Original Contributions
Structure–activity relationships of quercetin in antagonizing hydrogen peroxide-induced calcium dysregulation in PC12 cells1

https://doi.org/10.1016/S0891-5849(99)00119-7Get rights and content

Abstract

Oxidative stress can induce neurotoxic insults by increasing intracellular calcium (Ca2+), which has been implicated in various neurodegenerative diseases in aging. Previously, we showed that hydrogen peroxide induced calcium dysregulation in PC12 cells, as evidenced by (i) an increase in calcium baselines, (ii) a decrease in depolarization-induced calcium influx, and (iii) a failure to recover the Ca2+ levels. In the present experiments, we investigated whether a dietary flavonoid, quercetin, can antagonize the effects of hydrogen peroxide in the same cell model. We also investigated the possible structure–activity relationships of quercetin by comparing the results with four other flavonoids, each having a slightly different structure from quercetin. Our results indicated that two structural components, including (i) 3′, 4′-hydroxyl (OH) groups in the B ring and (ii) a 2,3-double bond in conjugation with a 4-oxo group in the C ring, along with the polyphenolic structures were crucial for the protection. These structural components are found in quercetin, and this compound was also the most efficacious in reducing both the H2O2-induced Ca2+ dysregulation in cells and oxidative stress assessed via the dichlorofluorescein assay. Collectively, these data indicated that the particular polyphenolic structural components of quercetin provided its strong antioxidant property of protecting cells against H2O2-induced oxidative stress and calcium dysregulation.

Introduction

Oxidative stress (OS) has been implicated in age-related neurodegenerative diseases such as Parkinson’s disease [1] and Alzheimer’s disease [2] as well as in neuronal injuries from trauma and ischemia–reperfusion [3], [4]. One detrimental neurotoxic insult from OS is a large increase in intracellular calcium. Sustained elevated calcium levels in cells may impair mitochondrial function and may activate phospholipases, proteases, and endonucleases, leading to irreversible membrane, organelle, and chromatin damage and eventually to cell death [5], [6].

Our previous work used a neuronal cell line (PC12 cells) to explore the ability of OS to alter calcium homeostasis and to identify putative neuroprotective agents [7]. Our results demonstrated that transient exposure of PC12 cells to H2O2 significantly induced (i) an increase in baseline calcium levels, (ii) a decrease in depolarization-induced calcium influx, and (iii) a decrease in calcium recovery after depolarization. Pretreatment with calcium channel blockers, such as nifedepine and conotoxin, or antioxidants, such as Trolox (K J Ross-Petersen A/S, Horsholm, Germany) (vitamin E analog) and n-tert-butyl-α-phenylnitrone (PBN, nitrone trapping agent) before exposure to H2O2 did not fully antagonize the effects of H2O2 on calcium recovery. Because these various agents tested so far have not provided protection against H2O2-induced calcium dysregulation, it became of interest to determine whether natural antioxidants could protect the cells. Therefore, we chose to test a common flavonoid, quercetin.

Quercetin is commonly found in fruits and vegetables [8] and is also the major ingredient in Ginkgo biloba [9], [10]. The extract of Ginkgo biloba has been shown to be neuroprotective [11] and to improve memory in mice [12] and in elderly patients with mild to moderate memory impairment [13] or dementia [14], [15]. One possible mechanism of action of this extract may be related to quercetin in protecting neurons from oxidative damage [16], [17], [18]. It is known that quercetin is a potent antioxidant as assessed via the oxygen radical absorbance capacity (ORAC) assay [19]. In addition, it has been shown to scavenge various reactive oxygen species, including superoxide, hydroxyl, peroxyl, and other free radicals [20], and can prevent free radical–induced lipid peroxidation [21], [22], [23].

In the present study, we were interested in testing whether quercetin was able to antagonize the H2O2-induced calcium dysregulation and also in delineating the possible structure–activity relationships of quercetin that may account for the protection. It has been known that three structural groups are important determinants for the antioxidant potential of flavonoids: (i) the o-dihydroxyl (OH) (catechol) structure in the B ring; (ii) the 2,3-double bond in conjugation with a 4-oxo function; and (iii) the additional presence of both 3- and 5- hydroxyl groups [20]. Quercetin fulfills these three structural requirements and has been shown to be a potent antioxidant in vitro [19], [24]. We were also interested whether these structural principles could confer the efficacy of quercetin in antagonizing the H2O2-induced calcium dysregulation and oxidative stress in living cells. To test this, we used four different flavonoids with structures similar to those of quercetin (Fig. 1) and compared their efficacies to that of quercetin on PC12 cells.

Section snippets

Cell cultures

PC12 cells were a gift from Dr. Arthur Tischler (Tufts University School of Medicine, Boston, MA, USA). They were grown in growth medium containing 85% RPMI-1640 with l-glutamine, 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. The cells were maintained in collagen-coated plates in 5% CO2/95% air at 37°C. The culture medium was changed twice weekly, and the cells were split 1:4 or 1:8 every week.

Chemicals

Quercetin, cyanidin

Results

Calcium homeostasis in the cells was evaluated by four parameters: (i) Ca2+ baseline, the resting calcium levels; (ii) percent increase of calcium on depolarization, which indicates the degree of excitability of cells; (iii) the depolarization response, which indicates the percentage of cells that depolarized ≥45% increase of calcium on depolarization; (iv) the calcium recovery, which reflects the cells’ ability to extrude or sequester calcium and recover the calcium levels after

Discussion

Oxidative stress–induced calcium dysregulation may lead to neuronal cell death. To protect neurons effectively from OS-induced damage, it is necessary to antagonize calcium dysregulation and maintain calcium levels. In the present study, we delineated the possible structure–activity relationships of quercetin that may account for its efficacy in antagonizing the H2O2-induced calcium dysregulation in PC12 cells by comparing quercetin with other flavonoids with similar structures. We also

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