www.elsevier.com/locate/lifescie

Life Sciences 73 (2003) 1517–1526

Nitric oxide-related toxicity in cultured astrocytes:

effect of Bacopa monniera

Alessandra Russoa,*, Francesca Borrellib, Agata Campisia, Rosaria Acquavivaa,Giuseppina Racitia, Angelo Vanellaa

aDepartment of Biological Chemistry, Medical Chemistry and Molecular Biology, University of Catania,

v.le A. Doria 6, 95125, Catania, ItalybDepartment of Experimental Pharmacology, University of Naples ‘Federico II’, via D. Montesano 49, 80131, Naples, Italy

Received 23 September 2002; accepted 6 March 2003

Abstract

There is growing evidence that high concentrations of nitric oxide (NO), generated by activated astrocytes,

might be involved in a variety of neurodegenerative diseases, such as Alzheimer’s disease, ischemia and epilepsy.

It has recently been suggested that glial cells may produce NO under superoxide radical stimulation by enzyme-

independent mechanism. This suggests that also natural antioxidants may have therapeutical relevance in

neurodegenerative diseases. Studies of Bhattacharya et al. have evidenced that Bacopa monniera (BM) (family

Scrophulariaceae), an Ayurvedic medicinal plant clinically used for memory enhancing, epilepsy, insomnia and as

a mild sedative, is able to reduce the memory-dysfunction in rat models of Alzheimer’s disease, but the molecular

mechanisms of this action are yet to be determined. In the present study, we examined the effect of a methanolic

extract of BM on toxicity induced by the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), in culture of

purified rat astrocytes. Our results indicate that, after 18 h of treatment, SNAP induced an increase in the

production of reactive species, but did not induce the rupture of cellular membrane. Conversely, this NO donor

induced a fragmentation of genomic DNA compared to control astrocytes. The extract of BM inhibited the

formation of reactive species and DNA damage in a dose dependent manner. This data supports the traditional use

of BM and indicates that this medicinal plant has a therapeutic potential in treatment or prevention of neurological

diseases.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Astrocytes; Bacopa monniera; DNA damage; Nitric oxide; Oxidants species

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0024-3205(03)00476-4

* Corresponding author. Tel.: +39-95-7384073; fax: +39-95-7384220.

E-mail address: alrusso@unict.it (A. Russo).

A. Russo et al. / Life Sciences 73 (2003) 1517–15261518

Introduction

Astrocytes, which provide mechanical support for neurons, play an essential role in maintaining

the metabolic function of these cells. But there is growing evidence that high concentrations of

nitric oxide (NO), generated by activated astrocytes, might be involved in a variety of neuro-

degenerative diseases, such as Alzheimer’s disease, ischemia and epilepsy (Colasanti and Suzuki,

2000). It is generally accepted that NO is generated in biological tissues by specific nitric oxide

synthases (NOS) which metabolize arginine to citrulline with the formation of NO. However, it has

also been reported that this gaseous free radical can be generated in vivo nonenzymatically under

different pathological conditions; under acidic conditions or in the presence of sufficient reducing

equivalents which occur in disease states, such as ischemia, nitrite can be reduced directly to NO

(Ignarro et al., 2002). Moreover, it has been demonstrated that L-arginine yields NO after reaction

with peroxides (Ignarro et al., 2002). The existence of this enzyme-independent mechanism of NO

formation is important in understanding the pathogenesis and treatment of these pathologies. It has

been suggested that astrocyte cells may produce NO under superoxide radical stimulation by

enzyme-independent mechanism (Manning et al., 2001). This suggests, therefore that also natural

antioxidants may have therapeutical relevance in neurodegenerative diseases.

Bacopa monniera (BM) (family Scrophulariaceae) is a creeping annual plant found throughout

the Indian subcontinent in wet, damp and marshy areas (Chopra et al., 1956). It is an Ayurvedic

medicinal plant, clinically used for memory enhancing, epilepsy, insomnia and as a mild sedative

(Singh and Dhawan, 1992). Pure chemical principles isolated from the plant include saponins,

bacosides A and B (Chatterjee et al., 1963, 1965; Garai et al., 1996; Chakravarty et al., 2001),

sigmasterol (Banerjee and Chakravarti, 1963), flavonoids, luteolin and luteolin � 7 glucoside and

different alkaloids, brahmine, nicotine and herpestine (Schulte et al., 1972). Studies by Bhattacharya

et al. showed that this plant is able to reduce the memory dysfunction in rat models of Alzheimer’s

disease and confirmed that the saponins present in the alcoholic extract of the plant are the major

active principles (Bhattacharya et al., 1998). However, the molecular mechanisms of the BM

pharmacological action remain conjectural.

Our previous results in cell-free systems demonstrate that methanolic extract of BM shows a direct

superoxide anion scavenging effect comparable to superoxide dismutase (SOD) enzyme activity, and

exhibits a greater protective effect than Trolox (water-soluble derivative of vitamin E) and ascorbic

acid on DNA cleavage, induced by hydroxyl radicals (.OH) generated from UV-photolysis of

hydrogen peroxide (H2O2) (Russo et al., in press). Based on these experimental results, the present

study was designed to examine the effect of a methanolic extract of BM herb on toxicity induced by

the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), in cultures of purified rat astrocytes.

Methods

Chemicals

SNAP, 2V,7V-dichlorofluorescein diacetate (DCFH-DA) was obtained from Sigma Aldrich Co. (St.

Louis, USA); mouse monoclonal antiserum to Glial Fibrillary Acidic Protein (GFAP) and h-Nicotinamide-adenine dinucleotide (NADH) were obtained from Boehringer Mannheim GmbH

A. Russo et al. / Life Sciences 73 (2003) 1517–1526 1519

(Germany). All other chemicals were purchased from GIBCO BRL Life Technologies (Grand

Island, NY, USA).

Plant material

Plant material was kindly donated by Laboratory C. Sessa (Milano).

One Kg of powdered plant material (Eur. Ph. sieve No. 355) was mixed with 3 litters methanol, stirred

for 30 min and allowed to stand overnight. After stirring for another 30 min, it was filtered under

vacuum through filter paper and the filtrate was evaporated to dryness in a Rotavapor. The extracted

drug was treated as above a further two times, with 3 litters of methanol each time, and the dry residues

obtained were mixed together. The dry starting material was 3700 g of Bacopa monniera herb. The

extraction yield was 200 g (5.40%).

Cell culture

Enriched astrocytes were prepared from newborn albino rat brains (1–2-day-old Wistar strain) as

described by Booher and Sensenbrenner (Booher and Sensenbrenner, 1972). Briefly, cells were

suspended in Dulbecco’s Modified Essential Medium (DMEM), supplemented with 20% heat-

inactivated fetal bovine serum (FBS), 4 mM glutamine, streptomycin (50 Ag/ml) and penicillin (50

U/ml), plated at density of 2 � 106/25 cm3 flask and maintained for two weeks at 37 jC in a

humidified atmosphere of 95% air and 5% CO2. The purified astrocytes were obtained shaking

culture flasks for 15 h (37 jC, 180 rpm) according to the method of McCarthy and de Vellis

(McCarthy and de Vellis, 1980). The cultures were characterized using the Glial Fibrillary Acidic

Protein (GFAP) (Bignami et al., 1972).

Treatments

Purified cells were treated for 18 h with SNAP (0.5–1–2 mM). At doses higher than 2 mM, the effect

of SNAP was not dose-dependent, as previously reported (Boullerne et al., 2001).

Even though the methanolic extract of BM was dissolved in ethanol, at the treatment stage the final

ethanol concentration was never higher than 0.05%. Under these conditions, ethanol was not toxic, had

no effect on the oxidant species levels and did not damage DNA. The methanolic extract of BM (3–6–

12 Ag/ml) was added to the cultures 10 min prior to addition of SNAP. After 18 h of treatment the

astrocyte cultures were collected for different assays.

Reactive species formation

Reactive species determination was performed by using a fluorescent probe 2V,7V-dichlorofluoresceindiacetate (DCFH-DA); 100 AM DCFH-DA, dissolved in 100% methanol was added to the cellular

medium and the cells were incubated at 37 jC for 30 min. Under these conditions, the acetate group is

not hydrolyzed (Hempel et al., 1999). After incubation, astrocytes were lysated and centrifuged at

10,000 g for 10 min. The fluorescence (corresponding to the radical species-oxidized 2V,7V-dichloro-fluorescein, DCF) was monitored spectrofluorometrically using a Hitachi F-2000 spectrofluorimeter

(Hitachi, Tokyo, Japan): excitation 488 nm, emission 525 nm. The values were expressed as

A. Russo et al. / Life Sciences 73 (2003) 1517–15261520

fluorescence intensity/mg protein. Protein concentration was measured according to Bradford et al.

(Bradford, 1976).

Lactic dehydrogenase (LDH) release

Lactic dehydrogenase (LDH) activity was spectrophotometrically measured in the culture medium

and in the cellular lysates at 340 nm by analyzing NADH reduction during the pyruvate-lactate

transformation (Murphy and Baraban, 1990). The percentage of LDH released was calculated as

percentage of the total amount, considered as the sum of the enzymatic activity present in the cellular

lysate and that in the culture medium. A Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan) was

used.

DNA analysis by COMET assay

The presence of DNA fragmentation was examined by single cell gel electrophoresis (COMET

assay), according to Singh et al. (Singh et al., 1991). Briefly, 0.8–1 � 105 cells were mixed with

75 Al of 0.5% low melting agarose (LMA) and spotted on slides. The ‘‘minigels’’ were maintained

in lysis solution (1% N-laurosil-sarcosine, 2.5 mM NaCl, 100 mM Na2EDTA, 1% Triton X-100,

10% DMSO, pH 10) for 1 h at 4 jC, and then denatured in a high pH buffer (300 mM NaOH, 1

mM Na2EDTA, pH 13) for 20 min, and finally electrophoresed in the same buffer at 18 V for 45

min. At the end of the run, the ‘‘minigels’’ were neutralized in 0.4 M Tris-HCl, pH 7.5, stained

with 100 Al of ethidium bromide (2 Ag/ml) for 10 min and scored using a Leika fluorescence

microscope (Leika, Wetzlar, Germany) interfaced with a computer. Software (Leica-QWIN) allowed

us to analyze and quantify DNA damage by measuring: a) tail length (TL), intensity (TI) and area

(TA); b) head length (HL), intensity (HI) and area (HA). These parameters are employed by the

software to determine the level of DNA damage as: i) the percentage of the fragmented DNA

(TDNA), and ii) tail moment (TMOM) expressed as the product of TD (distance between head and

tail) and TDNA.

Statistics

Means F SD are given. Statistical analysis between various experimental results was performed

using Student’s t-test.

Results

In order to examine the action of BM extract on NO-effect in rat astrocytes, we completely removed

microglial cells. The purified astroglial cells were identified as astrocytes by GFAP, a specific marker of

growth, maturation and differentiation for this cell type (Bignami et al., 1972). The degree of specificity

of purified astrocyte cultures was about 98% (Fig. 1).

Intracellular oxidants were determined using a fluorescent probe DCFH-DA. The probe diffuses

into the cells, intracellular esterases hydrolyze the acetate groups, and the resulting 2V,7V-dichloro-fluorescin (DCFH) then reacts with intracellular oxidants resulting in the observed fluorescence. The

Fig. 1. a) Morphological characterization of astrocyte cell cultures. b) Immunofluorescence staining for anti-GFAP in astrocyte

cell cultures.

A. Russo et al. / Life Sciences 73 (2003) 1517–1526 1521

intensity of fluorescence is proportional to the levels of intracellular oxidant species. The addition of

SNAP to cell cultures determined a dose dependent increase in the intracellular oxidants (Fig. 2). In

particular, when the astrocytes were treated with 2 mM SNAP, the fluorescence intensity was

Fig. 2. Intracellular oxidants in purified rat astrocytes untreated and treated for 18 h with SNAP at different concentration (0.5–

1–2 mM). Methanolic extract of BM (3–6–12 Ag/ml) was added to cultures 10 min prior to addition of 1 mM SNAP. Values

are the mean F SD of four experiments performed in duplicate. xsignificant vs. control untreated cells, *significant vs. SNAP

(1 mM) treated cells (p < 0.001).

Table 1

LDH released in purified rat astrocytes untreated and treated for 18 h with SNAP at different concentration

Treatment % LDH released

Control 10 F 1.2

SNAP

0.5 mM 10 F 1.7

1 mM 11 F 1.9

2 mM 9 F 0.76

Values are the mean F SD of four experiments performed in duplicate.

A. Russo et al. / Life Sciences 73 (2003) 1517–15261522

increased approximately 28-fold compared to the values obtained in controls. The cell-free wells

containing DCFH-DA did not give fluorescence, confirming that the diacetate-containing probe

requires interaction with cells to become fluorescent (data not shown).

Table 1 reports the results of LDH release. This assay was performed to evaluate the presence of cell

toxicity as a result of cell disruption subsequent to membrane rupture. LDH release in treated cells did

not differ from controls after of 18 h exposure to 0.5–1–2 mM SNAP.

DNA damage was analyzed using the COMET assay, a sensitive method for detecting DNA strand

breaks in individual cells and a versatile tool that is highly efficacious in human bio-monitoring of plant

antioxidant (Aruoma, 1999). The results of TDNA and TMOM (Table 2) clearly evidence a dose

dependent DNA damage to cells exposed to SNAP for 18 h, in particular at the highest concentration.

SNAP at 1 mM concentration was chosen to assay the effect of methanolic extract of BM (3–6–12 Ag/ml).

When added to the cells exposed to the stressing agent, this extract significantly reduced free radical

species production (Fig. 2) and prevented DNA damage, in particular at 12 Ag/ml concentration (Table 2).

Extract of BM alone did not affect the oxidant species levels and did not damage DNA at any of the

concentrations tested (data not shown).

Table 2

Comet assay of genomic DNA of purified rat astrocytes

Treatment TL TDNA TMOM

Control 1 F 0.3 27 F 3 45 F 11

SNAP 0.5 mM 7 F 1.2* 31 F 6 42 F 15

SNAP 1 mM 13 F 1.7* 99 F 7* 1285 F 58*

SNAP 2 mM 24 F 2* 169 F 4* 1365 F 35*

SNAP 1 mM + BM 3 Ag/ml 12 F 1.7*,

B

41 F 4*,B 841 F 40*,BSNAP 1 mM + BM 6 Ag/ml 8 F 2.2*,B 60 F 5*,B 486 F 120*,BSNAP 1 mM + BM 12 Ag/ml 3 F 1.6B 32 F 3.6B 127 F 34*,B

The astrocytes were treated for 18 h with SNAP at different concentration (0.5–1–2 mM).

Methanolic extract of BM (3–6–12 Ag/ml) was added to the cultures 10 min prior to addition of 1 mM SNAP.

Values are the mean F SD of four experiments performed in duplicate.B Significant vs. SNAP (1 mM) treated cells (p < 0.001).

*Significant vs. control untreated cells.

A. Russo et al. / Life Sciences 73 (2003) 1517–1526 1523

Discussion

The interactions between NO and reactive oxygen species (ROS) have assumed considerable

importance due to the simultaneous generation of both in various pathophysiological conditions. It

has been demonstrated that low-level NO acts as an antioxidant and higher-level NO as a pro-oxidant in

the presence of ROS mediated injury. NO has been shown to be cytoprotective through its reaction with

lipid alkoxyl and peroxyl radicals and iron (Joshi et al., 1999). Moreover, it has been demonstrated that

NO upregulates heme oxygenase (hsp32) and hsp70 which may also protect against ROS insult (Joshi et

al., 1999). It may also cause cellular injury via a reaction with O2� to form peroxynitrite (ONOO�),

which is highly cytotoxic as a result of initiating free radical-mediated lipid peroxidation, sulphydryl

oxidation and DNA single strand breaks (Joshi et al., 1999). It has also been demonstrated that ONOO�

can react rapidly with CO2 to give nitrosoperoxocarbonate anion (ONOOCO2�), an oxidant and nitrating

species, increasing the toxicity associated with the presence of either O2�. or NO alone (Joshi et al.,

1999). In addition, numerous data on the biochemical reactions of nitric oxide and its derived oxidants

suggests that the nitrogen dioxide (.NO2) and carbonate anion (CO3

�. ) radicals may play a role in

various pathophysiological processes (Augusto et al., 2002). Fukuto and Ignarro have recently suggested

that the ONOO� presence in biological systems is indirect and that other mechanisms may explain NO-

mediated toxicity under oxidative conditions in vivo (Fukuto and Ignarro, 1997). The broader chemistry

of NO, in fact, involves a redox array of species with distinctive properties and reactivities: NO+

(nitrosonium), NO.(NO radical) and NO� (nitroxyl anion). Interconversion of NO+, NO

.and NO� can

take place under cellular conditions, so all three species must be considered in order to fully explain the

complex biological activity of nitric oxide (Hughes, 1999). NO� generated from Angeli’s salt has been

reported to be cytotoxic and to cause DNA damage in cultured astrocytes; it can be converted to NO.or

ONOO� by reaction with oxygen. Thus it has been suggested that also these NO-derived species can be

involved in the toxicity mediated by Angeli’s salt (Ohshima et al., 1998). NO+ is the key species in the

process of nitrosation which is important in cell biochemistry. Compounds that act as NO+ donors are

effective triggers of apoptosis in Swiss 3T3 fibroblasts and neuronal PC12 cells (Hughes, 1999).

Moreover, it has been shown that SNAP, a NO./NO + donor compromises antioxidant defense systems,

including glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) in the rat

C6 glial cell line, by decreasing levels of activity, protein and mRNA of these enzymes (Dobashi et al.,

1997). Studies by Boullerne et al., evidence that this NO donor induces toxicity in mature oligoden-

drocytes, eliciting an influx of extracellular CA++, and suggest that NO+ rather than NO. mediates the

damage, as shown by significant protection with oxyhemoglobin (HbO2), a NO./NO+ scavenger, but not

with the specific NO.scavenger carboxy-PTIO (Boullerne et al., 2001).

Under our experimental conditions, SNAP determined an increase in the intracellular oxidants, as

demonstrated by oxidation of the marker 2V,7V-dichlorofluorescein diacetate, and DNA strand breaks.

The DNA damage was evaluated by COMET assay, widely considered a versatile and highly effective

tool in biomonitoring DNA integrity (Aruoma, 1999). However, our present data indicates that NO-

induced DNA damage occurred without an increase in cellular membrane breakage, as evaluated by

percentage of LDH release, even at the highest dosage of 2 mM. These results seem to confirm an

apoptotic cell death, characterized by cell shrinkage, membrane blebbing and formation of apoptotic

bodies without membrane disruption, chromatin condensation and oligonucleosomal fragmentation of

chromosomal DNA (Steller, 1995). Moreover, recent data (Godard et al., 1999) indicates that only

comets with high values of tail moments (TMOM) can be related to apoptosis (Table 2). On the other

A. Russo et al. / Life Sciences 73 (2003) 1517–15261524

hand, our results are not in contrast with those of Stanford et al., indicating that SNAP promotes

dendritic cell apoptosis by downregulating the expression of apoptosis protein cellular inhibitors,

thereby facilitating caspase activation and subsequent poly(ADP-ribose)polymerase (PARP) cleavage

(Stanford et al., 2001).

Preliminary data in a cell-free system indicated that, like carboxy-PTIO, methanolic extract of BM was

able to reduce the plasmid DNA single-strand breakage caused by Angeli’s salt, confirming a direct NO.

scavenging activity of this plant extract (Russo et al., unpublished data). The present study indicates, that

in astrocyte cells for the first time, methanolic extract of BM can inhibit the deleterious events induced by

high concentrations of nitric oxide that may play a role in neurodegenerative events occurring during

epilepsy, cerebral ischemia or Alzheimer’s disease (Colasanti and Suzuki, 2000). In fact, the addition of

this plant extract to astrocyte cells during SNAP treatment, reduced the intracellular oxidants and

consequently prevented genomic DNA damage, in particular at higher concentration (12 Ag/ml).

The antioxidant activity of flavonoids (Rice-Evans et al., 1996; Di Carlo et al., 1999; Russo et al.,

2000) and alkaloids (Herraiz and Galisteo, 2002) is widely reported. Kim et al. have shown that

ginsenoides from Panax ginseng protect cultured rat cortical cells from glutamate-induced neuro-

degeneration, inhibiting the overproduction of nitric oxide and preserving the level of SOD (Kim et al.,

1998). It has been reported that dammarane triterpene saponin from BM is able to inhibit the superoxide

anion in polymorphonuclear cells (Pawar et al., 2001). The extract used in the present study was in crude

form and probably contained many compounds, saponins, flavonoids and alkaloids (Chatterjee et al.,

1963; Chatterjee et al., 1965; Garai et al., 1996; Chakravarty et al., 2001; Banerjee and Chakravarti,

1963; Schulte et al., 1972) which have been isolated from this plant. At this stage it is not possible to say

which of these compounds are responsible for the observed effects. However, our experimental evidence

suggests that the protective effect of BM extract and its active principles, which probably act

synergistically against NO-induced DNA damage in astrocyte cells, may be attributable to their

superoxide anion, hydroxyl anion (Russo et al., in press) and NO.(Russo et al., unpublished data)

scavenging activity, and probably to their capacity to promote the enzymatic antioxidant systems, acting

at transcriptional level and protecting them from the nitrosation process. It was suggested that bacosides

induce membrane dephosphorylation, with a concomitant increase in protein and RNA turnover in

specific brain areas (Singh et al., 1990). Bhattacharya et al. report that extract of BM, rich in saponins, is

able to induce a dose-related increase in SOD, CAT and GSH-Px activities in rat frontal cortex, striatum

and hippocampus (Bhattacharya et al., 2000). In addition, recent studies by Dar and Channa which report

a calcium antagonistic activity of this Indian medicinal plant (Dar and Channa, 1999), suggest that the

capacity to contrast alterations in calcium homeostasis could contribute to neuroprotective effects

exhibited by Bacopa monniera.

In conclusion, data presented here supports the traditional use of BM and may explain, at least in part,

the cognition-facilitating and anti-aging effects reported in experimental animals (Bhattacharya et al.,

1998). Moreover, our results suggest that because of its ability to reduce NO-induced cellular alterations,

this Indian medicinal plant has a therapeutic potential in treatment or prevention of neurological diseases

and may justify further investigation of its other beneficial biological properties.

Acknowledgements

The authors would like to thank Dr. M. Wilkinson for proofreading the manuscript.

A. Russo et al. / Life Sciences 73 (2003) 1517–1526 1525

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