Protective role of maize purple plant pigment against oxidativestress in fluorosis rat brain

Boyan Li; Keyana Nozzari Varkani; Lu Sun; Bo Zhou; Xiaohong Wang; Lianying Guo; Han Zhang; Zhuo Zhang

doi:10.1515/tnsci-2020-0055

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Protective role of maize purple plant pigment against oxidativestress in fluorosis rat brain

Boyan Li , Keyana Nozzari Varkani , Lu Sun , Bo Zhou , Xiaohong Wang , Lianying Guo , Han Zhang and Zhuo Zhang

Published/Copyright: April 21, 2020

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From the journal Translational Neuroscience Volume 11 Issue 1

Abstract

In fluorosis-endemic areas, exposure to high levels of fluoride causesneurotoxicity such as lowered intelligence and cognitive impairment. Oxidativedamage is critical to pathophysiologic processes of fluoride intoxication, andneurotoxicity of fluoride may be associated with oxidative stress. In previousstudies, maize purple plant pigment (MPPP), which was rich in anthocyanins,showed a strong scavenging activity in vitro and invivo. The present study aimed to determine whether treatment withMPPP can alleviate fluoride-induced oxidative damage in rat brain. After 3months of experiment, brain tissues were assayed for oxidative stress variables,histological and Western blotting examinations. Our results showed that MPPPreduced the elevated malondialdehyde levels, increased superoxide dismutaseactivity, and further attenuated histopathological alterations and mitigatedneuronal apoptosis. Importantly, MPPP also reversed changes in Bax and Bcl-2.Therefore, it was speculated that MPPP protects brain tissue from fluoridetoxicity through its antioxidant capacity.

Keywords: fluoride; oxidative stress; anthocyanins; rats

1 Introduction

Given the widespread presence of fluorine in the natural environment, individuals areexposed to fluoride via food intake, inhalation, and dermal contact. Drinking waterrepresents the largest exposure source. In particular, in highly fluoridated regionsand in some developed areas that fluoridate the public water supply to reduce dentalcaries, fluoride may result in a health hazard [1,2].

Fluoride is required for normal growth and development of teeth and bones but canlead to fluorosis if taken excessively. Specifically, fluorosis can adversely affectthe skeleton and teeth, and may induce structural and functional changes in softtissues including brain tissue [3]. Epidemiological data show thatchronic exposure to high fluoride in water is closely associated with a lowerintelligence quotient in children [4,5,6]. In fluorosis-endemic areas, a certainhigh dose of fluoride intake is a potential risk factor for cognitive impairment inelderly people [7].Moreover, structural changes in nerve cells and brain functions in experimentalanimals subjected to chronic fluorosis have been described such as nuclearshrinkage, mitochondrial swelling, neurodegeneration, and deterioration of learningand memory [8,9,10]. These findings suggest a direct linkbetween excessive exposure to fluoride and brain function impairment, but little isknown about mechanisms underlying these phenomena.

Oxidative stress-induced neurotoxicity is considered a mechanism of brain impairmentcaused by fluorosis. Once fluoride has formed lipid-soluble complexes in the blood,it can cross the blood–brain barrier, penetrate brain cells, and accumulatein brain tissue, causing detrimental neurological effects [11]. Reactive oxygen species (ROS) andfree radicals can be generated when the fluoride content is high in the brain andcause oxidative damage and cell apoptosis in neurons [12], which may be controlled byapoptosis-related genes [13,14,15]. The literaturesuggests that increased ROS and lipid peroxidation (LPO) and decreased antioxidantenzyme activity occur in the brains of fluoride-intoxicated rats and thathistopathological changes can be observed, especially swelling of mitochondria andendoplasmic reticulum dilation in neurons [9,16]. Also, some studies confirm thatspecific antioxidants may protect against this damage [17].

Anthocyanins, the largest group of water-soluble pigments responsible for fruit andvegetable color, are flavonoids reputed to have biological antioxidant activity dueto their capacity as hydrogen donors [18]. They can also stabilize anddelocalize unpaired electrons, and their ability to chelate transition metal ionsmay be useful [19].Anthocyanin-rich maize purple plant pigment (MPPP) extracted from maize purple planthas been said to have antioxidant traits [3,20], but few reports of MPPP influoride-treated rat brains exist. Thus, we studied MPPP and any potentialneuroprotective effects against fluoride toxicity.

2 Materials and methods

2.1 Chemicals and reagents

Sodium fluoride (NaF, molecular weight 41.99) was procured from Sigma Chemical(St. Louis, MO, USA). Anti-Bax and anti-Bcl-2 antibodies were obtained fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). All other laboratory reagentsused were of analytical grade and obtained from Sigma, Invitrogen (Carlsbad, CA,USA) and Sangon Biotech Co., Ltd (Shanghai, China). MPPP extracted and separatedfrom maize purple plant was produced by Liaoning Dongya Seeds Co., Ltd(Shenyang, China). In our previous study, we confirmed that MPPP mainly contains45.96% cyanidin-3-glucoside, 12.99% 3′,4′-dihydroxyanthocyanin-3-glucoside, and 26.16% four other kinds of anthocyanins [21]. MPPP mixed withthe standard rodent diet was obtained from Shenyang Qianmin Animal Feeds Factory(Shenyang, China).

2.2 Animals and treatment

Eighty healthy weanling Wistar rats (50% male) were acclimated for 1 week beforeexperiments and fed a common basal pellet diet and water ad libitum. The ratswere randomized into four groups (N = 20/group) by body weightstratification. Group I (controls) received tap water and a common basal pelletdiet for 12 weeks. Group II (fluoride-treated rats) received 100 ppmfluoride ion (F⁻) in their drinking water and a common basalpellet diet for 12 weeks. Group III (experimental rats co-treated with fluorideand MPPP) received 100 ppm F⁻ in their drinking waterand pellet diet mixed with 5 g/kg MPPP for 12 weeks. Group IV(experimental rats co-treated with fluoride and MPPP) received 100 ppmF⁻ in drinking water and a pellet diet mixed with10 g/kg MPPP for 12 weeks. During treatment, daily water consumption,animal feed consumption and weight gain were recorded periodically. Fluoride andMPPP intake was calculated according to weekly average water and animal feedconsumption. All rats were kept in ventilated cages at 23–27°C,with 55–60% humidity and 12/12 h light/dark cycles. After 12weeks, the treatments were ended, and the rats were killed under light etheranesthesia.

All brain tissues were dissected carefully and blotted free of blood, and theirfresh weight was recorded. Brain somatic indices were calculated asg/100 g weight by the following formula: fresh weight of the brain/weightof the body × 100. Ten brain tissue samples were selected randomly fromeach group for the F⁻ assay and Western blotting; two otherbrain tissue samples from each group were fixed in 2.5% glutaraldehyde forultrastructural examination. The remaining brain tissues were homogenized inchilled potassium chloride and centrifuged at 3,000 × gfor 10 min at 4°C. The supernatant was used for biochemicalanalysis.

Ethical approval: This research related to animal usecomplied with the Guidelines for the Care and Use of Laboratory Animalsof the China National Institute of Health. The experimental protocolswere approved by the Ethics Committee for Animal Experiments of ShenyangMedical College (permit number S-2013-006). All surgeries were performedunder light ether anesthesia, and all efforts were made to minimizesuffering.

2.3 Determination of fluoride

Following published methods [22], brain tissue samples(50 mg) digested with lipase and protease were dissolved in an acidmixture (nitric acid and silver nitrate) in a closed compartment, which wasoverlaid with saturated sodium hydroxide. After neutralization for 24 h,fluorine reagent was added into the mixture, and F⁻ in brainswas calculated from a standard curve. Data were expressed as μgF⁻/kg brain tissue.

2.4 Ultrastructure of brain

Brain tissues were fixed with 2.5% glutaraldehyde for 2 h and 1% osmiumtetroxide for another 2 h. Subsequently, samples were dehydrated througha graded ethanol series and embedded in Spurr’s resin. Ultrathin sectionswere cut and stained with uranyl acetate and lead citrate and then observed andphotographed using a Hitachi H-7650 (Hitachi Ltd, Tokyo, Japan) transmissionelectron microscope.

2.5 Brain tissue oxidative stress markers

LPO was assessed via malondialdehyde (MDA) in rat brains. MDA, glutathione (GSH),glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) activity inbrain tissue was assayed using commercial kits (Jiancheng BioengineeringInstitute, Nanjing, China). Total proteins were measured using the Bradfordassay to normalize MDA, GSH, GSH-Px, and SOD [23]. Data are expressed as nmol/mgprotein for MDA, U/mg protein for SOD and GSH-Px, and mg/g protein for GSH inbrain tissues.

2.6 Bax and Bcl-2 expression in rat brains

Frozen brain tissue samples were placed in ice-cold lysis buffer, homogenized atlow temperature, and then centrifuged at 4°C at 12,000 ×g for 25 min. Protein in the supernatant wasquantified using the protein assay kit. Lysates with equal amounts of proteinwere separated on 10% SDS-PAGE and electrotransferred to a polyvinylidenedifluoride membrane (Millipore, Bedford, MA, USA), which was blocked with 5%non-fat dried milk in Tris-buffered saline with Tween 20 for 1.5 h atroom temperature. Thereafter, membranes were incubated with primary antibodiesagainst Bcl-2 and Bax (1:1,000) overnight at 4°C. Next, horseradishperoxidase-conjugated secondary antibody (1:6,000) was applied for 1 h atroom temperature. After rinsing with buffer, protein bands were visualized withan enhanced chemiluminescence reagent and analyzed by Gel-Pro Analyzer software.β-Actin was used as a protein-loading control.

2.7 Statistical analysis

All data were analyzed using SPSS v17.0 software (SPSS Inc., Chicago, IL, USA)and analyzed by one-way analysis of variance followed by Dunnett’s testto compare mean values between different treatment groups. Experimental resultsare expressed as mean ± standard error of mean (SEM), andp < 0.05 was considered to be statisticallysignificant.

3 Results

No clinical signs of toxicity were observed in any group of rats throughout thedosing period of 12 weeks. Body weight in fluoride-treated animals decreasedslightly as shown in Figure1. Brain fluoride in fluoride-treated groups increased significantlycompared with controls, and MPPP at both doses reduced this fluoride but notsignificantly. The brain somatic index showed no significant differences among allof the groups (Table1).

Figure 1

Effects of fluoride and MPPP on rat weight. Data are mean values of 20replicates.

Table 1

Levels of fluoride in brain and brain somatic index of rats(x ± SEM)

Groups	Levels of fluoride (μg/kg)	Brain somatic index (g/100 g)
Group I	210.28 ± 53.25	0.64 ± 0.04
Group II	800.21 ± 79.82*	0.67 ± 0.03
Group III	698.65 ± 111.32*	0.66 ± 0.03
Group IV	600.16 ± 37.40*	0.69 ± 0.03

Note: *p < 0.05 compared with the control group(group I). N = 10 in each group for levels of fluoride;N = 20 in each group for measurement of the brainsomatic index.

3.1 Ultrastructural observation of brain

Ultrastructural analysis of the experimental rat brains is shown in Figure 2. For controls(Figure 2a), oneoval nucleus with visible, clear nucleoli and double nuclear membranes, abundantmitochondria, and endoplasmic reticulum were found in neurons. Influoride-treated rats (Figure 2b), nerves were deformed, lacked a nuclear membrane, and hadchromatin condensation, swollen mitochondria, and broken cristae, and evidenceof apoptosis was present. In rats treated with fluoride and MPPP, brain cellshad swollen mitochondria but fewer abnormal mitochondria compared to group II,and pathological nuclear changes were reduced (Figure 2c and d).

$Figure 2 Ultrastructure of nerve cells in rats. (a) Controls; (b) rats fed100 mg/L fluoride; (c) rats fed 100 mg/L fluoride plus5 g/kg MPPP; and (d) rats fed 100 mg/L fluoride plus10 g/kg MPPP. Nu, nucleolus; NM, nuclear membrane; MD, membranedissolution; MS, mitochondrial swelling; CC, chromatin condensation; andMCF, mitochondrial crest fracture. Bar = 2 µm.$

Figure 2

Ultrastructure of nerve cells in rats. (a) Controls; (b) rats fed100 mg/L fluoride; (c) rats fed 100 mg/L fluoride plus5 g/kg MPPP; and (d) rats fed 100 mg/L fluoride plus10 g/kg MPPP. Nu, nucleolus; NM, nuclear membrane; MD, membranedissolution; MS, mitochondrial swelling; CC, chromatin condensation; andMCF, mitochondrial crest fracture. Bar = 2 µm.

3.2 Oxidation in rat brains

MDA, GSH, SOD, and GSH-Px activities were assessed, and the MDA level wassignificantly greater for the fluoride-treated rats than for the controls. MPPP(5 g/kg) reduced the elevated MDA after fluoride, and SOD increased(groups III and IV) compared with rats treated with fluoride alone(p < 0.05), as shown in Figure 3. GSH-Px activity and GSHwere only slightly increased in groups III and IV compared with thefluoride-treated rats group (data not shown).

Figure 3

LPO and antioxidant status in rat brains. (a) LPO production (MDA) and(b) SOD. *p < 0.05 compared with group I;#p < 0.05 compared with group II.

3.3 Bax and Bcl-2 expression in rat brains

Bax and Bcl-2 protein expression in brains as measured by Western blotting (Figure 4) showed thatBax increased in fluoride-treated rats compared with controls and also decreasedat both doses of MPPP. Bcl-2 protein expression in rat brains after MPPPtreatment was significantly elevated compared with fluoride-treated rats.

Figure 4

Bcl-2 and Bax protein in rat brains. (a) Western blot and (b) therelative densitometry of the bands. Relative protein expression wasnormalized to β-actin. Bars are mean ± SEM.*p < 0.05 compared with group I,#p < 0.05 compared with group II.

4 Discussion

Because soluble fluoride is absorbed easily from the gastrointestinal tract, highconcentrations of fluorine from drinking water may accumulate, but only thewater-soluble fluoride ion is relevant to human health [2]. Neurotoxic severity chiefly dependsupon the content of fluoride in drinking water when exceeding the WHO-recommendedvalue of 1.50 ppm [24,25].

Chronic ingestion of high concentrations of fluoride can induce excessive productionof oxygen free radicals with subsequent LPO in soft tissues [26]. Oxidative imbalance due to increasedfree radicals can cause oxidative damage in fluoride-intoxicated animals [27], especially thebrain, which has a high content of polyunsaturated fatty acids. Children from areasof endemic fluorosis have lower intelligence and elevated oxidative stress status,as measured by increased MDA [28]. MDA, an aldehydic product of membrane LPO, is often used as amarker of oxidative stress in tissues. We noted that MDA in brain tissues influoride-treated rats was significantly higher than in controls, as noted inprevious studies [29,30].Excessive fluoride intake can promote oxidative stress and disturb the antioxidantdefense system in brains of fluoride-intoxicated rats, so it has been suggested thatantioxidants (vitamins, resveratrol, and anthocyanins) and antioxidant-rich foods(such as rhodiola) may be useful for reducing such damage [17,31,32,33].

Anthocyanins, water-soluble natural plant pigments, have been reported to haveantioxidant effects [18]. Furthermore, one study suggests that consumption of anthocyanins canreduce free radicals in the body [34], likely via scavenging superoxideanion radicals [35],inhibiting LPO, and interfering with hydroxyl radical-generating systems [36]. Anthocyanin-richMPPP appeared to have antioxidant properties as we observed that MPPP (5 g/kgfeed) significantly reduced MDA in brains of fluoride-intoxicated rats, and SODactivity in rat brains after MPPP treatment (5 and 10 g/kg feed) wassignificantly elevated. Thus, MPPP may capture free radicals and enhance endogenousantioxidant activity.

Chronic fluorosis can cause brain structural and functional changes via oxidativestress after fluoride exposure [8,9,10], and we noted histological changes inthe brains of fluoride-treated rats characterized by cell nucleus deformation,chromatin condensation, and swollen mitochondria and typical morphologicalmanifestations of apoptosis. Similar observations were made by others who reportedcytomorphosis, intranuclear heterochromatin margination condensation, and shrinkageof the nucleus in the brains of fluoride-intoxicated mice [37]. MPPP may have alleviated the harmfuleffects of fluoride by increasing SOD activity and reducing MDA. Neuronal apoptosis,which has been reported in the presence of relatively high fluoride, was allegedlydue to increased oxidative stress [13]. Apoptosis can be coordinatelycontrolled by gene expression, so we measured the apoptosis-promoting Bax proteinand the inhibitory Bcl-2 protein, which are abundant in mitochondria, the nuclearmembrane, and endoplasmic reticulum. Others reported that Bax protein expression issignificantly upregulated in the brains of fluoride-treated rats, and a negativecorrelation was observed between fluoride concentrations in water and expression ofBcl-2 [37]. We reportthat Bax protein expression was significantly increased and expression of Bcl-2protein decreased in the brains of fluoride-treated rats. Also, MPPP prevented thedecrease in Bcl-2 and the increase in Bax expression after fluoride treatment,suggesting that MPPP alleviates apoptosis-mediated impairments.

MPPP did not modify fluoride ions in brain tissues; thus, reducing fluoride exposureis not a mechanism underlying its purported ability to reduce fluoride toxicity.

5 Conclusions

Oxidative stress plays a role in fluoride-induced toxicity and provokes pathologicalchanges and neuronal apoptosis in rat brains. Anthocyanin-rich MPPP may restorebrain health via its antioxidant properties. However, further research is requiredto understand how MPPP may be neuroprotective.

Phone: +86-24-62215717, Fax: +86-24-62215749, zhangzhuo@symc.edu.cn

Acknowledgments

This study was supported by the Department of Science and Technology of LiaoningProvince (grant number 2019-MS-306) and Shenyang Bureau of Science and Technology(grant number 18-013-0-48).

Conflict of interest: The authors state no conflict ofinterest.

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Received: 2019-08-28

Revised: 2020-02-19

Accepted: 2020-02-21

Published Online: 2020-04-21

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tnsci-2020-0055

Keywords for this article

fluoride; oxidative stress; anthocyanins; rats

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