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Pr otecti ve effect of x-3 pol yunsa tur ated fa tty acids (PUFA) on
sodium nitr ite induced nephr oto xicity and oxida ti ve damage
in rat kidne y
Md. Wasim Khan
1
, Na tar ajan A. Arivar asu, Shubha Priy amvada, Sar a Anees Khan,
Sheeba Khan , Ahad Noor Khan Yusufi
*
Departme nt of Bioc hemistr y, Faculty of Life Sciences , Aligarh Muslim Univer sity , Aligarh 202002, U.P ., India
ARTICLE INFO
Article his tory:
Receiv ed 30 No vember 2012
Receiv ed in re vised form
19 Februa ry 2013
Accepted 20 Februa ry 2013
Availa ble online 19 March 2013
Keywor ds:
Sodium nitrite
Fish oil
Flaxseed oil
Brush bord er membrane
Oxidativ e stress
Phosphate transport
ABSTRACT
Sodium nitrite (SNT) wid ely used as a curati ve agent in meat processing industry possesses
cell-transforming muta genic and cytog enic properties. Dietary omega-3 polyunsatura ted
fatty acids (x-3 PUF A) has been sho wn to reduce the se verity of certa in types of cancers,
car dio vascular and re nal diseases. The present stud y examined whe ther feeding of fish
oil (FO)/flaxseed oil (FXO) has protecti ve effect against SNT -induced toxicity . SNT signi fi-
cantly altered the activities of serum creatinine (Crt), blood urea nitroge n (BUN), metabolic
and brush bor der membrane (BBM) enzymes . SNT caused significant im balances in the
antioxida nt system associated with increased lipid peroxidation (LPO). Fe eding of FO and
FXO with SNT ameliorated the changes in various pa ramet ers caused by SNT . Nephrotox -
icity parameters lo wer ed and enzyme activities of carboh ydrate metabolism, BBM and
radioactiv ely labeled inor ganic phosphate (
32
Pi) transport wer e impro ved to near control
values. The results of the present stud y sugg est that x-3 PUF A-enriched FO and FXO from
sea-foods and plant sources respectiv ely are similarly effect ive in reducing SNT -induced
nephr otoxicity and oxidativ e da mage.
Ó2013 Else vier Ltd. All right s reserve d.
1. Intro duction
Nitrite (NO
2) occupies a unique position in human toxicology .
It is ubiquitou s in the en vir onment and can also be formed
fro m nitro genous compoun ds by micro or ganisms prese nt in
the soil, water , sali va and the gastro intestina l tr act. Contr ary
to being useful nitrite can cause ad verse cellular effects,
althoug h the extent and significance of health risk from its
exposur e is still unclear . The FAO/WHO , Jo int Expert Commit-
tee on Food Additi ve (JEFCA) has esta blish ed acce ptab le dail y
intakes (ADIs) of 0–0.07 mg/kg bod y weight for sodium nitrite
(SNT; expr essed as nitrite) (WHO , 2003 ) in rela tion to the
toxicological limit. SNT is widel y used in food and drug indus-
tries as a pre serva ti ve . Appr oximatel y 40% of ab sorbed nitrite
is excr eted unch ang ed in the urine and the fate of the rest is
not accur atel y kno wn (Cho w & Hong, 2002 ). Fr om resear ch
performed ove r the past decade , it is no w appare nt that
NO
2is ph ysiologicall y rec ycled in the bloo d and tissues to
form NO and other reacti ve nitr oge n species (RNS) (Lundbe rg,
Weitzber g, Lundber g, & Alving, 1994). NO
2has been impli-
cated in va rious pathoph ysiological conditions. The role of
SNT has been well documen ted in carc inogenicity ,mu tagen-
icty , cytotoxicity and methaemo globinemia (Ger , Kao , Shih, &
Deng, 1996; Okazaki et al., 2006; Spencer , Whiteman , Je nner ,&
1756-4646/$ - see front matter Ó2013 Else vier Ltd. All rights reserv ed.
http://dx.doi.org/ 10.1016/j.jf f.2013.02.009
* Corresponding author . Tel.: +91 571 2700741; fa x: +91 571 2706002.
E-mail address: wasimthesis@gmail.com (A.N.K. Yusufi).
1
Current address: Department of Cell Biology & Ph ysiology , CSIR-IICB , Jada vpur , Kolkata, India, 700032.
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967
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journal homepage: www.elsevier.com/locate/jff
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Halliw ell, 2002; Sun, Aoki, Wang, Guo , & Misumi, 2006). The
kidne y is a vital or gan, whic h pla ys an essentia l role in health,
disease and over all de velop ment and gro wth. A vast num ber
of en viron mental agents including certain drugs influence
these functions (Mahmood & Waters, 1994 ). Nitrite is excr eted
in the urine at a rate of appr oximatel y 20 ml/min, indicati ng
consider ab le renal tubular rea bsorption of this ion. Ho wever
ther e is minima l amount of liter ature on the effect of NO
2
on kidne y(Wennmalm et al., 1993 ).
Se veral appr oac hes ha ve been used to pre vent the un-
wanted and harmful ph ysiological effects of toxicants like
SNT . Muc h attention in this regar d has been paid to the pro tec-
ti ve effect s of natur al pro ducts (functional foods) suc h as oils
ric h in x-3 fatty acids. Dietar y fish oils, ric h in omeg a-3 PUF A,
are rapidl y incorpor ated into the membr ane phospholi pids of
cells, suggesting that the y are likel y to ha ve an effect on se ve ral
aspects of cell function (Simopoulo s, 2002 ). A nu mber of in ve s-
tigations ha ve demonstr ated that diet suppleme nted with fish
oil (FO) enric hed in x-3 fatty acids has pro found beneficial
health effect s ag ainst va rious pathologies including car dio vas-
cular disease s, respir ator y disease s, dia betes, de press ion, can-
cers, inflammator y and imm une renal disor ders (Caterina ,
Endr es, Kristense n, & Schm idt, 1994; Khandel wal et al., 2013).
Pre liminar y re ports indicate that FO pre ve nts ne phro toxicity
(Naqshb andi, Khan, Rizw an, Rehman, & Khan, 2012; Naqsh-
bandi, Khan, Rizw an, Yusufi, & Khan et al., 2012a,b ; Priy amv a-
da et al., 2008, 2010). Not lack
ing far behind plant sour ces of
PUF A suc h as flaxseed oil (>50% ALA) has sho wn gre at pro mise
in fighting ag ainst man y disease d conditions (Naqshb andi, Riz-
wan, & Khan, 2013). Dietar y suppleme ntation of flaxseed oil is
being used to reduce hyperlipid emia (Ristic-Me dic, Ristic, &
Tepsic, 2003) and is also being used as a car diopr otecti ve agent
(Espin, Soler -Ri vas, & Wic hers, 2000 ). Flaxsee d oil has rece ntly
been linked with bone de ve lopment in experim ental animals
(Tsujika wa et al., 2000 ).
Considerin g the potential clinical benefits of FO and FXO ,
the prese nt stud y was undertak en to examine the effect of
prior administ ratio n of FO and FXO on SNT -induced chan ges
on the enzyme s of renal brush bor der membr ane (BBM), carbo-
hydr ate metab olism, phosphate tr ansport and antioxidan t
par ameters. We hypothesize that FO and FXO wou ld pre ve nt
SNT -induced nep hro toxicity due to their intrinsic bioc hemic al
and antioxidan t prop erties that would lead to impr oved
meta bolism and antioxidan t defense mec hanism in the kid-
ne y. The results obtained indicate that dietar y supplemen ta-
tion with FO and FXO markedl y enhance d/amelior ated SNT -
induced nep hro toxicity par ameters. The activ ities of enzyme s
of carboh ydrat e meta bolism, BBM and phosphate tr ansport in
renal cortical BBM were significantly enhance d by feeding of
FO and FXO to SNT administ ered rats. Furthermo re, FXO
sho wed similar pro tecti ve effects as of FO and thus can be used
as a replac ement of FO for people with strict ve getarian ha bits.
2. Ma ter ials and methods
2.1. Chemicals and dr ugs
Fish oil (Menhaden, Sigma Chemical Co ., St Louis, MO , USA),
Flaxseed oil (Omega Nutrition, Vancouv er , Canada), Sodium
Nitrite (Sigma Chemical Co .), Gries rea gent (Sigma Chemica l
Co .). All other chem icals used were of anal ytical grad e and
were purc hased either fro m Sigma Chemical CO ., or Sisco
Reseac h Labo rato ry (Mumbai, India).
2.2. Diet
A nutri tionall y adequate labo rator y pellet diet was obtained
fro m Aashirw ad Industrie s (Chandigarh, India). Pellets were
crushed finely and mixed with (a) 15% Fish oil (b) 15% Flax-
seed oil and store d in airtight containers. Vitamin E as DL- a-
tocopher ol (270 mg/kg cho w) was added to eac h of the modi-
fied rat cho ws in or der to meet the incre ased meta bolic
requir ement for antioxidants on a diet high in pol yunsatu-
rated fatty acids and also to the normal rat diet in or der to
nu llify an y effect prod uced by vitamin E alone .
2.3. Experimental design
The animal experim ents were conducte d accor ding to the
guideline s of Committe e for Purpose of Contr ol and Supervi-
sion of Experimen ts on Animals (CPCSEA), Ministr y of En vi-
ronment and Fore sts, Go ve rnment of India (S.O .2778(E), [10/
11/2010]). Adult male Wistar rats (8 rats /gr oup) weighing
150–180 g fed with standa rd rat cho w (Aashirwaad Industries,
Chandig arh, India) and water ad libitum were condition ed for
one week befor e the start of the experim ent. Rats wer e di-
vided into four gro ups. The y were fed on a normal diet (con-
tr ol and SNT) or diet containin g 15% fish oil (SNT + FO) or 15%
flaxseed oil (SNT + FXO). After 15 da ys rats SNT was adminis-
tere d intr aperitoneall y (15 mg/kg bwt/da y) for 10 da ys to all
gro ups exce pt the contr ol. Contr ol animals rece iv ed an equi v-
alent vo lume of normal saline (0.9% sodium chloride). The
rats were sacrificed 24 h after the last injection under 1.9%
light ether anesthesia (0.08 ml per liter of vo lume of con-
tainer). Animals were exposed to ether prese nted on a cotton
ball or gauze pad inside a small cham ber , avo iding direct con-
tact of the animal with the cotton or gauze . Urine samples
were collected for 4 h in standar d meta bolic cages a da y be-
for e the sacrifice. Blood samples were collecte d and the kid-
ne y were harve sted and pro cessed for the pre par ation of
homogenates and brush bor der memb rane ve sicles (BBMVs)
as describe d belo w.
2.4. Ser um/urine che mistries
Serum/ urine par ameters – Serum samples were depr oteinat-
ed with 3% tric hlor oacetic acid in a ratio of 1:3, left for
10 min and then centrifuged at 2000 gfor 10 min. The prote in
fre e supernatan t was used to determine inor ganic phosphate
and cr eatinine . The pre cipitate was used to quanti tate total
phosphol ipids. Blood ure a nitro gen (BUN) and cho lester ol le v-
els were determined dir ectly in serum samples . Cre atinine in
serum and urine was estimated by the Ja ffe’s method (Jaf fe,
1886), briefly 0.4 M crea tinine and 0.75 M sodium hydr oxide
were rea cted with the sample to de ve lop a red color read at
520 nm. Cholester ol was estimated by the method of Zlatkis
and Bo yle (1953). Briefly serum was added to 0.05% ferric chlo -
ride in acetic-aci d solution mixed and allo wed to stand for 10–
15 min. Concentr ated sulphuric acid was added to all tubes
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967 957
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and mixed. The color was read again st the blank at 560 nm.
Phospho lipids in the serum was estimated by the method of
Conner ty , Briggs, and Eaton (1961). Glucose , inor ganic
phospha te and BUN were estimated by using commer cially
ava ilab le kit fro m Span dia gnostics, Mumbai, India follo wing
the manu factur er’s pro tocol.
2.5. Pr epar ati on of kidney homogena tes
Kidne y homogenate was pre pare d as described pre viously
(Naqshbandi et al., 2011 ).One part of the homogenate was
centrifu ged at 2000 g, for 10 min, at 4 °C and the supernat ant
was sa ved for assa ying the enzymes of carboh ydrat e metab o-
lism, second was centrifuged at 3000 gfor 15 min at 4 °C and
supernat ant was used for assa y of free- radical sca ve ngin g
enzyme s and the thir d was used for estimation of total thiol
(–SH) and lipid per oxidation.
2.6. Pr epar ati on of br ush bor der membr ane
Kidne y BBM was pre par ed fro m whole cortex homogenate
using the MgCl
2
pre cipitation method as described by Yusufi
and Dousa (1987). The final membr ane pre parat ions wer e sus-
pended in 300 mM mannito l, pH 7.4. Fr eshl y pre pare d BBM
Vesicles (BBMV) were used for the tr ansport of
32
Pi and some
portion was sa ve d and store d at 20 °C until further analy sis
for BBM enzyme s. Eac h sample of BBM was pre par ed by pool-
ing tissues fro m 2–3 rats.
2.7. Assa y of carboh ydr ate metabolism enzymes
The acti vities of the enzyme s in volvi ng oxidation of NA DH or
reduction of NADP were determin ed spectr ophotometr ically
on Cintr a 5 fixed for 340 nm using 3 ml of assa y in a 1-cm cuv-
ette at room temper atur e (28–30 °C). The enzyme assa ys of
Lactate deh ydrogenase (LDH, E.C.1.1.1.27), malate deh ydroge-
nase (MDH, E.C.1.1.1.37), malic enzyme (ME, E.C.1.1.1 .40), glu-
cose-6-p hosphate deh ydr oge nase (G6PDH, E.C.1.1.1.4 9),
glucose- 6-phospha tase (G6Pase , E.C.3.1 .3.3) and fructose -1,
6-bisph osphatase (FBPase , E.C.3.1.3 .11) activ ities were studied
as describe dbyKhundmir i, Asghar , Khan, Salim, and Yusufi
(2004). Hexokin ase was estimated by the method of Cra ne
and Sols (1953) and the remaining glucose was measur ed by
method of Nelson (1944).
2.8. Assa y of br ush bor der membr ane mar ker enzymes
and lysosomal mar ker enzymes
The activ ities of alkaline phosphata se (ALP), leucine amino
pe ptidase (LAP), c-glutam yl tr ansfera se (GGTase) sucr ase
and acid phospha tase (ACP ase) wer e determine d as described
by Far ooq, Yusufi, and Mahmoo d (2004).
2.9. Assa y of enzymes involved in fr ee radical scaveng ing
Super oxide dismu tase (SOD, E.C.1.15.1.1) was assa ye d by the
method of Marklund and Marklund (1974). Catalase (CAT,
E.C.1.1 1.1. 6) activ ity was assa ye d by the method of Giri, Iqbal,
and Athar (1996) and Glutathione perox idase was assa ye dby
the method of Flohe and Gunzler (1984).
2.10. Lipid per oxidation and total –SH gr oup estimation
Total SH gro ups wer e determine d by the method of Sedlak
and Lindsa y (1968). Lipid perox idation (LPO) was assa yed by
the method of Ohka wa, Ohishi, and Yagi (1979).
2.11. Transport of
32
Pi
Measur ement of
32
Pi (Bhabha Atomic Resear ch Center , Mum-
bai, India) uptake in BBMV(s) was carried out at 25 °C by rapi d
filtration tec hnique as describe dbyYusufi, Mur ayama , Gap-
stur , Szcze panska-K onkel, and Dousa (1994) either in the
prese nce or absence of Na-gr adient. Uptake was initiated by
addition of 30 ll of incubatio n medium containing
100 mmol/l mannito l, NaCl/KCl 100 mmol/l, 5 mmol/l Tris-
HEPES, pH 7.5, 0.1 mmol/l K
2
H
32
PO
4
to 15 ll BBM suspension
(50–100 lg pro tein) and incubated for the desir ed time inter -
val s (see results). The uptake was stopped by the addition of
3 ml ice cold stop solution (containing 135 mmol/l NaCl,
5 mmol/l Tris-HEPES and 10 mM sodium arsenate , pH 7.5)
and filtered immedia tely thr ough 0.45 lm DAWP Millipor e
(MA, USA) and washed thre e times with the stop solutio n
using a Cornw all type syringe (Wheaton, IL, USA). Corr ection
for non specific binding to filters was made by subtra cting
from all data the val ue of corre sponding blan k obtained by fil-
tratio n of the incubatio n buffer without vesicl es. The radi oac-
tivity of dried filters was measur ed by liquid scintillatio n
counting (Rackbeta, LKB , Wallac, Sw eden) with 10 ml scintil-
lation fluid (Cocktail T, SRL, Mumbai, India).
2.12. Determina tion of tissue nitrite
Tissue nitrite le ve ls were quantified using the Griess assa y
(Gr een et al., 1982 ). Kidne y homogenates wer e centrifuged
at 5000 gfor 10 min. 500 ll supernat ant was remo ve d and an
equal volum e of modified Griess reagent was added and the
samples were incubated in the dark for 20 min. Standar d
curve s were genera ted using 0–20 lM sodium nitrite . Absor -
bance was measur ed in standar ds and samples at 550 nm.
Concentr ations of nitrite were determin ed from the standa rd
curve , whic h was linear over the entir e concent ratio n range
tested. Values wer e expr essed as nanomols per gram tissue .
2.13. Statistical analyses
All data are expre ssed as Mean ± SEM for at least 4–5 differ ent
pre parat ions. Statistical eva luation was conducte d by one-
wa y ANO VA. A pro ba bility le vel of p< 0.05 was selected as
indicating statistica l significance. Most of the chang es be-
twee n var ious gro ups were compar ed with contro l va lues
for better understa nding and clarity .
3. Results
The pre sent work was undertaken to stud y detailed mec ha-
nism of SNT -induced ne phro toxicty and other deleterio us ef-
fects and its possib le prote ction by feeding x-3 fatty acids
enrich ed oils diets to the rats . To addr ess our hypothesis,
the effect of SNT alone and in combinat ion with fish oil (FO)
958 JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967
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or flaxseed oil (FXO) was determine d on ne phro toxicity
par ameters with serum bioma rkers enzyme s of oxidati ve
stre ss, brush bor der membr ane and carboh ydr ate meta bolism
in rat kidne y tissues. Na-de pendent phosphate tr ansport was
also determine d in ren al cortical BBM to assess the role of
kidne y in the conserv ation of Pi tr ansport under differ ent
experim ental conditions.
3.1. Effect of dietary fish oil (FO) and flaxseed oil (FXO) on
SNT induced nephr otoxicity par ameter s in ser um and urine
In gener al the rats remained clinicall y well thro ughout the
stud y. Ther e was no significant differ ence in dail y food intake
and bod y weig hts betw een contro l and other experim ental
rats (data not sho wn). SNT tr eatment to contr ol rats resul ted
in significant incr ease in serum creati nine (Scr), bloo d ure a
nitro gen (BUN), cho lester ol, glucose and phospholi pids but
decr ease in inor ganic phosphate (Pi) compar ed to contr ol rats
(Table 1). These changes wher e associa ted with pro found
phosphatu ria, pro tein uria and glucosu ria (Tab le 2) accompa -
nied by decr ease in cr eatinine clear ance (Tab le 2). Feeding of
FO or FXO diet to SNT administ ere d (FOSNT and FXOSNT ) rats
resulted in significant reversa l of va rious SNT elicited delete-
rious effects on serum and urine par ameters. Both FO and
FXO diets pre ve nted SNT -induced incre ase of Scr , BUN , glu-
cose and cholester ol and decr ease of serum Pi (Table 1).
FO/FXO diets appear to gre atly impr ove ren al functions in a
similar manne r as evident by the incre ase of cr eatinine clear -
ance and decre ase in the excr etion of pro tein, phospha te and
glucose in the urine (Table 2).
3.2. Effect of fish oil (FO) and flaxseed oil (FXO) on SNT
induced alter ations on metabolic enzymes in renal corte x and
medulla
The effect of SNT , FO/FXO diets and their combine d trea tment
was determin ed on the activ ities of va rious enzymes of carbo-
hydr ate metab olism in ren al cortex and medulla.
3.2.1. Effect on carbohyd rate metabolism in re nal cortex
As sho wn in Tab les 3a and b, SNT tr eatment to contr ol rats
significantly incre ased the acti vity of lactate deh ydrogenase
(LDH) but decre ased malate deh ydrogenase (MDH), hexoki-
nase (HK); glucose- 6-phospha tase (G6Pase) and fructose-1 ,
6-bisph osphatase (FBPase) activ ities in the renal cortex
(Tab le 3a). When SNT tr eatment was extended to FO and
FXO-fed rats , SNT -induced alter ations in metab olic enzyme
activ ities wer e not onl y pre vent ed by FO and FXO diets,
but G6P ase rem ained significantly higher in FOSNT and
FXOSNT compar ed to contr ol as well as SNT rats in the renal
cortex.
The effect of FO , FXO and SNT combin ed was also deter -
mined on glucose-6-ph osphate deh ydrogenase (G6PDH) and
NADP-malic enzyme (ME), sour ce of NA DPH pro duction
needed in various anab olic reacti ons (Table 3b). SNT trea t-
ment to contr ol rats significantly incr eased G6PDH but de-
cr eased ME activ ity . SNT elicited incre ase of G6PDH activ ity
was normalized to near contr ol va lues by both FO and FXO
diets. SNT induce d ME activ ity decre ase was arr ested by both
FO and FXO diet.
3.2.2. Effect on carbohyd rate metabolism in re nal medulla
The acti vities of LDH, MDH, HK, G6P ase and FBP ase in medul-
lar y homogenates were similarl y affected by SNT tr eatment
(Tab le 3a) as in the cortical homogenates. Feeding of FO and
FXO to SNT tr eated rats pre ve nted SNT induced alter ations
in va rious enzyme acti vities. Similar to cortex, G6PDH activ ity
incre ased wher eas ME activ ity decr eased by SNT in the me-
dulla (Tab le 3b). FO and FXO diet to SNT trea ted rats decre ased
G6PDH activ ity and incre ased ME activ ity .
3.3. Effect of dietary fish oil (FO) and flaxseed oil (FXO) on
SNT induced alter ations in biomar ker enzymes of BBM and
lysosomes
To assess the structur al inte grity of certain or ganelles e.g.,
plasma membr ane (BBM) and lyso somes, the effect of SNT
alone and in combinat ion with FO or FXO diets was deter -
mined on biomarker enzyme s of BBM and ly sosomes in the
homogenates of renal cortex and medulla and isolated BBM
pre par ations fro m ren al cortex.
3.4. Effect of SNT alone and with FO or FXO diet on
biomar ker s of BBM and lysosomes in the homogenates
The activ ites of alkalin e phosphata se (AlkPase), c-glutam yl
tr anspe ptidase (GGTase) and leucine aminope ptidase (LAP)
and acid phosphata se were determine d under differ ent
Table 1 – Effect of fish oil (FO) and flaxseed oil (FXO) on serum par ameters with SNT tr ea tment.
Grou p Serum creati nine
(mg/dl)
BUN
(mg/dl)
Cholester ol
(mg/dl)
Phospho lipid
(mg/dl)
Phospha te
(lmol/ml)
Glucose
(mg/dl)
Control 0.831 ± 0.591 26.32 ± 0.112 82.73 ± 0.921 24.29 ± 0.091 1.35 ± 0.523 89.91 ± 0.191
SNT 1.276 ± 0.09
*
(+54%)
48.69 ± 0.784
*
(+85%)
97.27 ± 0.124
*
(+18%)
35.36 ± 0.12
*
(+46%)
0.923 ± 0.763
*
(32%)
112.11 ± 0.091
*
(+25%)
FOSN T 0.934 ± 0.76
(+12%)
30.66 ± 0.651
(+16)
83.89 ± 0.873
(+1%)
26.77 ± 0.883
(+10%)
1.44 ± 0.275
(+7%)
83.78 ± 0.457
(7%)
FXOS NT 0.989 ± 0.841
*,
(+19%)
29.52 ± 0.327
(+12%)
80.76 ± 0.563
(2%)
28.82 ± 0.542
*,
(+19)
1.26 ± 0.665
(6%)
81.54 ± 0.112
(9%)
Results (specific act ivity expressed as lmol/ mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967 959
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experim ental conditions in the homogenates of renal cortex
and medulla (Tab le 4). SNT tr eatment to contro l rats caused
significant reduction in the specific activ ities of AlkP ase
(35%), GGT ase (57%) and LAP (52%) in cortic al homoge-
nate . The prior feeding of FO or FXO diet with SNT tr eatment
pre ve nted SNT elicited decr ease in BBM enzyme acti vities. As
can be seen from the data SNT induced decr ease in BBM en-
zyme activ ities were similarl y pre ve nted by FO or FXO diet.
The activ ity of acid phosphata se (ACP ase) was also decr eased
(45%) by SNT in cortical homogenates and FO/FXO diet was
able to pre ve nt the decre ase in enzyme activ ity in a similar
manne r(Tab le 4).
The acti vites of BBM enzymes similar to the cortex were
also lo were d in the medulla by SNT administ ration although
to a lo wer extent (Tab le 4). The consumpt ion of FO/FXO in
combinat ion with SNT treatm ent resulte d in the re ve rsal of
SNT induced decr ease in AlkP ase (19%), GGT ase (33%)
and LAP (42%) in the medulla . The activ ity of ACP ase was
not affected by SNT and SNT + FO . Ho weve r it was found to
be lo wer in SNT + FXO rats (18%) compar ed with the contr ol
rats .
3.5. Effect of SNT and SNT plus FO and FXO diet on BBM
mar ker s in isolated BBMV
The effect of SNT , FO and FXO on BBM marker enzymes was
further analy zed in BBMV pre par ations isolated fro m the re-
nal cortex (Fig. 1). The data sho ws a similar activ ity pattern
of BBM enzyme s as observ ed in cortical homogenates. Ho w-
eve r the ma gnitude of the effects was muc h mor e pro -
nounced in BBMV than in cortical homogenates. Acti vities
of AlkP ase (67%), GGT ase (53%) and LAP (51%) pro foundl y
declined by SNT treatm ent as compar ed to contr ol rats. Fi sh
oil (FO) and flaxseed oil (FXO) dietar y supplemen tation similar
to the effect in the homogenates appear ed to lo wer the se ve r-
ity of the SNT treatm ent. SNT -induce d decr ease in BBM en-
zyme activ ities was significantly pre vent ed by dietar y FO
and to gre ater extent by FXO diet.
Table 3a – Effect of fish oil (FO) and flaxseed oil (FXO) acti vities of LDH, MDH, G6P ase and FBP ase in homogena tes of
(a) cortex and (b) medulla with SNT tr ea tment.
Gr oup LDH MDH HK G6P ase FBP ase
(a)Cortex
Control 27.72 ± 0.01 94.71 ± 0.01 64.21 ± 0.76 0.56 ± 0.02 1.91 ± 0.02
SNT 35.61 ± 0.22
*
(+29%)
61.09 ± 0.61
*
(35%)
46.71 ± 0.29
*
(27%)
0.33 ± 0.07
*
(41%)
1.57 ± 0.08
*
(18%)
FOSNT 28.89 ± 0.77
(+4%)
97.61 ± 0.23
(+3%)
59.87 ± 0.77
*,
(7%)
0.65 ± 0.08
*,
(+16%)
1.82 ± 0.41
(5%)
FXOSNT 30.04 ± 0.07
(+8%)
90.66 ± 0.96
(4%)
67.78 ± 0.12
(+6%)
0.68 ± 0.11
*,
(+21%)
1.74 ± 0.23
(9%)
(b) Medu lla
Control 40.91 ± 0.09 86.41 ± 0.13 69.33 ± 0.39 0.80 ± 0.03 1.61 ± 0.11
SNT 49.69 ± 0.06
*
(+28%)
64.21 ± 0.07
*
(25%)
46.21 ± 0.77
*
(33%)
0.53 ± 0.24
*
(34%)
1.21 ± 0.05
*
(23%)
FOSNT 43.87 ± 0.54
(+7%)
88.22 ± 1.35
*,
(+19%)
60.03 ± 0.73
(13%)
0.71 ± 0.12
(11%)
1.84 ± 0.12
(+14%)
FXOSNT 42.87 ± 0.12
(+5%)
77.87 ± 0.66
(10%)
63.71 ± 0.45
(8%)
0.86 ± 0.81
(+8%)
1.513 ± 0.86
(6%)
Results (specific activity expressed as lmol/mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
Table 2 – Effect of fish oil (FO) and flaxseed oil (FXO) on ur ine par ameters of rats with SNT tr ea tment.
Gr oup Urine flow rate
(UFR)
(ml/day)
Cre atinine
clear ance
(ml/min/100 gm bod y wt.)
Phosphate
(lmol Pi/mg cr eatinine)
Pr otein
(mg/mmol cr eatinine)
Glucose
(mg/mg cr eatinine)
Control 33.67 ± 1.22 0.16 ± 0.43 0.68 ± 0.67 5.09 ± 0.75 8.87 ± 0.98
SNT 51.93 ± 8.3
*
(+54%)
0.09 ± 0.112
*
(44%)
1.08 ± 0.76
*
(+59%)
8.07 ± 0.78
*
(+59%)
13.75 ± 1.09
*
(+55)
FOSNT 38.40 ± 8.1
(+14)
0.22 ± 0.069
*,
(+37%)
0.49 ± 0.07
*,
(28%)
4.06 ± 0.65
*,
(20%)
9.33 ± 0.56
(+5%)
FXOSNT 41.61 ± 5.6
(+24%)
0.18 ± 0.012
(+13)
0.54 ± 0.041
*,
(20%)
4.56 ± 0.32
(10%)
10.69 ± 0.61
*
(+20%)
Results (specific activity expressed as lmol/mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
960 JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967
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3.6. Effect of dietary fish oil (FO) and flaxseed oil (FXO) on
SNT induced alter ati ons in antioxida nt defense par ameter s in
renal corte x and medulla
It is evident that reacti ve oxygen species genera ted by vari-
ous toxicants ar e import ant mediato rs of cellular injur y
and pathogenesis of va rious disease s. Primar y componen ts
of oxidati ve str ess and cellular injur y response include ele-
va tion of lipid per oxidation (LPO), dep letion of GSH and sup-
pre ssion of antioxid ant enzyme s. To ascertain the role of
antioxid ant system in SNT -induce d toxicity , the effect of
SNT observ ed on oxidati ve str ess param eters. SNT enhanced
lipid per oxidation (LPO) and significantly alter ed antioxidan t
enzyme s both in cortex and medulla , albeit differe ntly (Ta-
bl e 5). LPO measur ed in terms of thiobarbituric acid reacti ve
substance s (TBARS) re pro ted as malondiald eh yde (MDA
equi va lents) significantly enhance d in the cortex (+46%)
and medulla (+40%) to similar extent wher eas total-SH de-
clined in these tissue (29% to 37%). SNT treatm ent caused
marked incr ease in super oxide dism utase (SOD, +58%) and
Table 3b – Effect of fish oil (FO) and flaxseed oil (FXO) on acti vities of G6PDH, ME and ACP ase in homogena tes of (a) cortex
and (b) medulla with SNT tr ea tment.
Grou p G6PDH ME
(a) Cortex
Control 0.34 ± 0.02 1.42 ± 0.63
SNT 0.48 ± 0.02
*
(+42%)
1.15 ± 0.71
*
(19%)
FOSN T 0.38 ± 0.77
(+11%)
1.55 ± 0.67
(+9%)
FXOS NT 0.39 ± 0.01
*,
(+15%)
1.335 ± 0.34
(6%)
(b) Medulla
Control 0.25 ± 0.03 1.02 ± 0.21
SNT 0.31 ± 0.02
*
(+25%)
0.89 ± 0.01
*
(13%)
FOSN T 0.24 ± 0.09
(6%)
0.94 ± 0.87
(-8%)
FXOS NT 0.20 ± 0.99
(20%)
0.99 ± 0.71
(3%)
Results (specific act ivity expressed as lmol/ mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
Table 4 – Effect of fish oil (FO) and flaxseed oil (FXO) on biomarke rs of BBM and lysosomes in homogena tes of (a) cortex and
(b) medulla with SNT tr ea tment.
Grou p ALP
(lmol/mg pro tein/h)
GGT ase
(lmol/mg pro tein/h)
LAP
(lmol/mg prote in/h)
ACP ase
(lmol/mg pro tein/h)
(a) Cortex
Control 25.41 ± 0.512 34.62 ± 0.315 4.552 ± 0.291 12.21 ± 0.415
SNT 16.33 ± 1.241
*
(35%)
14.212 ± 0.079
*
(57%)
2.012 ± 0.617
*
(52%)
5.61 ± 0.312
*
(45%)
FOSN T 23.67 ± 0.564
(7%)
25.36 ± 0.09
*
(26%)
5.126 ± 0.481
(+13%)
10.63 ± 0.93
(13%)
FXOS NT 20.71 ± 0.223
*
(18%)
28.45 ± 0.427
*
(18%)
3.876 ± 0.09
*
(15%)
11.09 ± 1.08
(9%)
(b) Medulla
Control 11.121 ± 1.243 18.032 ± 0.049 4.143 ± 0.589 6.53 ± 0.130
SNT 9.038 ± 0.187
*
(19%)
12.063 ± 0.217
*
(33%)
2.384 ± 1.045
*
(42%)
5.995 ± 0.826
(8%)
FOSN T 13.765 ± 0.929
*
(+24%)
15.763 ± 0.459
*
(13%)
3.338 ± 0.671
(19%)
6.871 ± 0.772
(+5%)
FXOS NT 10.346 ± 0.38
(7%)
17.458 ± 0.612
(3%)
3.985 ± 0.883
(4%)
5.332 ± 0.336
*
(18%)
Results (specific act ivity expressed as lmol/ mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967 961
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glutathio ne per oxidase (GSH-Px, +30%) activ ities but de-
cr ease in catalase (16%) activ ity in the renal cortex . In me-
dulla ho wever the activ ity of SOD (58%) and catalase
(28%) significantly decre sed but in the acti vitiy of GSH-Px
(+45%) was significantly increa sed by SNT administr ation
alone .
A
B
Fig. 1 – Effect of FO and FXO on acti vities of AlkP ase, GGT ase and LAP in (A) cortical homogen ates and (B) BBM with SNT
tr ea tment. Results (lmol per mg pro tein per hour) ar e Mean ± SEM for five differ ent pr epar ati ons.
*
Significantly differ ent fro m
contr ol,
significantl y differ ent fro m SNT : at p< 0.05 by one way ANO VA.
Table 5 – Effect of fish oil (FO) and flaxseed oil (FXO) on enzymatic and non enzymatic antioxidant parameters in
homogenates of (a) cortex and (b) medulla with SNT treatment.
Gr oup Lipid per oxidation
(nmol/gm tissue)
Total SH
(lmol/gm tissue)
Super oxide dism utase
(Units/mg prote in)
Catalase
(lmol/mg pro tein/min)
GSH-Px
(lmol/mg pro tein/min)
(a) Corte x
Control 177.94 ± 5.88 9.53 ± 0.76 9.75 ± 0.66 205.41 ± 1.09 0.38 ± 0.07
SNT 259.51 ± 4.33
*
(+46%)
6.78 ± 0.56
*
(29%)
15.45 ± 0.61
*
(+58%)
172.14 ± 3.31
*
(16%)
0.27 ± 0.98
*
(+30%)
FOSNT 195.36 ± 8.13
(+10%)
8.81 ± 1.85
(7%)
13.87 ± 0.32
*
(+42%)
267.88 ± 0.23
*,
(+30%)
0.40 ± 0.10
(+6%)
FXOSNT 201.14 ± 0.99
(+13%)
9.02 ± 0.12
(5%)
14.11 ± 0.67
*
(+45%)
224.09 ± 0.77
(+9%)
0.41 ± 0.77
(+10%)
(b) Medu lla
Control 104.25 ± 6.49 8.01 ± 0.45 36.29 ± 1.11 134.21 ± 5.32 0.65 ± 0.45
SNT 146.35 ± 0.67
*
(+40%)
5.35 ± 0.42
*
(37%)
15.19 ± 0.54
*
(58%)
195.61 ± 3.93
*
(+45%)
0.47 ± 0.24
*
(28%)
FOSNT 101.68 ± 1.23
(3%)
8.95 ± 0.34
(+11%)
27.48 ± 0.85
*,
(25%)
154.64 ± 0.71
*,
(+15%)
0.79 ± 0.01
*,
(+22%)
FXOSNT 102.39 ± 0.713
(2%)
7.43 ± 0.71
(7%)
29.98 ± 0.71
*,
(17%)
161.82 ± 0.99
*,
(+20%)
0.70 ± 0.55
(+8%)
Results (specific activity expressed as lmol/mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
962 JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967
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3.7. Effect of dietary fish oil and flaxseed oil on SNT
induced alter ations in Na-gr adient dependent tr ansport of
32
Pi
in BBMV(s) isolated fr om renal corte x
The bulk of filtered Pi in the kidne y is rea bsorbed by its pro x-
imal tubule . The Na-gr adient dep endent {Na
outside
(Na
o
)>-
Na
inside
(Na
i
)} tran sport of Pi in renal proxim al tubule acro ss
its luminal brush bor der membr ane (BBM) is an initial and
regu lator y ste p. The Pi is transp orted by secondar y acti ve
tr ansport mec hanism, requir es expend iture of ener gy in the
form of ATP that is genera ted by cellular metab olism. The up-
take of
32
Pi was determin ed in the prese nce and ab sence of
Na-gr adient in the initial uphill phase (30 s) and after equilib-
rium at 120 min in BBM pre par ations. The rate of concentr a-
ti ve uphill uptake of
32
Pi in the pre sence of a Na-gr adient
(NaCl in the medium) was markedl y decr eased by SNT
tr eatment (Tab le 6). Ho wever , the uptake of
32
Pi at the
equilibriu m phase (120 min) when Na
o
= Na
i
was not signifi-
cantl y differ ent betw een the tw o gro ups. Also Na-inde pen-
dent uptake (in the ab sence of a Na grad ient, when NaCl in
the medium was repl aced by KCl wher eK
o
>K
i
) of
32
Pi at
30 s and 120 min was also not affected by SNT trea tment indi-
cating specific alter ations onl y when Na-gr adient was pre s-
ent. When SNT tr eatment was extended to FO and FXO
feeding rats, SNT induced decre ase in
32
Pi tran sport was not
observ ed.
3.8. Effect of dietary fish oil and flaxseed oil on SNT -
induced alter ati ons in nitrite concentr ation in renal corte x and
medulla
SNT incre ased the tissue concentr ations of nitrite as sho wn in
Fig 2A and B, the accum ulation of nitrite being greate r in renal
medulla . Both FO and FXO impr oved the va lues to near con-
tr ol in renal medulla but onl y partiall y in renal cortex.
4. Discussion
Nitrite is a widel y used agent in the food pro cessing and col-
oring industr y. When used in combinat ion with salts (in the
form of SNT), nitrites serv e as important antimic robia l agents
in meat to inhibit the gro wth of bacterial spore s that cause
botulism, a deadly food-borne illness. Additio nall y, nitrites
ha ve been emplo ye d as a vasod ilator or a cir culator y depr es-
sant to relie ve smooth muscl e spasms and an antidote for
tr eating cyanid e poisoning (Nicke rson, 1970 ). Perhaps the
most commo n adv erse effect of nitrite toxicity in humans is
the formation of methemog lobin, a substance that interfer es
with the ability of red bloo d cells to carr y oxygen when con-
centr ations reach 30–40% of total hemoglobin concentr ation,
whic h can be fatal (Ger et al., 1996 ). Nitrites ma y also react
with certain amines in food to pro duce carc inogenic N-nitr o-
so compound s, nitro samines man y of whic h are kno wn to
Table 6 – Effect of fish oil (FO) and flaxseed oil (FXO) on uptake of
32
Pi in brush bor der membr ane ve sicles (BBMV) fro m
whole cortex with SNT tr ea tment.
(A) Na
+
-gr ad ient dependent uptake (Na
o
> Na
i
) (pmol/mg protein)
Group 30 s 120 min
Control 2750.73 ± 10.53 422.50 ± 17.89
SNT 1263.72 ± 16.30
*
(62%)
372.09 ± 15.92
(12%)
FOSN T 2778.57 ± 15.98
(+1%)
482.14 ± 12.66
(+14%)
FXOS NT 2702.3 ± 33.87
(2%)
472.91 ± 12.82
(+12%)
(B) K
+
-gr adie nt dependent uptake (K
o
>K
i
) (pmol/mg protein)
Group 30 s 120 min
Control 184.37 ± 6.73 354.96 ± 4.26
SNT 189.48 ± 4.228
(+3%)
366.06 ± 17.20
(+3%)
FOSN T 197.88 ± 15.56
(+7%)
360.81 ± 15.25
(+2%)
FXOS NT 186.33 ± 18.72
(1%)
354.36 ± 11.28
(0.2%)
(C) Net Na
+
-gradi ent depend ent uptake at 30 s
Group Na
+
-gradient dependent upt ake K
+
-gradient dependent uptake Net uptake (Na
+
-K
+
)
Control 2750.73 ± 10.53 184.37 ± 6.73 2566.36 ± 25.04
SNT 1263.72 ± 16.30 189.48 ± 4.228 1074.24 ± 11.24
*
(57%)
FOSN T 2778.57 ± 15.98 197.88 ± 15.56 2580.69 ± 20.11
(+1%)
FXOS NT 2702.3 ± 33.87 186.33 ± 18.72 2515.97 ± 19.25
(2%)
Results (specific act ivity expressed as lmol/ mg protein/h) are Mean ± SEM for five different pre parations.
* Significantly different from control.
Significantly different from SNT at p< 0.05 by one wa y ANO VA. Values in parentheses re present percent change from control.
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967 963
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cause cancer (Van Maanen , Pache n, Dallinga , & Kleinjans,
1998). A high dietar y intake of nitrite has been implicated as
a risk factor for human cancer and formation of nitr oso com-
pounds in the stomac h during inflammation is corr elated
with nitrite in food and water (Mirvish, 1995 ). A num ber of
bioc hemical chan ges, functio nal impairm ents and histopa-
thological lesions ha ve been observ ed in nitrite trea ted rats.
Ora l administ ration of SNT caused imm unosuppr esion in
mice (Abuharfeil, Sarsour , & Hassune h, 2001 ). Anemia and
ne phritis can occur due to pro longed exposur e to nitrites
(Dollahite & Ro we, 1974 ).
Since nitrites are a part of the ver y en vironm ent in whic h
we liv e it is virtuall y impossib le to eliminate them completel y
fro m the bod y, besides the y also pla y vital roles in the normal
functio ning of the human bod y when prese nt at their ph ysio-
logical concentr ations. Ho wever , their indiscr iminate use in
agricultur e as fertilize rs and in va rious industries, it has been
found that nitrites are normal ly prese nt at higher concent ra-
tions in the bod y. Se veral appr oache s ar e being attempted to
minimize the nitrite toxicity withou t altering the normal
ph ysiological functio ns of the bod y, one suc h attempt being
the use of dietar y vitamin E and selenium (Cho w & Hong,
2002). Recent studies ha ve sho wn that x-3 fatty acids reta rd
the progr ession of va
rious forms of cancers, de press ion,
arthritis, asthma,car dio va scular and renal disor ders (Caterina
et al., 1994). Recentl y it has been documen ted that x-3 fatty
acids pro tects ag ainst gentamicin , ura nyl nitra te cisplatin
and nitric oxide donors/m etabo lites-induce d nep hro toxicity
(Khan et al., 2012a, 2012b; Naqshband i et al., 2011, 2012; Pri-
yamv ada et al., 2008, 2010). Ome ga-3 PUF A ha ve intrinsic anti-
oxidant pro perties due to the prese nce of eicsope ntaenoic
acid (EPA), docosahe xaenoic acid (DHA) and alpha-linole nic
acid (ALA) (Simopoulo s, 2002 ). DHA was sho wn to incre ase
glutathione synthesis (Ar ab , Rossar y, Souler e, & Stegh ens,
2006). Massar o et al. (2002) report ed the rea ctiv e oxygen spe-
cies (ROS) quenc hing ab ilities of oleate and linked them to
its purported anti-ather oscler otic activ ities. Fr om a mec hanis-
tic vie wpoint, NA D(P)H oxidase is one of the major contrib u-
tors to endothel ial free radical prod uction and it has been
sho wn to be inhibited by DHA and (presumab ly) other PUF As
(Massar o et al., 2006 ). This might gr eatl y explain the observ ed
effect of PUF A on RO S pro duction. DHA-m ediated inhibitio n of
inteleukin 1-alpha (IL-1a) induced ROS pro duction, whic h
wou ld contribute to the anti-inflammator
y actions of ome ga
3 fatty acids at the endothel ial le ve l has also been report ed
(Massar o et al., 2002 ). One other potent ial mech anism of
action of PUF As would be that the y act as a ‘‘sink’’ to trap free
radica ls without getting oxidized themselv es (Massar o et al.,
2002). Fin all y, PUF As, especial ly those of the omeg a 3 series,
are pre cursors of anti-inflammator y lipid mediators suc h as
resol vins, pro tectins, and neur opr otectins (Serhan, Arita,
Hong, & Gotlinger , 2004; Yacoubian & Serhan, 2007).
A
B
Fig. 2 – Effect of FO and FXO on tissue nitrit e lev els in (A) cortica l and (B) medullary homogena tes with SNT tr ea tment. Results
(nanomol per gram tissue) ar e Mean ± SEM for five differ ent pr epar at ions.
*
Significantl y differ ent fro m contr ol,
significantl y
differ ent fro m SNT : at p< 0.05 by one wa y ANO VA.
964 JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967
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The prese nt work was under taken to stud y detailed mec h-
anism of SNT -induced ne phro toxic alter ations and the possi-
ble action mec hanism of FO and FXO in pre ve nting those
alter ations in rat kidne y and whether FXO is equall y potent
as FO . The results sho w that SNT administr ation prod uced a
typical pattern of nep hrotox icity as indicate d by incre ased
Scr , BUN accompan ied by massi ve pro tein uria, glucosu ria
and phospha turia. SNT administ ratio n to contro l rats also re-
sulted in significant increa se of LDH and G6PDH activ ities and
decr ease in the acti vities of MDH (TCA cycle); G6P ase, FBP ase
(gluconeogenesis) and ME in both renal cortex and medulla.
These results indicate a shift in the ener gy metab olism alter -
nati ve ly from aer obic to anaer obic most likely due to mito-
cho ndrial dysfunction as evident by significant incre ase in
LDH and decr ease in MDH activ ities (Banda y, Faro oq, Priy am-
va da, Yusufi, & Khan, 2008). FO and FXO when giv en for
15 da ys prior to and follo wing SNT administr ation pro tected
ag ainst SNT induced deleterio us effects in va rious bloo d/ur -
ine par ameters and enzyme activ ities of va rious pathw ays.
SNT -induced incre ase of Scr/BUN and hyper cholester olemia,
phosphol ipidosis, pro teinu ria, glucosu ria and phosphatu ria
were all absent in FO/FXO-fed SNT tr eated rats . Dietar y FO/
FXO were also ab le to pre ve nt SNT -induced decre ase/incr ease
in the acti vities of certain enzymes in vo lv ed in carboh ydrat e
meta bolism both in renal cortex and medulla. These pro tec-
ti ve effects can be attribu ted to the fact that x-3 fatty acids
are ab le to effecti ve ly enhance the activ ities of enzyme s of
carboh ydrat e metab olism in renal tissue s similar to those re-
ported in rat liv er (Yilmaz, Songur , Ozyurt, Zar arsiz, & Sarsil-
maz, 2004).
The inte grity of the BBM was assessed by the status of its
biomarke r enzyme s. SNT caused significant decr ease in the
activ ities of AlkP ase , GGT ase and LAP in cortical homogenates
and to a muc h greate r extent in BBMV . The decr ease in BBM
enzyme activ ities might ha ve occurr ed due to loss of BBM en-
zyme and other components into lumen follo wed by their
excessi ve excr etion in the urine as rep orted earlier (Abu-Spe-
tan & Abdel-Ga yo um, 2001). A marked redu ction observ ed in
Na-de pendent tr ansport of
32
Pi by SNT also supports nitrite
induced se vere dama ge to BBM of renal pro ximal tubules. It
has been sho wn that dietar y fatty acids incorpor ate in the cel-
lular membr anes altering structur al inte grity and functional
capacitie s of plasma membr ane and other or ganelles leading
to alter ed cellular metab olic acti vities (Ca ve, 1991 ). Taken to-
gether the pre sent results sho w that SNT trea tment indeed
significantly alter ed the structur al inte grity and functiona l
capacity of BBM and mitoc hondria reflected by marked de-
cr ease in the acti vities of BBM enzymes: AlkP ase , GGT ase ,
LAP , BBM Na-de pendent tr ansport of
32
Pi and certain enzyme s
of carboh ydrat e metab olism respecti ve ly . Dietar y fish/flax-
seed oils giv en together with SNT not onl y pre vent ed/r e-
tar ded SNT -induced decre ase of BBM enzymes but the
activ ity of AlkP ase rem ained higher in FXSNT than contr ol
rats. FO/FXO also resulte d in normalizat ion of
32
Pi transp ort
acro ss the renal BBM in SNT tr eated rats.
RNS are consider ed to be important mediators of SNT -in-
duced toxicity (Cho w & Hong, 2002 ). Nitrite is a ke y oxidation
pro duct as well as a read y sour ce of nitric oxide , which in turn
reacts rapidl y with super oxide to form the highly reacti ve
species, per oxynitrite . Incr eased formation of per oxynitrite
is likel y to be the critical eve nt responsib le for the toxic effect
of nitrites. The pre sent results confirm earlier findings and
sho w that SNT administr ation to contr ol rats caused se ve re
dama ge to renal tissues most likely by RNS gener ation as
appar ent by perturbatio n in the antioxid ant enzymes (SOD,
Catalase and GPx-SH) and total-SH content that lead to in-
cr eased lipid per oxidation. The feeding of FO/FXO diet to
SNT tr eated rats pre vented SNT -induced augment ation of
LPO and alter ation of antioxidan t enzyme activ ities. Results
also demonstr ate that ther e is an incre ase in the tissue accu-
mu lation of nitrite after SNT trea tment. This ma y be due to
the inabil ity of the kidne y to filter out excess nitrite fro m
the bod y. This ina bility of the kidne yma y be a result of the
dama ge done by SNT to the structur e of kidne y. FO/FXO diets
significantly reduce tissue accum ulation of nitrite by pro tect-
ing the structur al inte grity of the kidne y.
The pro tection ag ainst SNT effected by FO/FXO can be
attributed to their intrinsic bioc hemical and natur al antioxi-
dant pro perties. As report ed earlier , feeding of FO alone
caused significant incr ease of SOD , catalase and GSH-Px
activ
ities accompani ed by lo wer LPO va lues renal tissues (Pri-
ya mv ada et al., 2008). Thus it appears that FO/ FXO enric hed
in x-3 fatty acids enhanced resistance to free radical attac k
gener ated by SNT administr ation similar ly as demonstr ated
in lupus nep hritis and other pathologies (Chandr asekar & Fer -
nandes, 1994; Donadio , 2001; Xi & Chen, 2000). Dietar yx-3
fatty acids suppleme ntation has also been sho wn to stre ngth-
en antioxidan t defense mec hanism in the plasma of normal
rats (Er dogan et al., 2004 ). Our resul ts thus support the ratio-
nale that x-3 fatty acids enric hed FO/FXO ma y be effecti ve
dietar y supplemen tation in the mana gement of SNT nep hro -
toxicity and other pathologies in whic h antioxidant defense
mec hanism are deceler ated.
5. Conclusion
We conclud e that while SNT elicited deleteriou s ne phro toxic
effects by causing se ve re dama ge to ren al mitoc hondria,
brush bor der membr ane and other or ganelles and by sup-
pre ssing antioxidan t defense mech anism, dietar y supple-
mentatio n with fish/flaxseed oil enric hed in x-3 fatty acids
caused impr ove ment in nu trition/ene rgy meta bolism, BBM
inte grity ,
32
Pi tr ansport capacity and antioxidan t defenses
and thus pre ve nted SNT induce d va rious deleterio us effects.
It was also observ ed that flaxseed was equall y pro tecti ve
ag ainst SNT induced ne phro toxicity as fish oil. This comes
as a relie f to the billions of ve getarians world wide who are un-
ab le to consum e fish oil/pr oducts. Based on our pre sent obser -
va tions and alre ad y kno wn health benefits we pro pose that
dietar y fish/flaxseed oil suppleme ntation ma y pro vide a cush-
ion for a pro long ed ther apeutic option again st SNT ne phro p-
ath y without harmful side effects.
Conflict of inter est
The authors state no conflict of inter est betw een them.
JOURNAL OF FUNCTIONAL FOODS 5 (2013) 956–967 965
Author's personal copy
Ack no wledgements
Indian Council of Medical Resear ch (ICMR), Ne w Delhi, India
is ack no wledged for the awar d of JRF (Junior Resear ch Fello w-
ship)/Senior Resear ch Fello wship to SP and WK. Council of
Scienti fic and Industrial Resear ch (CSIR), Ne w Delhi, India
for the awar d of JRF to SK. Financial support to the de part-
ment fro m Uni ve rsity Gr ants Commissio n (UGC-DRF), De part-
ment of Scienc e and Techn ology (DST-FIST) and a resear ch
gr ant (58/21/2001-BMS) fro m ICMR to ANKY is also gra tefull y
ack no wledged.
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