oxidative stress.
GFR (mL−1min−1g 100−1g B.W.)
0.2 1 0.8 0.6
Summary points
**
0.4
Cyclosporine A (CsA), although widely used as an immunosuppressive drug, exerts frequent and dosedependent cytotoxic effects, probably related to ROS overproduction. l Hydroxytyrosol (DOPET), the powerful olive oil antioxidant, could counteract CsA cytotoxicity through its scavenging properties. l In vitro studies with rat renal tubular cells, the main target of CsA cytotoxic activity, have demonstrated that DOPET effectively counteracts ROS production and lipoperoxidation. However, these effects are not paralleled by an equivalent decrease in CsA cytotoxicity. l The in vivo protective effect of DOPET has been assayed in rats. While exerting a significant protection toward CsA-induced oxidative stress, this polyphenol l
0.2 0 Control
DOPET
CsA
CsA + DOPET
Figure 136.5 Effect of hydroxytyrosol on the glomerular filtration rate (GFR) measured in rats treated with cyclosporin A. GFR was measured by inulin clearance. Inulin concentration in plasma and urine was calculated by the colorimetric method. **p 0.001 vs control. The figure has been reproduced with permission from Capasso et al., (2008).
In conclusion, the in vitro and in vivo effective DOPET protection from CsA-induced oxidative stress is only associated with mild effects on histological damage and does not affect the altered glomerular function and the hypertension,
1252
does not influence renal histological and hemodynamic alterations. l In conclusion, DOPET protection is only associated with mild effects on histological damage and does not affect the altered glomerular function and the hypertension. l The data support the view that kidney injury by CsA is mainly related to pathogenetic mechanisms independent from oxidative stress.
Acknowledgments The authors gratefully acknowledge the Oxford University Press for the kind permission to use Figures 1, 6 and 7 from Capasso G. et al., Nephrol. Dial. Transplant. 23, 1186–1195.
References Atessahin, A., Ceribasi, A., Yilmaz, S., 2007. Lycopene, a carotenoid, attenuates cyclosporine-induced renal dysfunction and oxidative stress in rats. Basic Clin. Pharm. Toxicol. 100, 372–376. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A.M., Segura-Carretero, A., Fernández-Gutiérrez, A., Lercker, G., 2007. Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules 12, 1679–1719. Carluccio, M.A., Siculella, L., Ancora, M.A., Massaro, M., Scoditti, E., Storelli, C., Visioli, F., Distante, A., De Caterina, R., 2003. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler. Thromb. Vasc. Biol. 23, 622–629. Capasso, G., Di Gennaro, C.I., Della Ragione, F., Manna, C., Ciarcia, R., Florio, S., Perna, A., Pollastro, R.M., Damiano, S., Mazzoni, O., Galletti, P., Zappia, V., 2008. In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats. Nephrol. Dial. Transplant. 23, 1186–1195. Covas, M.I., de la Torre, K., Farré-Albaladejo, M., Kaikkonen, J., Fitó, M., López-Sabater, C., Pujadas-Bastardes, M.A., Joglar, J., Weinbrenner, T., Lamuela-Raventós, R.M., de la Torre, R., 2006. Postprandial LDL phenolic content and LDL oxidation are modulated by olive oil phenolic compounds in human. Free Radic. Biol. Med. 40, 608–616. D’Angelo, S., Ingrosso, D., Migliardi, V., Sorrentino, A., Donnarumma, G., Baroni, A., Masella, L., Tufano, M.A., Zappia, M., Galletti, P., 2005. Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells. Free Radic. Biol. Med. 38, 908–919. Deiana, M., Aruoma, O.I., Bianchi, M.P., Spencer, J.P.E., Kaur, H., Halliwell, B., Banni, S., Dessi, M.A., Corongiu, F.P., 1999. Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol. Free Radic. Biol. Med. 26, 762–769. de la Puerta, R., Ruiz Gutierrez, V., Hoult, R.S., 1999. Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. Biochem. Pharmacol. 57, 445–449.
Section | III Tyrosol and Hydroxytyrosol
Dell’Agli, M., Maschi, O., Galli, G.V., Fagnani, R., Dal Cero, E., Caruso, D., Bosisio, E., 2008. Inhibition of platelet aggregation by olive oil phenols via cAMP-phosphodiesterase. Br. J. Nutr. 99, 945–951. Della Ragione, F., Cucciolla, V., Borriello, A., Della Pietra, V., Pontoni, G., Racioppi, L., Manna, C., Galletti, P., Zappia, V., 2000. Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis. Biochem. Biophys. Res. Commun. 278, 733–739. Della Ragione, F., Cucciolla, V., Criniti, V., Indaco, S., Borriello, A., Zappia, V., 2002. Antioxidants induce different phenotypes by a distinct modulation of signal transduction. FEBS Lett. 532, 289–294. Durak, I., Cetin, R., Candir, O., Devrim, E., Kiliçoglù, B., Avci, A., 2007. Black grape and garlic extracts protect against cyclosporine A nephrotoxicity. Immunol. Invest. 36, 105–114. Fabiani, R., Rosignoli, P., De Bartolomeo, A., Fuccelli, A., Morozzi, G., 2008. Inhibition of cell cycle progression by hydroxytyrosol is associated with upregulation of cyclin-dependent protein kinase inhibitors p21WAF1/Cip1 and p27Kip1 and with induction of differentiation in HL60 cells. J. Nutr. 138, 42–48. Galletti, P., Di Gennaro, C.I., Migliardi, V., Indaco, S., Della Ragione, F., Manna, C., Chiodini, P., Capasso, G., Zappia, V., 2005. Diverse effects of natural antioxidants on cyclosporin cytotoxicity in rat renal tubular cells. Nephrol. Dial. Transplant. 20, 1551–1558. González-Santiago, M., Martín-Bautista, E., Carrero, J.J., Fonollá, J., Baró, L., Bartolomé, M.V., Gil-Loyzaga, P., López-Huertas, E., 2006. Onemonth administration of hydroxytyrosol, a phenolic antioxidant present in olive oil, to hyperlipidemic rabbits improves blood lipid profile, antioxidant status and reduces atherosclerosis development. Atherosclerosis 188, 35–42. Goya, L., Mateos, R., Bravo, L., 2007. Effect of the olive oil phenol hydroxytyrosol on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert-butylhydroperoxide. Eur. J. Nutr. 46, 70–78. Manna, C., Della Ragione, F., Cucciolla, V., Borriello, A., D’Angelo, S., Galletti, P., Zappia, V., 1999. Biological effects of hydroxytyrosol, a polyphenol from olive oil endowed with antioxidant activity. Adv. Exp. Med. Biol. 472, 115–130. Manna, C., Galletti, P., Maisto, G., Cucciolla, V., D’Angelo, S., Zappia, V., 2000. Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 470, 341–344. Maiuri, M.C., De Stefano, D., Di Meglio, P., Irace, C., Savarese, M., Sacchi, R., Cinelli, M.P., Carnuccio, R., 2005. Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation. Naunyn Schmiedebergs Arch. Pharmacol. 371, 457–465. Mustafi-Pour, Z., Zal, F., Monabati, A., Vessal, M., 2008. Protective effects of a combination of quercetin and vitamin E against cyclosporine A-induced oxidative stress and hepatotoxicity in rats. Hepatol. Res. 38, 385–392. Rezzani, R., 2006. Exploring cyclosporine A-side effects and the protective role-played by antioxidants: the morphological and immunohistochemical studies. Histol. Histopathol. 21, 301–316. Trichopoulou, A., Dilis, V., 2007. Olive oil and longevity. Mol. Nutr. Food Res. 51, 1275–1278. Visioli, F., Galli, C., 2002. Biological properties of olive oil phytochemicals. Crit. Rev. Food Sci. Nutr. 42, 209–211. Wongmekiat, O., Leelarugrayub, N., Thamprasert, K., 2008. Beneficial effect of shallot (Allium ascalonicum L.) extract on cyclosporine nephrotoxicity in rats. Food Chem. Toxicol. 46, 1844–1850.
Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity Vincenzo Zappia1, Patrizia Galletti1, Caterina Manna1, Stefania D’Angelo1,2, Daniela Napoli1, Maria Luigia De Bonis1 and Giovambattista Capasso3 1
Department of Biochemistry and Biophysics, School of Medicine, Second University of Naples, Italy Faculty of Motor Sciences, Parthenope University, Naples, Italy 3 Chair of Nephrology, Department of Internal Medicine, Second University of Naples, Italy 2
136.1 Introduction Olive oil represents the typical lipidic source of the Mediterranean diet that has been associated with a low incidence of several pathologies, including cardiovascular diseases and neurological disorders (Bendini et al., 2007; Trichopoulou and Dilis, 2007). The beneficial properties of olive oil have been mainly attributed to its high content of monounsaturated oleic acid, however, in recent years converging evidence indicates that the non-glyceride fraction of olive oil, rich in vitamin and non-vitamin antioxidants including polyphenols, significantly contributes to its benefits on human health (Visioli and Galli, 2002). Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET) is the main ortho-diphenolic compound of olive oil and is mainly responsible for the antioxidant properties of this nutrient. Indeed, it has been shown to function as an efficient scavenger of peroxyl radicals in several biological systems and contributes to increase the shelf-life of the oil, preventing its auto-oxidation (Visioli and Galli, 2002).
136.2 Biological effects of hydroxytyrosol The biological activities of DOPET have been explored by several groups and are summarized in Table 136.1 (for a comprehensive review see Bendini et al., 2007). Even though the majority of them can be directly ascribed to its antioxidant activity (Manna et al., 1999; Visioli and Galli 2002), emerging evidence (Della Ragione et al., 2002) supports the view that some effects of this molecule are independent from its scavenging properties. DOPET inhibits in vitro low-density lipoprotein oxidation and modulates the Olives and Olive Oil in Health and Disease Prevention. ISBN: 978-0-12-374420-3
oxidative/antioxidative balance in plasma (Covas et al., 2006). Experiments from our group demonstrated that DOPET, which effectively permeates cell membranes via passive diffusion (Manna et al., 2000), counteracts the cytotoxic effects of reactive oxygen species (ROS) in various human systems. Preincubation of intestinal Caco-2 cells with DOPET prevents the typical damages of oxidative stress (Manna et al., 1999). Similarly, this polyphenol exerts a protective effect against the H2O2-induced oxidative hemolysis and lipid peroxidation in red blood cells (Manna et al., 1999). Moreover, it has been demonstrated that the molecule counteracts the up-rise of specific markers of oxidative stress in UVA-irradiated melanoma cells. In fact it prevents the formation of abnormal L-isoaspartyl residues in proteins and reduces lipid peroxidation in irradiated M14 cells (D’Angelo et al., 2005). Furthermore, pretreatment of human hepatocarcinoma HepG2 cells with micromolar concentrations of DOPET completely prevents the decrease of reduced glutathione and the rise of malondialdehyde induced by tert-butyl hydroperoxide in this cell line (Goya et al., 2007). As already mentioned, it has been proposed that the antioxidant effect of DOPET probably contributes to the prevention of some degenerative diseases and supports brain cell survival after oxidative injuries (Bendini et al., 2007; Trichopoulou and Dilis, 2007). The effects of DOPET on inflammation/atherogenesis have also been thoroughly investigated. It has been demonstrated that this antioxidant inhibits the expression of adhesion molecules in a human endothelial cell line (HUVEC) exposed to proinflammatory cytokines (Carluccio et al., 2003). Moreover, DOPET administration in hyperlipidemic rabbits is able to reduce the size of atherosclerotic lesions of the aortic arch (González-Santiago et al., 2006). The ability of DOPET to counteract inflammation is also
1245
Copyright © 2010 Elsevier Inc. All rights of reproduction in any form reserved.
1246
Section | III Tyrosol and Hydroxytyrosol
Table 136.1 Biological effects of hydroxytyrosol. Affected biological systems
Experimental models
References
LDL oxidation in vitro and in vivo*
Human
Covas et al., 2006
Cytokine-induced endothelial activation and macrophage activation*
Human umbilical vein endothelial cells (HUVEC)
Carluccio et al., 2003
Bovine aortic endothelial cells (BAEC)
Maiuri et al., 2005
Murine macrophages (J774) Platelet and leukocytes arachidonate lipoxygenases*
Rat
de la Puerta et al., 1999
PMA-induced respiratory burst*
Human neutrophils
Visioli and Galli, 2002
Peroxynitrite-dependent tyrosine nitration*
Neuronal hybridoma cell line N-18-RE-105
Deiana et al., 1999
Platelet aggregation*
Human
Dell’Agli et al., 2008
Cell proliferation* and accumulation of cells in the G0/G1 phase**
Human myeloid leukemia cells (HL-60)
Fabiani et al., 2008
Apoptosis**
Human myeloid leukemia cells (HL-60)
Della Ragione et al., 2000
Human carcinoma colon cell line (HT-29)
Fabiani et al., 2008
Atherosclerotic lesion development (*)
Rabbit
González-Santiago et al., 2006
ROS-mediated citotoxicity (*)
Human carcinoma colon cell line (Caco-2)
Manna et al., 1999
Human erythrocytes
Manna et al., 1999
Human melanoma cell line (M14)
D’Angelo et al., 2005
Human hepatocarcinoma cell line (HepG2)
Goya et al., 2007
*
Inhibition. Induction.
**
supported by the evidence that extra virgin olive oil administration significantly decreases the levels of known markers of this process, such as thromboxane B2 (Bendini et al., 2007) and 5- and 12-lipoxygenases (de la Puerta et al., 1999). On the other hand, the anti-inflammatory properties of the phenolic fraction of olive oil are widely documented in the literature, DOPET being a powerful inhibitor of neutrophil respiratory burst (Visioli and Galli, 2002). The prevention of abnormal cell proliferation and the induction of apoptosis, both events involved in carcinogenesis, have been described as effects of DOPET not directly attributable to its antioxidant properties (Bendini et al., 2007). Indeed, it has been recently demonstrated that this compound, in a micromolar concentration range, inhibits the proliferation and induces apoptosis in different human cell lines (Della Ragione et al., 2000; Fabiani et al., 2008). In detail, the polyphenol alters HL60 cell cycle progression, inducing
an accumulation of cells in the G0/G1 phase (Manna et al., 1999; Fabiani et al., 2008). In the same cell line, it has been recently demonstrated that this effect is associated with an up-regulation of cyclin-dependent protein kinase inhibitors and with an induction of cell differentiation (Fabiani et al., 2008). In this respect, it has been proposed that DOPET may affect the expression of genes involved in the regulation of tumor cell proliferation and differentiation, such as p27Kip1 and p21WAF/Cip1 (Fabiani et al., 2008). It is worthwhile mentioning that tyrosol (4-hydroxyphenylethanol), the DOPET analogue which lacks the ortho-diphenolic moiety, does not show any antioxidant activity and fails to exert any inhibitory effect on cell growth, indicating that the two ortho-hydroxyl groups are critical for both antioxidant and antiproliferative effects (Manna et al., 1999). The prevention of DNA damage, responsible for mutagenesis and carcinogenesis, can be envisioned as another
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
mechanism associated with the DOPET anticancer effect. This phenol, indeed, exerts an inhibitory effect on peroxynitrite-dependent DNA base modifications as well as on tyrosine nitration (Deiana et al., 1999).
1247
Table 136.2 Key features of cyclosporine nephrotoxicity. 1. Cyclosporine A (CsA), is a cyclic undecapeptide produced by the fungus Tolypocladium inflatum Gams
136.3 Cyclosporine and ROS
2. CsA is an immunosuppressant drug primarily used in humans to prevent organ transplant rejection
Cyclosporine A (CsA), a cyclic undecapeptide of fungal origin, is the most widely used immunosuppressive drug in organ transplantation and in the treatment of autoimmune disorders (Table 136.2). However, its clinical use is often limited by the frequent and dose-dependent severe side effects, including nephrotoxicity, hypertension, hepatotoxicity, lymphoproliferative disorders, atherosclerosis of the graft and interstitial myocardial fibrosis (Rezzani, 2006). Nephrotoxicity is the main untoward consequence of CsA treatment; moderate to severe renal dysfunction has been, indeed, documented in 30% of CsA-treated patients. On the basis of the length of treatment, two forms of CsA nephrotoxicity have been described: acute CsA treatment induces reversible reduction of both glomerular filtration rate (GFR) and renal blood flow, related to the afferent arteriolar vasoconstriction. This latter effect is associated with an increase in vasoconstrictor factors such as endothelin, thromboxane A2, angiotensin II and/or a decrease in vasodilators, such as prostacyclin and nitric oxide. Thus, an imbalance in the release of vasoactive substances may account for the reported renal vasoconstriction. On the other hand, when chronically administered, CsA can lead to irreversible renal failure due to the synergical effect of several factors, such as renal vasoconstriction, tubulointerstitial fibrosis, tubular atrophy and glomerular sclerosis (Rezzani, 2006). Even though the mechanisms of CsA toxicity are not fully understood, in recent years several experimental evidences have been collected on the involvement of oxidative stress in the toxic effects of CsA treatment. It has been reported that this drug induces membrane lipid peroxidation in several in vitro and in vivo experimental models, as well as in transplanted patients. In vivo, CsA increases lipoperoxidation in the rat kidney and liver, depletes the hepatic and renal pool of glutathione and impairs antioxidant defenses. Moreover, it has been reported that mRNA and protein levels of heme oxygenase-1, an enzyme responsive to changes in the redox status, vary after treatment with CsA (Rezzani, 2006). Several hypotheses have been proposed in order to correlate CsA treatment and oxidative stress, such as up-regulation of the kidney cytochrome P450-dependent ROSproducing system, perturbation of the balance between vaso dilation–vasoconstriction, in turn responsible for tubular hypoxia–reoxygenation, increased formation of renal thromboxane A2, induction of nitric oxide production. In addition, a possible direct interference of CsA with the intracellular homeostasis of glutathione has been suggested (Rezzani, 2006).
3. CsA inhibits interleukin-2 gene transcription and the transition of T-lymphocytes from the G0 to G1 phase of the cell cycle 4. CsA, although extensively used in kidney transplantation, causes important renal adverse effects, including acute and chronic renal dysfunction, hemolytic-uremic syndrome, hypertension, electrolyte disturbances and defects in urinary concentrating ability 5. Prolonged CsA administration may lead to structural changes which are no longer dose-dependent and reversible and cause end-stage renal failure 6. Although the mechanisms of nephrotoxicity have not been well defined, some evidence suggests that reactive oxygen species play a causative role
The hypothesis that CsA toxicity is mediated by ROS led investigators to use antioxidant molecules such as taurine, lipoic acid, melatonin and N-acetylcysteine to prevent or ameliorate its adverse effects (Rezzani, 2006). Since many plant-derived ‘phytonutrients’ are becoming increasingly known for their antioxidant activity, the use of plant-derived antioxidants against nephrotoxicity induced by treatment with CsA has been thoroughly explored in animal models. The administration of shallot (Allium ascalonicum) extract along with CsA counteracts its deleterious effects on renal dysfunction, oxidative stress markers and morphological changes (Wongmekiat et al., 2008). Lycopene, the carotenoid pigment found in tomatoes and other red fruits, ameliorates the CsA-induced pathological alterations including: tubular necrosis, degeneration, thickened basement membranes and intertubular fibrosis (Atessahin et al., 2007). Resveratrol, a naturally occurring phenolic compound abundantly present in grapes and red wine, protects against CsA-induced nephrotoxicity through a nitric oxide-dependent mechanism (Rezzani, 2006). Comparable results have been obtained using curcumin, the principal component of the Indian curry spice turmeric (Rezzani, 2006). Similarly, provinol, a polyphenolic extract obtained from red wine, prevents the increase of systolic blood pressure and nephrotoxicity in rat, through a mechanism that involves reduction of oxidative stress and iNOS expression, via nuclear factor-B pathway (Rezzani, 2006).
1248
6
Fluorescence intensity/106 cells
Both dried black grape and aqueous garlic counteract CsA nephrotoxicity, reducing the malondialdehyde level in the kidney tissue, possibly by preventing oxidative reactions (Durak et al., 2007). Vitamin E protects renal function and structure when administered in vivo to CsA-treated rats (Rezzani, 2006). Moreover, it has been demonstrated that the combination of quercetin and vitamin E plays a protective role against the imbalance elicited by CsA between the production of free radicals and the antioxidant defense systems, suggesting that a combination of these two antioxidants may find clinical application where cellular damage is a consequence of ROS (Mustafi-Pour et al., 2008).
Section | III Tyrosol and Hydroxytyrosol
5 4 3
*
2 1 0
A
Control
DOPET
CsA
CsA + DOPET
Control
DOPET
CsA
CsA + DOPET
The aim of the in vitro study (Galletti et al., 2005) was to investigate the possible protective effect of DOPET on CsA-induced nephrotoxicity using immortalized renal proximal tubule cells (RPTc) from normotensive Wistar–Kyoto rats. This cell line has been frequently selected as a model system, since tubular cells represent in vivo the major target of CsA-induced nephrotoxicity both in humans and in animal models (Galletti et al., 2005 and references therein). In the RPT cellular model system, CsA toxicity is a relatively late event, since 95% of RPTc are still largely viable up to 8 h in the presence of 25 M CsA. Conversely, prolonged incubation time leads to a dramatic decrease in cell viability, with 30% cell death after 20 h exposure to CsA. When RPTc have been incubated with CsA in the presence of 10 M DOPET, ROS formation was measured using the dichlorofluorescein (DCF) assay. As shown in Figure 136.1A, CsA treatment results in a significant increase in ROS formation. Moreover, the CsA-induced fluorescence signal is totally quenched by DOPET treatment. However, this phenol appears completely ineffective in preventing the toxic effect of the drug and in restoring cell viability (Figure 136.1B). The effect of DOPET on CsA-induced membrane lipoperoxidation has also been evaluated (Figure 136.2). RPTc incubation in the presence of CsA significantly increases the lipoperoxidation products (TBARS) (30% above basal), confirming that CsA treatment exposes cells to an oxidative microenvironment. Again, DOPET effectively counteracts the increase in TBARS formation. The pivotal role played by glutathione (GSH) in cellular protection against free radical damage is well known. The effect of DOPET on CsA-induced imbalance of the GSH redox state is shown in Table 136.3. CsA treatment induces in RPTc a significant increase in oxidized gluta thione (GSSG), resulting in a 50% reduction in the [GSH]/ [GSSG] ratio. This result supports a direct interference of
100 80 60 40 20 0
B
Figure 136.1 Effect of hydroxytyrosol (DOPET) on cyclosporine A (CsA)-induced reactive oxygen species (ROS) production (A) and cytotoxicity (B) in renal proximal tubule cells (RPTc). RPTc were exposed for 20 h to 25 M CsA in the presence or absence of 10 M DOPET. ROS production was detected by means of the fluorescent indicator dichlorofluorescein, as reported by Galletti et al. (2005). Cell viability was evaluated by the trypan blue exclusion method. Data are expressed as means SD (n 3) *p 0.001 vs CsA. 3
TBARS nmol−1 µg protein
136.4 Effects of hydroxytyrosol on cyclosporine cytotoxicity in rat renal tubular cells
Cell viability (% of control)
120
2
* 1
0 Control
DOPET
CsA
CsA + DOPET
Figure 136.2 Effect of hydroxytyrosol (DOPET) on cyclosporine A (CsA)-induced lipid peroxidation in renal proximal tubule cells (RPTc). RPTc were incubated for 20 h with 25 M CsA in the presence or absence of 10 M DOPET. Data are expressed as means SD (n 3) *p 0.05 vs CsA.
the drug with the intracellular GSH homeostasis. However, DOPET fails to provide any appreciable protection (Table 136.3) against the CsA-induced alterations in gluta thione metabolism.
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
Table 136.3 Effect of hydroxytyrosol (DOPET) on glutathione redox state in CsA-treated RPTc. GSSG nmol mg1 protein
GSH nmol mg1 protein
GSH/GSSG molar ratio
Control
1.32 0.65
15.9 4.2
12 3.8
CsA 25 M
2.85 1.14*
17 3.8
6.0 3.4*
CsA 25 M DOPET 10 M
2.8 0.15*
18.3 0.54 6.5 0.15*
Effect of cyclosporine-A (CsA) on the glutathione redox state in RPTc. Control samples received CsA vehicle. The glutathione (GSH), the oxidized glutathione (GSSG) and the GSH/GSSG ratio values are reported as mean SD. Significance (t-test)/ANOVA: *p 0.01 vs control.
The reported data indicate that DOPET effectively counteracts both CsA-induced ROS production and membrane lipoperoxidation in RPTc; however, the protection against the CsA-induced oxidative stress is not paralleled by an equivalent decrease in CsA cytotoxicity. On the basis of these findings, ROS generation induced by CsA does not appear strictly related to its nephrotoxi city; therefore, the generalization that antioxidants might exert a protective effect against the adverse effects of CsA, proposed by several authors and shared by nephrologists, is called into question by the reported in vitro approach.
136.5 Cyclosporine nephrotoxicity in rats: effect of hydroxytyrosol in vivo To further elucidate whether oxidative stress is responsible for CsA toxicity, a recent study assayed the protective effect of DOPET on oxidative stress, renal histology and hemodynamic alterations induced in rats by chronic CsA treatment (Capasso et al., 2008). In order to evaluate CsA-induced superoxide production within cells of the abdominal aorta and renal artery, the changes in fluorescence resulting from the oxidation of dihydroethidium (DHE) were monitored. The red fluorescence generated by the binding of ethidium–DNA complex is considered an appropriate indicator of superoxide production within the cells. As reported in Figure 136.3, the abdominal aorta of CsA-treated animals presents a red fluorescent signal significantly brighter than controls. When the rats were treated with CsA plus DOPET the fluorescence intensity was similar to controls, indicating that the polyphenol is able to completely prevent the production of superoxide.
1249
This experimental approach indicates that also in vivo DOPET is able to quench CsA-induced ROS production both in aorta and renal artery. These results are fully in agreement with the data of the in vitro study previously reported. TBARS were measured in the rat kidney to evaluate the CsA-induced oxidative alteration of cellular components. A significant increase (p 0.01) of TBARS was observable after CsA treatment (Table 136.4); the simultaneous administration of DOPET and CsA restored TBARS to control level, thus demonstrating that, also when administered in vivo, the polyphenol completely prevents the effect of CsA on lipid peroxidation. Moreover, chronic CsA treatment significantly increased the GSSG level (p 0.01 versus control). Also in this case, the co-administration of DOPET completely reversed this effect. Accordingly, CsA treatment lowered the cellular [GSH]/[GSSG] ratio by 44%, while DOPET completely prevented this alteration. As already mentioned, clinical observations and experimental evidence (Capasso et al., 2008 and references therein) indicate that CsA administration is associated with major side effects, including hypertension and renal failure related with fibrosis and vasoconstriction. It has been proposed that ROS overproduction induced by CsA may lead to the inhibition of NO synthesis with the consequent appearance of hypertension. Consistently, as shown in Figure 136.4, starting from the second week of CsA treatment, an increase of about 15 mmHg in both systolic blood pressure (BP) and diastolic BP can be observed. According to literature data, CsA increased both systolic BP and diastolic BP by 16–17 mmHg at the end of the treatment period. However, the administration of DOPET did not yield any observable effect on CsA-induced hypertension. A severe impairment of renal hemodynamics is another side effect of chronic administration of CsA. Among the implicated mechanisms, there is general agreement that the major factor of the CsA effect on GFR is mediated by its action on afferent arteriolar resistance. In this study, GFR was measured by means of inulin clearance. The effect of DOPET on chronic CsA-induced renal failure is reported in Figure 136.5. DOPET per se did not exert any change on glomerular function (0.93 0.05 mL min1 100 g1 b.w.). CsA treatment significantly decreased GFR compared to control animals (0.51 0.03 versus 0.94 0.05 mL min1 100 g1 b.w., respectively) (p 0.01), while DOPET, in combination with CsA, did not exert any protection (0.46 0.02 mL min1 100 g1 b.w.). Therefore, in contrast with its ROS-quenching effect, DOPET is unable to prevent both the increase in BP and the decrease in GFR. It is worth underlining that DOPET alone had almost no detrimental effect, confirming the low toxicity of this antioxidant phenol. The lack of a protective effect of DOPET on BP and kidney hemodynamics fits very nicely with the histological data, indicating that the antioxidant was unable to act on the arteriolopathy.
1250
Section | III Tyrosol and Hydroxytyrosol
Figure 136.3 Effect of cyclosporine A (15 mg kg1 day1) and hydroxytyrosol (20 mg kg1 twice daily) on the production of superoxide in the abdominal aorta. Superoxide concentrations within cells of the abdominal aorta were monitored by measuring the changes in fluorescence resulting from the oxidation of dichlorofluorescein. The figure has been reproduced with permission from Capasso et al., (2008).
Table 136.4 Oxidative stress markers evaluation after cyclosporine A (CsA) treatment. Control
DOPET (20 mg kg1)
CsA (15 mg kg1)
CsA (15 mg kg1) DOPET (20 mg kg1)
TBARS (nmol/g tissue)
20.8 3.4
23.8 2.5
37.2 7.4a**
28.0 2.3b*
GSSG (nmol GSH/g tissue)
1.66 0.11
1.50 0.10
2.17 0.29a**
1.60 0.19b*
GSH/GSSG ratio
31.09 3.4
29.62 2.3
17.08 2.1a**
33.57 2.4b*
Lipid peroxidation was measured in kidney tissues as thiobarbituric acid reacting substances (TBARS); oxidized glutathione (GSSG) was assessed by enzymatic assay. Control group received CsA vehicle, CsA group received CsA 15 mg kg1 day1 and DOPET group received DOPET 20 mg kg1 twice daily. Significance (t-test)/ANOVA: a vs control; b vs CsA; *p 0.05; **p 0.01.
136.6 Conclusions All together these observations lead to the conclusion that CsA-induced kidney injury is only partially due to oxidative stress. Moreover, the in vivo results are in agreement with in vitro data indicating that DOPET is able to completely prevent CsA-induced oxidative stress in rat tubular cells, but is ineffective in ameliorating the associated reduction of cell viability. The most reasonable interpretation of the data obtained in vivo is that the process leading to BP increase and GFR decrease is not necessarily related to ROS-dependent mechanism(s). Thus, even when CsA-induced oxidative stress is completely reverted by DOPET, renal failure and hypertension cannot be prevented, probably because
of other underlying mechanisms, such as artheriolopathy. Such a hypothesis seems to be in contrast with literature data showing that the administration of ‘antioxidant drugs’ like vitamin E (Rezzani, 2006) and lycopene (Atessahin et al., 2007) is able to reduce oxidative stress and ameliorate renal function after CsA treatment. It is highly likely that these two effects are mutually independent. Indeed, it should be underlined that both compounds, beside their antioxidant activity, exert key functions such as modulation of enzymatic activities and alteration of gene expression, which could account for their protective effect against CsA treatment. In this respect, it has been demonstrated that the overexpression of superoxide dismutase 1 by gene delivery, 3 days prior to the in vivo CsA administration, can partially reduce CsA-induced pathological alterations and inhibition of renal function (Rezzani, 2006).
mmHg
Chapter | 136 Effects of Hydroxytyrosol on Cyclosporine Nephrotoxicity
1251
Systolic blood pressure
140 135 130 125 120 115 110 105 100 0
5
10
20
15
25
Time (days)
mmHg
A
CsA+DOPET
CsA
DOPET
Control
Diastolic blood pressure
125 120 115 110 105 100 95 90 85 0
5
10
15
20
25
Time (days) CsA+DOPET
CsA Control
DOPET
B
Figure 136.4 Effect of cyclosporine A (15 mg kg day1) and hydroxytyrosol (20 mg kg1 twice daily) on systolic and diastolic pressure in rats. The rats were treated as described in the text. Diastolic and systolic blood pressure were measured on conscious rats using the tail method. Panel A shows the systolic pressure trend during the treatment period. Panel B shows the diastolic pressure results. †p 0.05, ††p 0.01, †††p 0.001 versus day 0; *** p 0.001 versus control. The figure has been reproduced with permission from Capasso et al., (2008). 1
thus supporting the view that kidney injury by CsA is mainly related to pathogenetic mechanisms independent from oxidative stress.
GFR (mL−1min−1g 100−1g B.W.)
0.2 1 0.8 0.6
Summary points
**
0.4
Cyclosporine A (CsA), although widely used as an immunosuppressive drug, exerts frequent and dosedependent cytotoxic effects, probably related to ROS overproduction. l Hydroxytyrosol (DOPET), the powerful olive oil antioxidant, could counteract CsA cytotoxicity through its scavenging properties. l In vitro studies with rat renal tubular cells, the main target of CsA cytotoxic activity, have demonstrated that DOPET effectively counteracts ROS production and lipoperoxidation. However, these effects are not paralleled by an equivalent decrease in CsA cytotoxicity. l The in vivo protective effect of DOPET has been assayed in rats. While exerting a significant protection toward CsA-induced oxidative stress, this polyphenol l
0.2 0 Control
DOPET
CsA
CsA + DOPET
Figure 136.5 Effect of hydroxytyrosol on the glomerular filtration rate (GFR) measured in rats treated with cyclosporin A. GFR was measured by inulin clearance. Inulin concentration in plasma and urine was calculated by the colorimetric method. **p 0.001 vs control. The figure has been reproduced with permission from Capasso et al., (2008).
In conclusion, the in vitro and in vivo effective DOPET protection from CsA-induced oxidative stress is only associated with mild effects on histological damage and does not affect the altered glomerular function and the hypertension,
1252
does not influence renal histological and hemodynamic alterations. l In conclusion, DOPET protection is only associated with mild effects on histological damage and does not affect the altered glomerular function and the hypertension. l The data support the view that kidney injury by CsA is mainly related to pathogenetic mechanisms independent from oxidative stress.
Acknowledgments The authors gratefully acknowledge the Oxford University Press for the kind permission to use Figures 1, 6 and 7 from Capasso G. et al., Nephrol. Dial. Transplant. 23, 1186–1195.
References Atessahin, A., Ceribasi, A., Yilmaz, S., 2007. Lycopene, a carotenoid, attenuates cyclosporine-induced renal dysfunction and oxidative stress in rats. Basic Clin. Pharm. Toxicol. 100, 372–376. Bendini, A., Cerretani, L., Carrasco-Pancorbo, A., Gómez-Caravaca, A.M., Segura-Carretero, A., Fernández-Gutiérrez, A., Lercker, G., 2007. Phenolic molecules in virgin olive oils: a survey of their sensory properties, health effects, antioxidant activity and analytical methods. An overview of the last decade. Molecules 12, 1679–1719. Carluccio, M.A., Siculella, L., Ancora, M.A., Massaro, M., Scoditti, E., Storelli, C., Visioli, F., Distante, A., De Caterina, R., 2003. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler. Thromb. Vasc. Biol. 23, 622–629. Capasso, G., Di Gennaro, C.I., Della Ragione, F., Manna, C., Ciarcia, R., Florio, S., Perna, A., Pollastro, R.M., Damiano, S., Mazzoni, O., Galletti, P., Zappia, V., 2008. In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats. Nephrol. Dial. Transplant. 23, 1186–1195. Covas, M.I., de la Torre, K., Farré-Albaladejo, M., Kaikkonen, J., Fitó, M., López-Sabater, C., Pujadas-Bastardes, M.A., Joglar, J., Weinbrenner, T., Lamuela-Raventós, R.M., de la Torre, R., 2006. Postprandial LDL phenolic content and LDL oxidation are modulated by olive oil phenolic compounds in human. Free Radic. Biol. Med. 40, 608–616. D’Angelo, S., Ingrosso, D., Migliardi, V., Sorrentino, A., Donnarumma, G., Baroni, A., Masella, L., Tufano, M.A., Zappia, M., Galletti, P., 2005. Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells. Free Radic. Biol. Med. 38, 908–919. Deiana, M., Aruoma, O.I., Bianchi, M.P., Spencer, J.P.E., Kaur, H., Halliwell, B., Banni, S., Dessi, M.A., Corongiu, F.P., 1999. Inhibition of peroxynitrite dependent DNA base modification and tyrosine nitration by the extra virgin olive oil-derived antioxidant hydroxytyrosol. Free Radic. Biol. Med. 26, 762–769. de la Puerta, R., Ruiz Gutierrez, V., Hoult, R.S., 1999. Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. Biochem. Pharmacol. 57, 445–449.
Section | III Tyrosol and Hydroxytyrosol
Dell’Agli, M., Maschi, O., Galli, G.V., Fagnani, R., Dal Cero, E., Caruso, D., Bosisio, E., 2008. Inhibition of platelet aggregation by olive oil phenols via cAMP-phosphodiesterase. Br. J. Nutr. 99, 945–951. Della Ragione, F., Cucciolla, V., Borriello, A., Della Pietra, V., Pontoni, G., Racioppi, L., Manna, C., Galletti, P., Zappia, V., 2000. Hydroxytyrosol, a natural molecule occurring in olive oil, induces cytochrome c-dependent apoptosis. Biochem. Biophys. Res. Commun. 278, 733–739. Della Ragione, F., Cucciolla, V., Criniti, V., Indaco, S., Borriello, A., Zappia, V., 2002. Antioxidants induce different phenotypes by a distinct modulation of signal transduction. FEBS Lett. 532, 289–294. Durak, I., Cetin, R., Candir, O., Devrim, E., Kiliçoglù, B., Avci, A., 2007. Black grape and garlic extracts protect against cyclosporine A nephrotoxicity. Immunol. Invest. 36, 105–114. Fabiani, R., Rosignoli, P., De Bartolomeo, A., Fuccelli, A., Morozzi, G., 2008. Inhibition of cell cycle progression by hydroxytyrosol is associated with upregulation of cyclin-dependent protein kinase inhibitors p21WAF1/Cip1 and p27Kip1 and with induction of differentiation in HL60 cells. J. Nutr. 138, 42–48. Galletti, P., Di Gennaro, C.I., Migliardi, V., Indaco, S., Della Ragione, F., Manna, C., Chiodini, P., Capasso, G., Zappia, V., 2005. Diverse effects of natural antioxidants on cyclosporin cytotoxicity in rat renal tubular cells. Nephrol. Dial. Transplant. 20, 1551–1558. González-Santiago, M., Martín-Bautista, E., Carrero, J.J., Fonollá, J., Baró, L., Bartolomé, M.V., Gil-Loyzaga, P., López-Huertas, E., 2006. Onemonth administration of hydroxytyrosol, a phenolic antioxidant present in olive oil, to hyperlipidemic rabbits improves blood lipid profile, antioxidant status and reduces atherosclerosis development. Atherosclerosis 188, 35–42. Goya, L., Mateos, R., Bravo, L., 2007. Effect of the olive oil phenol hydroxytyrosol on human hepatoma HepG2 cells. Protection against oxidative stress induced by tert-butylhydroperoxide. Eur. J. Nutr. 46, 70–78. Manna, C., Della Ragione, F., Cucciolla, V., Borriello, A., D’Angelo, S., Galletti, P., Zappia, V., 1999. Biological effects of hydroxytyrosol, a polyphenol from olive oil endowed with antioxidant activity. Adv. Exp. Med. Biol. 472, 115–130. Manna, C., Galletti, P., Maisto, G., Cucciolla, V., D’Angelo, S., Zappia, V., 2000. Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells. FEBS Lett. 470, 341–344. Maiuri, M.C., De Stefano, D., Di Meglio, P., Irace, C., Savarese, M., Sacchi, R., Cinelli, M.P., Carnuccio, R., 2005. Hydroxytyrosol, a phenolic compound from virgin olive oil, prevents macrophage activation. Naunyn Schmiedebergs Arch. Pharmacol. 371, 457–465. Mustafi-Pour, Z., Zal, F., Monabati, A., Vessal, M., 2008. Protective effects of a combination of quercetin and vitamin E against cyclosporine A-induced oxidative stress and hepatotoxicity in rats. Hepatol. Res. 38, 385–392. Rezzani, R., 2006. Exploring cyclosporine A-side effects and the protective role-played by antioxidants: the morphological and immunohistochemical studies. Histol. Histopathol. 21, 301–316. Trichopoulou, A., Dilis, V., 2007. Olive oil and longevity. Mol. Nutr. Food Res. 51, 1275–1278. Visioli, F., Galli, C., 2002. Biological properties of olive oil phytochemicals. Crit. Rev. Food Sci. Nutr. 42, 209–211. Wongmekiat, O., Leelarugrayub, N., Thamprasert, K., 2008. Beneficial effect of shallot (Allium ascalonicum L.) extract on cyclosporine nephrotoxicity in rats. Food Chem. Toxicol. 46, 1844–1850.