Ethanol and age enhances fluoride toxicity through oxidative stress and mitochondrial dysfunctions in rat intestine

Ethanol and age enhances fluoride toxicity through oxidativestress and mitochondrial dysfunctions in rat intestine

Shailender Singh Chauhan • Akhtar Mahmood •

Sudarshan Ojha

Received: 8 June 2013 / Accepted: 30 August 2013 / Published online: 11 September 2013

� Springer Science+Business Media New York 2013

Abstract Fluoride toxicity and alcohol abuse are the two

serious public health problems in many parts of the world.

The current study was an attempt to investigate the effect

of alcohol administration and age on fluoride toxicity in rat

intestine. Six and 18 months old female Sprague Dawley

rats were exposed to sodium fluoride (NaF, 25 mg/kg),

30 % ethanol (EtOH, 1 ml/kg), and NaF?EtOH (25 mg/

kg?1 ml/kg) for a period of 20, 40, and 90 days. The

levels of lipid peroxidation were increased, while the

content of reduced glutathione, total, and protein thiol was

decreased with NaF treatment. Under these conditions,

animals showed an age-related decline in the activities of

superoxide dismutase, catalase, glutathione peroxidase,

glutathione reductase, and glutathione-S-transferase which

were further aggravated upon NaF or/and EtOH treatment.

Mitochondrial respiration rate and the activities of com-

plexes I, II, and IV enzymes of electron transport chain

were decreased, while the levels of nitric oxide and cit-

rulline were increased with age and NaF or/and EtOH

treatment. Histological examination revealed large reactive

lymphoid follicles, excess of lymphocytes in lamina pro-

pria of villi, villous edema, focal ileitis, necrosis of villi,

and ulceration in NaF- or/and EtOH-treated animals in both

the age groups. These findings suggest that fluoride medi-

ate its toxic effects on intestine through oxidative stress and

mitochondrial dysfunctions which are further augmented

with alcohol consumption and advancing age.

Keywords Ethanol � Fluoride � Oxidative stress �Mitochondrial functions

Introduction

Humans are exposed to fluoride inevitably because it is a

ubiquitous contaminant of the environment. Among vari-

ous sources, drinking water is the highest contributor of

fluoride to humans [1]. The toxic effects of fluoride are not

restricted to bone and teeth, but it also targets soft tissues,

including gastrointestinal tract [2]. The mammalian small

intestine is exposed to fluoride concentrations several times

higher than those attained in other tissues. In addition,

concurrent exposures to fluoride and other xenobiotics may

further influence their toxic effects on intestine by under-

going some antagonistic or synergistic interactions. Alco-

hol consumption is common among human population

across the globe, but little attention has been paid to

evaluate toxic effects of fluoride together with ethanol

ingestion. Interactions between fluoride and ethanol are an

important problem in modern toxicology since both pose a

risk to human and animal health. Co-exposures to fluoride

and ethanol are common among alcoholics residing in high

fluoride endemic areas.

A growing body of evidences suggests that exposure to

fluoride or ethanol cause toxic effects by generating reactive

oxygen species [3–5]. Free radical generation, lipid peroxi-

dation, and changes in the antioxidants have been reported in

the intestine of animals treated with fluoride [6] or ethanol

[7]. Inkielewicz et al. [8] showed that fluoride-induced lipid

peroxidation in liver, kidney, brain, and serum of male rats is

S. S. Chauhan (&)

Department of Gastroenterology, Post Graduate Institute of

Medical Education and Research (PGIMER), Sector-12,

Chandigarh 160012, India

e-mail: [email protected]

A. Mahmood � S. Ojha

Department of Biochemistry, Panjab University,

Chandigarh 160014, India

123

Mol Cell Biochem (2013) 384:251–262

DOI 10.1007/s11010-013-1804-6

enhanced in the presence of ethanol. We have previously

reported for the first time that fluoride and ethanol have

synergistic effects on lipid peroxidation and antioxidant

defense systems in rat intestine [9]. However, the effects of

fluoride together with ethanol on mitochondria in intestine

are not yet reported. Impairment in mitochondrial functions

leads to increased production of free radicals (superoxide

and hydroxyl radicals) and is associated with many chronic

degenerative diseases [10]. Fluoride is known to interfere

with cellular energy production by inhibiting enzymes of

electron transport chain through the effect of peroxynitrite

[3, 11]. Vani and Reddy [12] reported suppression of both

antioxidant and energy producing enzymes in female mice

treated with 20 mg of fluoride/kg body weight for 14 days.

Similarly, ethanol affects mitochondrial functions and cause

damage to its DNA. Hoek et al. [13] showed that ethanol-

induced mitochondrial DNA damage, if not adequately

repaired, impairs mitochondrial functions, which further

increases oxidative stress in the cell. Taking these aspects

into consideration and the fact that little is known, we

investigated combined effects of these toxicants on mito-

chondrial functions and oxidative stress in rat intestine.

Materials and methods

Chemicals

Sodium fluoride (NaF) and ethanol (EtOH) were procured

from Sisco Research Laboratories (SRL) Pvt. Ltd. Mum-

bai, India and Changshu Yangyuan Chemicals, China,

respectively. All other analytical grade chemicals and

reagents were purchased from Merck (Germany), Sigma or

SRL Chemicals (India). Ultrapure water prepared by lab-

PURE-Series Analytica6 & Ultraplusuf (BIO-AGE, Mo-

hali, India) was used throughout the experimental period.

Animals

Six- and 18-month-old female Sprague Dawley rats

weighing 200–220 and 280–300 g, respectively, were

procured from the Central Animal House of Panjab Uni-

versity, Chandigarh, India. They were housed in propylene

cages and maintained at 22� ± 3 �C, on a 12:12 h light

dark cycle and a minimum 40 % RH. Standard pellet diet

(Ashirwad Industries, India) and water were given ad libi-

tum. After 1 week of acclimatization, animals from each

age group were separately subjected to random group

division; i.e., Control (untreated); NaF-treated (25 mg/kg);

30 % EtOH-treated (1 ml/kg); and NaF?EtOH co-treated

(a combination of NaF and EtOH as mentioned above).

There were 12 animals in each group and all the treatments

were given orally using Ryle’s tube daily at 9 am for

90 days. Body weight gain along with food and water

intake was recorded during the experimental period. The

experimental protocol was approved by the Institute’s

Ethical Committee, in accordance with the guidelines

issued for the use of laboratory animals. Four overnight-

fasted 6- or 18-month-old rats from each group were

euthanized after 20, 40, and 90 days under light ether

anesthesia. Intestine was removed, rinsed with ice-cold

isotonic saline (0.9 % w/v NaCl) and processed for the

preparation of tissue homogenate and post-mitochondrial

supernatant (PMS) as described previously [9].

Isolation of mitochondria

Intestinal mitochondria were isolated [14]. Briefly, 10 %

(w/v) tissue homogenate was prepared in buffer A (0.44 M

sucrose, 10 mM Tris, 10 mM EDTA, 0.1 % BSA, pH 7.4)

using mechanically driven Teflon-fitted Potter-Elvehjam

type homogenizer and centrifuged at 6009g for 15 min.

The pellet was discarded and supernatant was further

centrifuged at 14,0009g for 15 min. The crude mito-

chondrial pellet was separated and washed with buffer A

and spun at 7,0009g for 15 min. The final mitochondrial

pellet was suspended in 5 ml of buffer B (0.44 M sucrose,

10 mM Tris–HCl, pH 7.4).

Oxidative stress

Parameters relating to oxidative stress including lipid per-

oxidation (LPO); reduced glutathione (GSH); total (T-SH)

and protein thiols (Pt-SH); superoxide dismutase (SOD);

catalase (CAT); glutathione peroxidase (GPx); glutathione

reductase (GR); glutathione-S-transferase (GST); and pro-

tein concentration were assayed using standard protocols as

described previously [9].

Mitochondrial functions

NADH dehydrogenase (complex I)

Requisite amount of mitochondrial preparation was added

to the reaction mixture containing 0.2 M glycyl glycine

buffer (pH 8.5), 6 mM NADH, 1 mM cytochrome Coxidized

and 0.02 M NaHCO3. The increase in the absorbance was

followed at 550 nm for 3 min. The results were expressed

as nmoles NADH oxidized/min/mg protein using molar

extinction coefficient of reduced cytochrome C at 550 nm

(19.6 mM-1 cm-1) [15].

Succinate dehydrogenase (complex II)

The reaction mixture contained 0.2 M sodium phosphate

buffer (pH 7.8), 1 % (w/v) BSA, 0.6 M sodium succinate

252 Mol Cell Biochem (2013) 384:251–262

123

(prepare freshly), and 0.03 M potassium ferricyanide (amber

bottle). The reaction was initiated by adding suitable amounts

of mitochondrial preparation. The change in absorbance was

read at 420 nm for 3 min. The results were expressed as

nmoles succinate oxidized/min/mg protein [16].

Cytochrome oxidase (complex IV)

Oxidized cytochrome C (0.3 mM) was reduced by the

addition of sodium borohydride and then neutralized to pH

7.0 by 0.1 N HCl. 0.3 mM reduced cytochrome C was

added to 0.075 M sodium phosphate buffer pH 7.4 and the

reaction was initiated by the addition of appropriate

amount of mitochondrial preparation. The decrease in

absorbance was followed at 550 nm for 3 min. The results

were expressed as nmoles cytochrome c oxidized/min/mg

protein using molar extinction coefficient of reduced

cytochrome C at 550 nm (19.6 mM-1 cm-1) [17].

Nitric oxide (NO)

Briefly, to the 100 ll of the sample, 200 ll of Griess

reagent [prepared by mixing fresh solution of n-napthyl

ethylenediamine dihydrochloride (0.15 % solution) in dis-

tilled water and sulfanilamide (1.5 % solution) in 1 N HCl

in a ratio of 1:2] was added in the wells of the ELISA strip.

After keeping the ELISA strip in dark for 30 min, the pink

color so observed was read for its absorbance at 540 nm on

an ELISA reader. Concentrations were determined from a

linear standard curve prepared by using sodium nitrite

(1.25–10 nmol). The results were expressed as nmoles of

NO2 accumulated in the sample/well [18].

Citrulline

To the 200 ll of sample, 50 ll of 30 % ZnSO4 solution was

added and mixed well to precipitate proteins and the contents

were centrifuged. 20 ll of the supernatant was diluted to

480 ll with 0.1 N HCl and 1.5 ml of chromogenic solution

[freshly prepared by mixing 85 % H3PO4 and 0.5 % (w/v)

diacetylmonoxime ? 0.1 % (w/v) thiosimocarbazide solu-

tions in the ratio of 2:1 immediately before use] was added

and vortexed. The mixture was kept in boiling water bath for

5 min. After cooling, the absorbance was measured at

530 nm. Blank and standards (1 mM L-citrulline) were run

simultaneously. L-Citrulline was used as a standard to cal-

culate the citrulline levels and results were expressed as lM

of citrulline/mg of protein [19].

Mitochondrial respiration rate

Reduction of MTT 3-(4,5-dimethylthiazolyl-2)-2,5-

diphenyltetrazolium bromide was used to assess the

activity of the mitochondrial respiratory chain activity in

mitochondria [20]. The reaction mixture containing

mitochondrial preparation (50 lg protein) and MTT

(0.1 mg/ml) was incubated at 37 �C for 30 min and then

centrifuged to form formazan pellet. The pellet was

dissolved in 1 ml of absolute ethanol and the mixture

was re-centrifuged and absorbance of the supernatant

was measured at 570 nm. Results were expressed as lg

formazan formed/min/mg protein by using blue formazan

as standard.

Tissue morphology

A portion of the intestine was fixed in 10 % formaldehyde

for histological studies. After fixation, tissues were

embedded in paraffin; solid sections were cut at 5 lm and

stained with hematoxylin and eosin. The sections were

examined under light microscope and photomicrographs

were taken.

Statistical analysis

All grouped data were statistically evaluated with SPSS/

14.0 software package program for windows. Hypothesis

testing methods included two-way analysis of variance

(ANOVA) followed by least significant difference (LSD)

and post hoc Dunnett test. p \ 0.05 were considered to

indicate statistical significance. All the results were

expressed as mean ± SD for four animals in each group.

Results

Effect on body weight gain

Exposure to NaF or EtOH, separately or together produced

a significant reduction in the body weight gain (Fig. 1a).

The observed decline in body weight gain of treated groups

was evident from day 28 and 49 onwards in 6- and

18-month-old animals, respectively. In 6-month-old rats,

the gain in body weight after 90 days was highest (48 %)

in control group and lowest (10 %) in NaF and EtOH co-

treated group. However, administration of NaF or EtOH

alone, showed 30 and 16 % gain in the body weight,

respectively (Fig. 1a). In 18-month-old animals, the gain in

body weight was continuous for control (32 %) and NaF

(22 %) treated groups throughout the experimental dura-

tion. Exposure to EtOH alone or together with NaF showed

a sharp decline in the body weight gain from day 63

onwards. After 90 days, there was 14 and 11 % gain in

body weight of EtOH and NaF?EtOH treated animals,

respectively (Fig. 1a).

Mol Cell Biochem (2013) 384:251–262 253

123

Effect on food intake

Food intake in treated animals was significantly decreased

as compared to controls (Fig. 1b). In 6-month-old animals

a time-dependent decline in food intake was observed from

day 28, 35, and 49 onwards in NaF?EtOH, EtOH, and NaF

treated groups, respectively. After 90 days, food intake was

increased by 22 % in control animals, whereas it was

decreased in NaF (29 %), EtOH (21 %), and NaF?EtOH

(36 %) treated rats (Fig. 1b). A similar trend in food intake

was revealed by 18-month-old animals receiving NaF or

EtOH alone, or in combination. However, the observed

decline was evident from day 14 onwards in treated groups,

compared to controls. After 90 days, control animals

exhibited 45 % increase in food intake while rats treated

with NaF, EtOH, and NaF?EtOH showed 37, 34, and

40 % decrease, respectively (Fig. 1b).

Effect on water intake

There was a significant reduction in water intake in 6- and

18-month-old animals from day 7 onwards (Fig. 1c). In

6-month-old animals, water intake recorded after 90 days

showed 16 % increase in control group followed by 35, 23,

and 24 % reduction in NaF, EtOH, and NaF?EtOH treated

groups, respectively (Fig. 1e). Water intake exhibited by

18-month-old control animals after 90 days was 20 %

high, compared to initial values. Individual exposure to

EtOH for 90 days showed 26 % reduction in water intake

while animals receiving NaF alone or together with EtOH

resulted in 31–32 % decline in water consumption under

these conditions (Fig. 1c).

Effect on non-enzymatic parameters relating

to oxidative stress

The levels of lipid peroxidation marker malondialdehyde

(MDA) were high and T-SH, GSH, and Pt-SH content were

low in 18-month-old animals than in 6-month-old rats

(p \ 0.05). Exposure to NaF or EtOH, separately or toge-

ther resulted in a significant elevation in MDA levels along

with reduced T-SH, GSH, and Pt-SH content in the intes-

tine of 6- and 18-month-old rats (Table 1). After 90 days,

MDA levels in rats receiving NaF or EtOH alone were

increased by 212 and 234 %, respectively, in 6-month-old

animals and by 225 and 269 %, respectively, in 18-month-

old rats. However, MDA levels following administration of

NaF together with EtOH for 90 days showed 363 %

increase in 6-month-old and 379 % in 18-month-old

animals.

At the end of the experimental period NaF-treated ani-

mals showed 29 and 44 % reduction in the intestinal T-SH

content in 6- and 18-month-old rats, respectively. Admin-

istration of EtOH decreased the levels of T-SH in 6-month-

Fig. 1 Effect of chronic fluoride and ethanol co-exposure on body

weight (a), food intake (b), and water intake (c) in 6- and 18-month-

old rats. Values are mean ± SD (n = 4); * p \ 0.05 versus control

group; � p \ 0.05 versus NaF treated group; # p \ 0.05 versus EtOH

treated; a p \ 0.05 versus 20 days; b p \ 0.05 versus 40 days;c p \ 0.05 versus 6-month old

254 Mol Cell Biochem (2013) 384:251–262

123

old (48 %) and 18-month-old (60 %) rats under these

conditions. However, rats receiving NaF together with

EtOH for 90 days showed 54 and 77 % decline in the

intestinal T-SH content in 6- and 18-month-old animals,

respectively.

After 90 days, GSH levels in NaF-treated rats were

decreased by 53 % in 6-month-old and by 63 % in

18-month-old animals. EtOH feeding for 90 days produced

46 and 50 % reduction in the intestinal GSH content in 6-

and 18-month-old animals, respectively. However, the

observed decline in the tissue GSH content in rats receiving

NaF together with EtOH for 90 days was 58 % in 6-month-

old and 69 % in 18-month-old animals.

Exposure to NaF for 90 days resulted in 27 and 42 %

decrease in the intestinal Pt-SH content in 6- and

18-month-old animals, respectively. EtOH feeding under

these conditions reduced the levels of Pt-SH in the intestine

of 6-month-old (49 %) and 18-month-old (61 %) old rats

after 90 days. However, rats co-exposed to NaF and EtOH

for 90 days exhibited 54 and 78 % reduction in the intes-

tinal Pt-SH content in 6- and 18-month-old animals,

respectively.

Effect on enzymatic parameters relating to oxidative

stress

The activities of enzymes (SOD, CAT, GPx, GR, and GST)

relating to oxidative stress in intestine were low (p \ 0.05)

in 18-month-old animals than in 6-month-old rats. Expo-

sure to NaF or EtOH, separately or together resulted in a

significant decline in the activities of these enzymes in both

the age groups (Table 2). The observed decrease in SOD

activity after 90 days of NaF treatment was 65 % in

6-month-old and 73 % in 18-month-old rats. Exposure to

EtOH for similar experimental duration reduced the intes-

tinal SOD activity by 61 % in 6-month and by 71 % in

Table 1 Effect of chronic fluoride and ethanol co-exposure on non-enzymatic parameters relating to oxidative stress in intestine of 6- and

18-month-old rats

Parameter Age

(months)

Treatment

(days)

Groups

Control NaF treated EtOH treated NaF?EtOH treated

LPO 6 20 0.31 ± 0.03 0.55 ± 0.04* 0.64 ± 0.04* 0.93 ± 0.07*�#

40 0.44 ± 0.06a 1.27 ± 0.06*a 1.43 ± 0.05*�a 1.94 ± 0.08*�#a

90 0.52 ± 0.02ab 1.62 ± 0.03*ab 1.73 ± 0.04*�ab 2.40 ± 0.04*�#ab

18 20 0.91 ± 0.03c 1.83 ± 0.02*c 2.20 ± 0.07*� 3.06 ± 0.05*�#

40 1.39 ± 0.03ac 4.19 ± 0.08*ac 4.66 ± 0.34*�ac 5.90 ± 0.06*�#ac

90 1.82 ± 0.05abc 5.92 ± 0.07*abc 6.73 ± 0.08*�abc 8.74 ± 0.13*�#abc

T-SH 6 20 14.90 ± 0.11 11.70 ± 0.11* 9.54 ± 0.06*� 8.28 ± 0.17*�#

40 14.40 ± 0.14a 10.80 ± 0.14*a 8.49 ± 0.14*�a 7.30 ± 0.16*�#a

90 13.90 ± 0.13ab 9.80 ± 0.14*b 7.17 ± 0.22*�ab 6.33 ± 0.32*�#ab

18 20 12.60 ± 0.05c 8.52 ± 0.23*c 7.19 ± 0.05*�c 6.17 ± 0.10*�#c

40 9.46 ± 0.15ac 6.14 ± 0.11*ac 4.64 ± 0.16*�ac 3.79 ± 0.14*�#ac

90 8.23 ± 0.22abc 4.59 ± 0.13*abc 3.28 ± 0.32*�abc 1.86 ± 0.09*�#abc

GSH 6 20 1.41 ± 0.02 0.96 ± 0.03* 1.11 ± 0.02*� 0.83 ± 0.03*�#

40 1.30 ± 0.02 0.77 ± 0.02*a 0.83 ± 0.02*�a 0.61 ± 0.02*�#a

90 1.00 ± 0.02ab 0.44 ± 0.04*ab 0.54 ± 0.03*�ab 0.42 ± 0.01*#ab

18 20 1.23 ± 0.02 0.73 ± 0.02*c 0.81 ± 0.01*� 0.63 ± 0.03*�#c

40 1.05 ± 0.02a 0.49 ± 0.03*ac 0.56 ± 0.01*�ac 0.39 ± 0.03*�#ac

90 0.88 ± 0.03abc 0.32 ± 0.01*abc 0.44 ± 0.04*�abc 0.27 ± 0.03*#abc

Pt-SH 6 20 13.50 ± 0.11 10.70 ± 0.14* 8.43 ± 0.07*� 7.45 ± 0.16*�#

40 13.00 ± 0.13a 10.10 ± 0.16*a 7.67 ± 0.14*�a 6.69 ± 0.16*�#a

90 12.90 ± 0.14a 9.35 ± 0.12*�ab 6.62 ± 0.21*�ab 5.91 ± 0.32*�#ab

18 20 11.40 ± 0.05c 7.79 ± 0.21*c 6.38 ± 0.05*�c 5.54 ± 0.12*�#c

40 8.41 ± 0.14ac 5.65 ± 0.12*ac 4.08 ± 0.16*�ac 3.41 ± 0.15*�#ac

90 7.36 ± 0.24abc 4.26 ± 0.13*abc 2.84 ± 0.34*�abc 1.58 ± 0.10*�#ab

Values are mean ± SD (n = 4). Units: lipid peroxidation (LPO) nmol malondialdehyde/mg protein; total thiols (T-SH) lmol/mg protein;

reduced glutathione (GSH) lmol/mg protein; protein thiols (Pt-SH) lmol/mg protein

* p \ 0.05 versus control group; � p \ 0.05 versus sodium fluoride (NaF) treated group; # p \ 0.05 versus ethanol (EtOH) treated; a p \ 0.05

versus 20 days; b p \ 0.05 versus 40 days; c p \ 0.05 versus 6-month-old

Mol Cell Biochem (2013) 384:251–262 255

123

18-month-old animals under these conditions. However,

groups receiving NaF together with EtOH for 90 days

showed 78 and 83 % decline in the intestinal SOD activity

in 6- and 18-month-old animals, respectively.

After 90 days, the observed decrease in intestinal CAT

activity following individual exposure to NaF or EtOH was

44 and 49 %, respectively, in 6-month-old and 52 and

56 %, respectively, in 18-month-old animals. Co-exposure

to NaF and EtOH for 90 days decreased the intestinal CAT

activity by 63 % in 6-month-old and by 68 % in 18-month-

old animals.

Intestinal GPx activity recorded after 90 days in NaF

treated group was reduced by 49 and 58 % in 6- and

18-month-old animals, respectively. EtOH feeding for

90 days showed decreased GPx activity in the intestine of

6-month-old (63 %) and 18-month-old (68 %) animals

under these conditions. However, the observed decrease in

the intestinal GPx activity after 90 days in NaF and EtOH

co-treated group was 69 % in 6-month-old and 80 % in

18-month-old animals.

After 90 days, animals treated with NaF showed 47 and

56 % decrease in the intestinal GR activity in 6- and

Table 2 Effect of chronic fluoride and ethanol co-exposure on enzymatic parameters relating to oxidative stress in intestine of 6- and 18-month-

old rats

Parameter Age

(months)

Treatment

(days)

Groups


SOD 6 20 13.50 ± 0.82 10.50 ± 0.17* 10.80 ± 0.84* 7.50 ± 0.75*�#

40 15.80 ± 0.82a 9.22 ± 0.90* 9.85 ± 0.74* 6.89 ± 0.68*�#

90 17.40 ± 0.45ab 6.09 ± 0.61*ab 6.73 ± 0.65*ab 3.91 ± 0.25*�#ab

18 20 10.40 ± 0.66c 6.27 ± 0.92*c 5.50 ± 0.90*c 2.35 ± 0.78*�#c

40 11.80 ± 0.72ac 4.84 ± 0.66*ac 4.99 ± 0.11*c 4.06 ± 0.45*c

90 13.70 ± 0.15abc 3.71 ± 0.16*abc 3.90 ± 0.16*abc 2.39 ± 0.26*�#bc

CAT 6 20 2.33 ± 0.07 1.63 ± 0.06* 1.77 ± 0.03*� 1.41 ± 0.02*�#

40 1.95 ± 0.04a 1.23 ± 0.07*a 1.14 ± 0.09*a 0.84 ± 0.03*�#a

90 1.51 ± 0.04ab 0.84 ± 0.01*ab 0.76 ± 0.01*�ab 0.56 ± 0.04*�#ab

18 20 1.77 ± 0.10c 1.19 ± 0.07* 1.26 ± 0.11* 0.98 ± 0.05*�#

40 1.46 ± 0.08ac 0.87 ± 0.04*a 0.76 ± 0.04*a 0.57 ± 0.04*�#ac

90 1.25 ± 0.07abc 0.60 ± 0.06*abc 0.54 ± 0.10*abc 0.40 ± 0.04*�abc

GPx 6 20 17.20 ± 0.24 13.40 ± 0.24* 12.50 ± 0.41*� 10.40 ± 0.27*�#

40 16 ± 0.35a 10.30 ± 0.43*a 7.70 ± 0.39*�a 5.98 ± 0.37*�#a

90 15.70 ± 0.32a 7.99 ± 0.21*ab 5.88 ± 0.44*�ab 4.92 ± 0.34*�#ab

18 20 14.20 ± 0.46c 10.30 ± 1.02*c 9.33 ± 0.40*c 7.20 ± 0.68*�#c

40 12.10 ± 0.38ac 7.08 ± 0.16*ac 4.68 ± 0.23*�ac 3.43 ± 0.21*�#ac

90 10.80 ± 0.58abc 4.54 ± 0.23*abc 3.50 ± 0.40*�abc 2.18 ± 0.06*�#abc

GR 6 20 34.70 ± 0.91 23.70 ± 0.46* 26 ± 0.73*� 20.20 ± 0.43*�#

40 32.60 ± 0.84a 21.40 ± 0.73*a 23.40 ± 0.82*�a 17.50 ± 0.46*�#a

90 30.10 ± 0.96ab 15.90 ± 0.47*ab 15 ± 0.82*ab 13.50 ± 0.44*�#ab

18 20 24.60 ± 0.27c 15.30 ± 0.27*c 16.30 ± 0.65*c 12.70 ± 0.58*�#c

40 22.10 ± 0.33ac 13 ± 0.33*ac 12.60 ± 0.56*ac 10.30 ± 0.62*�#ac

90 20.10 ± 0.48abc 8.81 ± 0.48*abc 7.79 ± 0.64*abc 6.50 ± 0.30*�#abc

GST 6 20 20.40 ± 0.64 15.40 ± 0.51* 16.50 ± 0.52*� 13.40 ± 0.36*�#

40 19.10 ± 0.50a 13.30 ± 0.42*a 14.40 ± 0.45*�a 10.90 ± 0.52*�#a

90 17.70 ± 0.18ab 11.30 ± 0.19*ab 11.50 ± 0.31*ab 8.12 ± 0.43*�#ab

18 20 17.50 ± 1.02c 9.99 ± 0.36*c 12.20 ± 0.42*�c 8.95 ± 1.21*#c

40 15.80 ± 0.80ac 7.93 ± 0.33*ac 9.86 ± 0.54*�ac 7.34 ± 0.55*#c

90 13.40 ± 0.62abc 6.80 ± 0.74*abc 6.57 ± 0.60*abc 4.31 ± 0.67*�#abc

Values are mean ± SD (n = 4). Units: superoxide dismutase (SOD) units/mg protein; catalase (CAT) mmol H2O2 decomposed/min/mg protein;

glutathione peroxidase (GPx) nmol NADPH oxidized/min/mg protein; glutathione reductase (GR) nmol NADPH oxidized/min/mg protein;

glutathione-S-transferase (GST) nmol GSH-CDNB conjugate formed/min/mg protein



256 Mol Cell Biochem (2013) 384:251–262

123

18-month-old animals, respectively. EtOH feeding under

these conditions reduced the enzyme activity in intestine of

6-month-old animals by 50 % and in 18-month-old rats by

61 %. The observed decrease in the intestinal GR activity

following co-exposure to NaF and EtOH for 90 days was

55 and 68 % in 6-month-old and 18-month-old animals,

respectively.

Intestinal GST activity after 90 days administration of

NaF was decreased by 36 % in 6-month-old and by 49 %

in 18-month-old animals. EtOH feeding for 90 days

Table 3 Effect of chronic fluoride and ethanol co-exposure on parameters relating to mitochondrial functions in intestine of 6- and 18-month-

old rats

Parameter Age

(months)

Treatment

(days)

Groups


Complex I 6 20 14.67 ± 0.63 13.32 ± 0.17 14.12 ± 0.48 12.66 ± 0.39*#

40 14.53 ± 0.54 11.50 ± 0.14*a 12.27 ± 0.19*�a 10.28 ± 0.18*�#a

90 14.38 ± 0.23 9.31 ± 0.34*ab 9.98 ± 0.43*ab 7.63 ± 0.30*�#ab

18 20 10.18 ± 0.06c 9.45 ± 0.45*c 9.95 ± 0.41c 8.16 ± 0.15*�#c

40 10.10 ± 0.12c 8.32 ± 0.21*ac 8.35 ± 0.19*ac 7.26 ± 0.21*�#ac

90 9.79 ± 0.29ac 5.51 ± 0.13*abc 6.04 ± 0.25*�abc 3.75 ± 0.15*�#abc

Complex II 6 20 182.56 ± 3.82 155.36 ± 5.53* 168.07 ± 4.76*� 155.50 ± 5.28*#

40 177.91 ± 4.66 140.99 ± 1.17*a 147.90 ± 4.65*a 135.51 ± 5.50*#a

90 175.58 ± 5.42 117.96 ± 4.69*ab 123.15 ± 4.60*ab 113.05 ± 4.66*ab

18 20 142.60 ± 4.14c 115.56 ± 5.53*c 127.94 ± 4.67*�c 113.14 ± 4.59*#c

40 135.63 ± 5.49c 98.18 ± 4.89*ac 105.65 ± 5.46*ac 97.93 ± 4.67*ac

90 125.34 ± 5.48abc 61.33 ± 1.08*abc 67.98 ± 4.73*abc 57.72 ± 5.00*#abc

Complex IV 6 20 252.52 ± 1.36 231.43 ± 2.45*� 218.89 ± 1.70*� 222.37 ± 2.15*�

40 244.38 ± 3.74a 200.83 ± 1.74*a 193.86 ± 3.50*�a 207.57 ± 3.25*�#a

90 234.99 ± 3.06ab 160.33 ± 1.68*ab 152.27 ± 3.46*�ab 171.61 ± 2.62*�#ab

18 20 206.33 ± 1.70c 196.17 ± 2.08*c 192.42 ± 1.59*�c 187.75 ± 1.23*�#c

40 187.99 ± 3.35ac 177.01 ± 1.39*ac 162.33 ± 1.53*�ac 174.42 ± 1.75*#ac

90 181.84 ± 3.37abc 133.46 ± 2.47*abc 124.91 ± 1.92*�abc 143.17 ± 1.86*�#abc

Nitric oxide 6 20 3.72 ± 0.10 4.58 ± 0.04* 4.58 ± 0.04* 4.70 ± 0.10*

40 4.16 ± 0.09a 5.22 ± 0.03*a 5.46 ± 0.09*�a 6.18 ± 0.09*�#a

90 4.27 ± 0.05a 7.23 ± 0.05*ab 7.57 ± 0.17*�ab 8.18 ± 0.06*�#ab

18 20 5.16 ± 0.07c 6.60 ± 0.06*c 6.79 ± 0.12*�c 6.84 ± 0.07*�c

40 5.74 ± 0.16c 7.43 ± 0.04*ac 7.94 ± 0.08*�ac 9.13 ± 0.11*�#ac

90 5.93 ± 0.08abc 9.31 ± 0.19*abc 9.84 ± 0.27*�abc 11.50 ± 0.08*�#abc

Citrulline 6 20 24.17 ± 0.70 25.49 ± 0.96 27.23 ± 0.51*� 25.89 ± 0.74*

40 26.06 ± 0.32a 35.27 ± 1.23*a 41.22 ± 0.94*�a 38.53 ± 0.51*�#a

90 29.61 ± 0.77ab 45.50 ± 1.10*ab 48.41 ± 0.89*�ab 52.62 ± 1.04*�#ab

18 20 31.82 ± 0.71c 34.40 ± 0.54*c 36.76 ± 0.67*�c 39.28 ± 0.70*�#c

40 32.62 ± 0.83c 41.87 ± 0.42*ac 45.23 ± 0.78*�ac 52.46 ± 0.93*�#ac

90 34.11 ± 0.78ac 52.03 ± 1.36*abc 58.55 ± 1.05*�abc 63.11 ± 1.24*�#abc

MTT reduction 6 20 7.34 ± 0.04 7.27 ± 0.12 7.26 ± 0.06 7.03 ± 0.08*�#

40 7.27 ± 0.08 6.79 ± 0.07*a 6.23 ± 0.05*�a 6.60 ± 0.08*�#a

90 7.14 ± 0.04ab 5.69 ± 0.23*ab 4.34 ± 0.08*�ab 5.02 ± 0.09*�#ab

18 20 6.42 ± 0.03c 6.06 ± 0.08*c 5.75 ± 0.06*�c 5.52 ± 0.07*�#c

40 6.35 ± 0.02ac 5.54 ± 0.09*ac 5.01 ± 0.09*�ac 4.85 ± 0.10*�#ac

90 6.16 ± 0.04abc 4.27 ± 0.06*abc 3.87 ± 0.04*�abc 3.39 ± 0.08*�#abc

Values are mean ± SD (n = 4). Units: complex I (nmol NADH oxidized/min/mg protein); complex II (nmol succinate oxidized/min/mg pro-

tein); complex IV (nmol cytochrome c oxidized/min/mg protein); nitric oxide (units/mg protein); citrulline (lmol citrulline/min/mg protein);

LSS (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reduction (lmol formazan formed/mg protein)



Mol Cell Biochem (2013) 384:251–262 257

123

showed reduced GST activity in the intestine of 6-month

(35 %) and 18-month (51 %) old animals under these

conditions. Groups administered NaF together with EtOH

showed 54 and 68 % decline in the intestinal GST activity

in 6- and 18-month-old animals, respectively, after

90 days.

Effect on parameters relating to mitochondrial

functions

Exposure to NaF or EtOH, separately or together lead to a

significant reduction in the mitochondrial respiration rate,

activities of complexes I, II, and IV along with elevated

levels of NO and citrulline in the intestinal mitochondria of

6- and 18-month-old animals (Table 3). Alterations in the

mitochondrial functions were more pronounced in rats co-

exposed to NaF and EtOH in both the age groups. NaF

administration for 90 days showed 35 and 44 % decrease

in the mitochondrial complex I activity in the intestine of

6- and 18-month-old animals, respectively. Groups given

EtOH for 90 days reduced the enzyme activity by 31 % in

6-month-old and by 38 % in 18-month-old animals.

However, co-exposures to NaF and EtOH for 90 days

exhibited 47 and 62 % decrease in the mitochondrial

complex I activity in the intestine of 6- and 18-month-old

animals, respectively.

Complex II activity in the intestinal mitochondria

recorded after 90 days of NaF treatment was decreased by

33 % in 6-month-old and by 51 % in 18-month-old ani-

mals. Administration of EtOH for 90 days also exhibited

reduced complex II activity in the intestinal mitochondria

of 6-month-old (30 %) and 18-month-old (46 %) animals

under these conditions. The observed decrease in mito-

chondrial complex II activity following NaF and EtOH co-

treatment for 90 days was 36 and 54 % in 6- and

18-month-old animals, respectively.

Mitochondrial complex IV activity in the intestine was


18-month-old animals exposed to NaF for 90 days. Feed-

ing EtOH for similar experimental duration resulted in

decreased complex IV activity in the intestinal mitochon-

dria of 6-month (35 %) and 18-month (31 %) old animals.

However, rats co-exposed to NaF and EtOH for 90 days

showed 27 and 21 % decrease in the mitochondrial com-

plex IV activity in the intestine of 6- and 18-month-old

animals, respectively.

Mitochondrial NO levels in the intestine of rats

administered NaF or EtOH separately for 90 days were

elevated by 69 and 77 %, respectively, in 6-month-old and

by 57 and 66 %, respectively, in 18-month-old rats.

However, rats co-treated with NaF and EtOH for 90 days

increased mitochondrial NO levels in the intestine by 92 %

in 6-month-old and by 94 % in 18-month-old animals.

Mitochondrial citrulline levels following 90 days treat-

ment to NaF were increased by 53–54 % in the intestine of

6- and 18-month-old animals. Feeding EtOH for similar

experimental duration mitochondrial citrulline levels in the

intestine were elevated by 63 % in 6-month-old and by

72 % in 18-month-old rats. Administration of NaF together

with EtOH for 90 days exhibited 78 and 85 % increase in

the mitochondrial citrulline levels in the intestine of 6- and

18-month-old animals, respectively.

Mitochondrial respiration rate in the intestine were


18-month-old animals following NaF administration for

90 days. EtOH feeding for similar experimental duration

also exhibited reduced mitochondrial respiration rate in

6-month (39 %) and 18-month (37 %) old animals under

these conditions. Co-exposures to NaF and EtOH for

90 days resulted in 30 and 45 % reduction in the mito-

chondrial respiration rate in the intestine of 6- and

18-month-old rats, respectively.

Effect on tissue morphology

The effect of NaF or EtOH ingestion, separately or together

on the intestinal morphology in 6- and 18-month-old ani-

mals is presented in Fig. 2. The intestinal morphology in

6-month-old NaF-treated animals after 90 days depicted

moderately excess of lymphocytes, swelling and separation

of villi in the lamina propria of the ileum region, whereas

18-month-old animals exhibited severe lymphocyte infil-

tration in the villi with patchy loss of lining cells. The

group receiving EtOH for 90 days showed a marked

swelling of villi by edema and lymphocyte infiltration,

which are fairly deep in the mucosa of 6-month-old ani-

mals. There was ulceration of the mucosa, loss of villi, and

diffused lymphocyte infiltration in 18-month-old EtOH-

treated animals after 90 days. Co-exposure to NaF and

EtOH showed area of inflammatory infiltration and

necrosis in the ileum region of 6-month-old animals, while

18-month-old animals showed patchy loss of villi, lym-

phocyte infiltration, and severe ileitis.

Discussion

The dose of sodium fluoride used in this study corresponds

to those of human exposures, living in high fluoride

endemic areas [1]. Also the ethanol dose employed of the

rats reflected general alcohol intake by humans. The data

presented indicate a progressive decline in body weight

gain; food and water intake; antioxidant defense resulting

in tissue oxidative stress; and mitochondrial functions with

age, which are further enhanced by NaF or/and EtOH

treatment. Fluoride-treated animals exhibit retarded growth

258 Mol Cell Biochem (2013) 384:251–262

123

and decline in organo-somatic index, which might be due

to excessive loss or breakdown of tissue proteins under

these conditions, leading to decreased body weight gain

[12]. Ethanol-related decrease in the body weight gain may

be attributed to its effect on digestion, absorption, storage,

utilization, and excretion of essential nutrients [21]. A

concurrent decline in food consumption noticed among

treated animals could additionally contribute to poor

growth and body weight gain [22, 23].

In the present study, LPO levels in intestine were ele-

vated, while, T-SH and Pt-SH content along with the

activities of SOD, CAT, GPx, GR, GST were decreased

with age and exposure to fluoride or/and ethanol. Inter-

estingly, GSH content was unaltered with age under these

conditions. This suggests its primary role in intestinal

antioxidant defense system. Fluoride exposure stimulates

LPO in membranous structures but its mechanism is not

fully understood [2]. Available data suggests a multidi-

rectional mechanism involving a decrease in GSH levels,

T-SH and diminished activities of antioxidant enzymes.

This can induce a peroxidative state in biological systems

and, in turn, lead to peroxidation of polyunsaturated fatty

acids. It is well known that ethanol metabolism by alcohol

dehydrogenase yields acetaldehyde and NADH, which

subsequently increases the NADH/NAD? ratio. Aldehyde

oxidase acts upon acetaldehyde and NADH with the for-

mation of superoxide anion radical [24]. This could be one

of the contributing factors for elevated LPO levels in eth-

anol-treated rats. The effect of fluoride on intestinal LPO

levels was more pronounced in the presence of ethanol.

This may be attributed to fluoride-related depletion of

antioxidants and ethanol-induced oxygen radical genera-

tion through microsomal cytochrome P450 system or

xanthine oxidase pathway [25].

The moderate effect of age on intestinal thiol content

might be attributed to the fact that the efficiency of S-thi-

olation as a mechanism of antioxidant defense is affected

with age, which creates an increased risk of irreversible

oxidation of -SH groups of proteins. Glutathione depletion

has been reported in some, but not all the tissues of aged

mice and rats [26] which are similar to the present findings.

A progressive decline in the intestinal thiol content of

treated animals might be attributed to fluoride- or ethanol-

related escalation in tissue LPO levels and oxidative stress.

The reduction in T-SH might be due to depletion of GSH or

changes in the Pt-SH content. The decrease in GSH levels

may also result from concurrent decline in the activity of

GR, which is a crucial enzyme for maintaining -SH/-SS-

ratio in the cell.

The observed decline in intestinal SOD and CAT

activity is in accord with the previous studies reporting

decreased enzyme activities with age [27] and following

exposure to fluoride [6, 12] or ethanol [28]. Fluoride causes

inhibition of SOD activity by binding to active site of

copper on enzyme [29]. Reduced SOD activity in rats

exposed to ethanol alone might be implicated to irrevers-

ible inactivation of the enzyme as a result of enhanced

levels of free radicals by ethanol metabolism [30]. The

observed decline in the intestinal CAT activity was more

pronounced in rats co-treated with fluoride and ethanol.

Several factors may contribute to this phenomenon:

(i) fluoride-associated inhibition of SOD and ethanol-

Old

Old

6 M

onth

18 M

onth

100 m 100 m 100 m 100 m

100 m 100 µm 100 m 100 µ

µ µ µ µ

µ µ µ µµ

Fig. 2 Histology of the rat small intestine after 90 days treatment, magnification 940; arrows indicate changes in the morphology

Mol Cell Biochem (2013) 384:251–262 259

123

related loss of NADPH, (ii) excess of H2O2 production,

(iii) enhanced LPO levels, and (iv) a combination of these

factors. In contrast to our results, Inkielewicz et al. [8]

reported antagonistic effect fluoride and ethanol co-

administration on CAT activity in male rats. These dis-

crepancies may be attributed to differences in organs, sex,

species, and ages of animals studied, under different

experimental conditions.

The observed decrease in intestinal GPx activity of rats

treated with fluoride or/and ethanol may be attributed to

increased LPO levels or solely due to the decreased bio-

availability of GSH under these conditions. GR is required

to maintain high -SH/-SS- ratio, while GST catalyze the

addition of tripeptide glutathione to endogenous and

xenobiotic substrates, which have electrophilic functional

groups. The observed decline in the activities of these

enzymes with age is in agreement with previous findings,

reporting age-related reduction in GR and GST activities in

rats [31]. The decline in intestinal GR and GST activities in

treated animals is indicative of enhanced oxidative stress,

which makes tissue more vulnerable to fluoride- or/and

ethanol-induced oxidative injury. The results obtained from

the earlier studies suggest that GSH depletion lead to a

decrease in GR activity [32]. Reduction in GST activity in

treated animals may be attributed to low GSH levels,

because it is required as a substrate [33].

Since mitochondria are the main sites of cellular energy

supply, modulation of their functional activity is important

for preserving cell viability under normal conditions and

during metabolic stress. Fluoride or/and ethanol exposure

by modifying the activity of mitochondrial enzymes, may

alter the mitochondrial respiratory rate. The activities of

complexes I, II, and IV in intestinal mitochondria were

significantly reduced with age and with fluoride or/and

ethanol treatment. This would result in impaired mito-

chondrial function as a consequence of inhibited electron

flow from NADH to oxygen. An age-dependent impair-

ment of mitochondrial function may be due to either

decreased electron transfer, or increased H? permeability

of the inner membrane, or decreased H?-driven ATP

synthesis [34]. The decrease in mitochondrial complex I

activity observed in treated animals may be due to fluoride-

or ethanol-induced depletion of reducing equivalents

NADH and NADPH. Thus, a decline in the levels of

reducing equivalents decreases mitochondrial GSH content

and thereby may lead to decreased complex IV activity

[35]. One of the most important effects of fluoride is

inhibition of cellular energy production [36]. Fluoride can

pass through the inner mitochondrial membrane and

inhibits complexes II and IV activities [37]. Impaired

mitochondrial functions in ethanol fed rats are in agree-

ment with the findings of Verma et al. [38], who described

a significant decline in the rate of mitochondrial respiration

together with reduced activities of complexes I, II, and IV

enzymes upon ethanol treatment.

An increase in NO levels in rats exposed to fluoride or

ethanol have been shown by others [39, 40]. NO regulates

mitochondrial function by binding to cytochrome c oxi-

dase. It competes with O2, inhibiting the activity of the

enzyme [41] and thus, negatively regulates mitochondrial

oxidative phosphorylation. The elevation in NO levels in

treated animals may be attributed to the fluoride- or etha-

nol-induced increase in the nitric oxide synthase activity.

Previously, it was reported that fluoride increased nitric

oxide synthase activity, which plays a major role in

degenerative diseases, primarily by damaging mitochon-

drial energy production, inhibiting glutamate reuptake, and

stimulating lipid peroxidation [42].

MTT reduction assay, a marker of mitochondrial respi-

ration is commonly used to assess any impairment in the

mitochondrial functioning. The reduced MTT metabolism,

as observed in the present study, suggests that mitochon-

drial respiratory functions and activity of dehydrogenase

are compromised with age, which are further aggravated by

fluoride or/and ethanol administration. The present findings

are in agreement to earlier reports, which indicated a

reduction in mitochondrial respiration rate following fluo-

ride [43] or ethanol [44] exposure. Thus, the decrease in

complexes I, II, and IV activities; elevated NO and cit-

rulline levels; and reduced MTT metabolism suggest an

overall perturbation of the electron transfer pattern leading

to absolute mitochondrial dysfunction as a result of age and

fluoride- or/and ethanol-related damage to intestine.

Altered mitochondrial respiration may further disrupt the

supply of oxygen to intestinal cells and generate a state of

hypoxia. Such a metabolic stress could cause cell damage

by increasing the production of reactive oxygen species

and increased oxidative stress.

Histopathological examination of intestine revealed that,

treatment to fluoride or ethanol exhibited similar patho-

logical changes but differ only in their degree of severity.

The morphological changes observed in intestine of fluo-

ride-treated animals in this study are similar to the findings

of Sondhi et al. [45], who have reported widespread infil-

tration of lymphocytes in sub-mucosa and lamina propria

upon 100 ppm sodium fluoride treatment for 30 days.

Studies in human subjects have also revealed damage to

gastro-duodenal mucosa with ingestion of fluoride [46].

These changes could be attributed to the fact that ingested

fluoride forms hydrofluoric acid in the stomach which has a

corrosive effect on gastrointestinal tract leading to aber-

ration of its structure and function. Ethanol treated rats

showed mild villous edema and large lymphoid follicles in

the ileal region of the small intestine. Histological changes

were further magnified with fluoride and ethanol co-treat-

ment, resulting in focal ileitis and necrosis of villi. This

260 Mol Cell Biochem (2013) 384:251–262

123

could be due to synergistic effects of fluoride and ethanol

in this tissue.

In conclusion, the data described herein shows that

fluoride mediates its toxic effects on intestine through

oxidative stress and by impairing mitochondrial functions

which are further augmented with ethanol consumption and

advancing age. These findings if extrapolated to humans

living in high endemic fluoride areas would imply high risk

to such individuals to age-related intestinal disorders.

Acknowledgments We gratefully acknowledge the financial assis-

tance from University Grants Commission (UGC), New Delhi, India.

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Documents

Ethanol and age enhances fluoride toxicity through oxidative stress and mitochondrial dysfunctions in rat intestine