Effects of elevated dietary iron on the gastrointestinalexpression of Nramp genes and iron homeostasis in rainbowtrout (Oncorhynchus mykiss)

Raymond W. M. Kwong •

Charmain D. Hamilton • Som Niyogi

Received: 22 May 2012 / Accepted: 6 August 2012 / Published online: 15 August 2012

� Springer Science+Business Media B.V. 2012

Abstract Diet is the primary source of iron (Fe) for

freshwater fish, and the absorption of Fe is believed to

occur via the Nramp family of divalent metal trans-

porters (also called DMT1). Presently, the homeostatic

regulation of dietary Fe absorption in fish is poorly

understood. This study examined the gastrointestinal

mRNA expression of two Nramp isoforms, Nramp-band Nramp-c, in the freshwater rainbow trout (On-

corhynchus mykiss), following exposure to elevated

dietary Fe [1,256 mg Fe/kg food vs. 136 mg Fe/kg

food (control)] for 14 days. The physiological perfor-

mance, plasma Fe status and tissue-specific accumu-

lation of Fe were also evaluated. In general, the mRNA

expression level of Nramp was higher in the intestine

relative to the stomach. Interestingly, fish fed on a

high-Fe diet exhibited a significant induction in

Nramp expression after 7 days, followed by a decrease

to the level observed in control fish on day 14. The

increase in Nramp expression correlated with the

elevated gastrointestinal and plasma Fe concentra-

tions. However, the hepatic Fe concentration remained

unchanged during the entire exposure period, indicat-

ing strong homeostatic regulation of hepatic Fe level

in fish. Fish appeared to handle increased systemic Fe

level by elevating the plasma transferrin level, thereby

enhancing the Fe-binding capacity in the plasma.

Overall, our study provides new interesting insights

into the homeostatic regulation of dietary Fe uptake

and handling in freshwater fish.

Keywords Diet � Homeostasis � Iron � Nramp �Rainbow trout � Transferrin

Introduction

Iron (Fe) is an essential micronutrient for vertebrates

including fish, and it is a component of many vital

proteins and enzymes such as hemoglobin, myoglobin

and mitochondrial electron transport chain enzymes.

However, excess Fe can be toxic because it can

generate free radical species and cause oxidative

damage (Crichton et al. 2002). Therefore, the main-

tenance of Fe homeostasis in the body is critical.

Teleost fish can acquire Fe from the water as well as

from the food; however, diet is believed to be the

major source of Fe (Bury and Grosell 2003). The

precise daily dietary Fe requirement for most fish is

R. W. M. Kwong

Toxicology Centre, University of Saskatchewan,

Saskatoon, SK S7N 5B3, Canada

Present Address:R. W. M. Kwong

Department of Biology, University of Ottawa,

30 Marie Curie, Ottawa, ON K1N 6N5, Canada

C. D. Hamilton � S. Niyogi (&)

Department of Biology, University of Saskatchewan,

Saskatoon, SK S7N 5E2, Canada

e-mail: som.niyogi@usask.ca

123

Fish Physiol Biochem (2013) 39:363–372

DOI 10.1007/s10695-012-9705-2

not known. It has been suggested that a range of

30–170 mg Fe/kg in the diet is required for fish to

prevent Fe deficiency syndromes (Watanabe et al.

1997).

The mechanisms regulating the absorption of

dietary Fe in fish have not been fully elucidated.

Previous studies have suggested that ferric Fe (Fe3?),

the major form of inorganic Fe in the diet, is first

reduced to ferrous Fe (Fe2?) (Carriquiriborde et al.

2004; Kwong et al. 2010), and the absorption of Fe2?

into enterocytes is mediated by the Nramp family of

divalent metal transporters (also called DMT1)

(Kwong et al. 2010; Kwong and Niyogi 2008). In

freshwater rainbow trout (Oncorhynchus mykiss),

three different Nramp mRNA isoforms (Nramp-a,

Nramp-b and Nramp-c) have been identified to date

(Cooper et al. 2007; Dorschner and Phillips 1999;

Kwong et al. 2010). However, when expressed in

Xenopus oocytes, only Nramp-b and Nramp-c proteins

were found to mediate Fe2? uptake (Cooper et al.

2007). We have previously reported that both Nramp-

b and Nramp-c isoforms are expressed in the stomach

and intestinal tract of rainbow trout (Kwong et al.

2010). Using intestinal sac preparations from rainbow

trout, the absorption of Fe was shown to occur along

the entire (anterior, mid and posterior) intestinal tract

(Kwong and Niyogi 2008). However, the spatial

differences in Nramp expression between the stomach

and intestine, and the possible isoform-specific

responses to dietary Fe level, have not been investi-

gated. The precise mechanisms by which Fe2? trans-

ported across the basolateral membrane are also

poorly characterized, but the mammalian ortholog of

iron-regulated protein-1 (IREG1) has been suggested

to mediate the Fe2? extrusion (Donovan et al. 2000).

Following extrusion, Fe2? is oxidized to Fe3? by

hephaestin prior to its binding to transferrin in the

plasma, and Fe is subsequently delivered and stored in

the liver (Carriquiriborde et al. 2004; Walker and

Fromm 1976).

In vertebrates, Fe homeostasis is believed to be

regulated primarily by the absorption process, and

excretion plays only a minor role (Andrews 2000). In

mammals, the maintenance of Fe balance during high

or low dietary Fe exposure can be regulated by

modulating the Nramp2 (mammalian ortholog of

rainbow trout Nramp-b and Nramp-c) gene expression

in the gut, thereby regulating Fe acquisition (Gunshin

et al. 1997; Zoller et al. 2001). Changes in the Nramp

gene expression in response to dietary Fe level have

also been demonstrated in teleost fish. For example,

zebrafish (Danio rerio) and European sea bass

(Dicentrarchus labrax) fed with a low-Fe diet exhib-

ited an increase in the gene expression of Nramp2

ortholog in the intestine (Cooper et al. 2006; Neves

et al. 2011), presumably as an adaptive response to

up-regulate Fe uptake. However, Craig et al. (2009)

reported that treatment with a high-Fe diet also

induced the expression of Nramp gene in zebrafish

although Fe burden remained unchanged. Similarly,

Kwong et al. (2011) showed that rainbow trout fed on

an elevated Fe diet exhibited no marked changes in

tissue-specific Fe burden after 4 weeks. However, the

potential alterations in the temporal expression pattern

of Nramp genes in relation to systemic Fe regulation

have not been examined. In addition, it is yet to be

fully understood how fish are able to maintain Fe

homeostasis during exposure to high dietary Fe level

(Carriquiriborde et al. 2004; Kwong et al. 2011).

The present study was designed to examine the

Nramp gene expression and Fe homeostasis in fresh-

water fish following exposure to elevated dietary Fe,

using rainbow trout as a model species. The specific

objectives of this study were threefold: (1) to evaluate

the physiological status of fish fed with a high-Fe diet;

(2) to examine the expression of Nramp-b and Nramp-

c genes in the gastrointestinal tract (stomach and

intestine) of fish; and (3) to examine Fe homeostasis

by evaluating Fe handling in the plasma and tissue-

specific Fe accumulation in fish.

Materials and methods

Fish

Freshwater rainbow trout (O. mykiss; *200 g wet

weight) were obtained from the Fort Qu’Appelle Fish

Culture Station in Fort Qu’Appelle, Saskatchewan,

Canada. Fish were acclimated for at least 4 weeks in

the laboratory conditions and supplied with aerated,

dechlorinated water [hardness 157 mg/L, alkalinity

109 mg/L (both as CaCO3), pH 8.1–8.2, dissolved

organic carbon (DOC) 2.2 mg/L, Fe 5.5 lg/L] in a

flow-through system. The water temperature was

maintained at 15 �C with a photoperiod of 14:10 light

to dark cycle. Fish were fed daily with commercial

trout chow (Martin Mills Inc., Elmira, Canada;

364 Fish Physiol Biochem (2013) 39:363–372

123

comprised of 41.0 % crude protein, 11.0 % crude fat,

3.5 % crude fiber, 1 % calcium, 0.85 % total phos-

phate and 0.45 % total sodium). A week prior to the

beginning of the experiment, fish were transferred to a

control diet (see below for details) at 2.5 % daily

ration (dry feed weight/wet fish body weight).

Diet preparation

To prepare the high-Fe diet, the commercial trout

chow was first rehydrated with approximately 25 %

(v/w) Nanopure water and was ground to powder using

a blender. A known amount of Fe (as FeSO4�7H2O)

(dissolved in Nanopure water; EM Science, USA) was

added to the mix and blended for about 10 min to

ensure homogenous mixing. The mix was then

extruded through a pasta maker and cut into small

pellets. The pellets were kept at -20 �C until frozen

and was subsequently dried in a freeze dryer (Lab-

conco Freezone, USA). The dried pellet was stored at

4 �C until use. The control diet (normal Fe diet) was

prepared in the same fashion without the addition of

Fe. The Fe concentration in the food was verified using

a flame atomic absorption spectrometry (see below for

details) (Analyst 800, PerkinElmer, USA), and their

concentrations and daily doses are presented in

Table 1.

Experimental treatment and sampling

A total of 30 fish were divided equally into two tanks,

and each tank received continuous aeration and water

flow at a rate of 1 L/min. Fish in each tank were fed

with a different diet (control and Fe-enriched diet, see

Table 1) for 14 days at a daily ration of 2.5 % (dry

feed weight/wet fish weight), divided equally into two

rations per day. Fecal matters were removed daily 1 h

post-feeding using a siphon. No fish mortality

occurred in any treatments during the exposure period.

Water samples were collected from each treatment

tank three times a week 1 h post-feeding for the

measurement of Fe (see below for details), and no

elevated Fe concentration was recorded in any of the

water samples.

An initial six fish were randomly sampled on the

day the experiment was started (day 0), and six fish

from each tank were sampled on days 7 and 14. Fish

were immediately euthanized with an overdose of

MS-222 (Syndel Laboratories Ltd., Canada) and

measured individually for weight and length. Blood

samples were taken immediately by caudal puncture

with a 5-mL syringe pre-rinsed with heparin (Sigma-

Aldrich, USA) and centrifuged for 4 min at 10,000 g to

collect plasma. The stomach and intestine were

dissected out, cut open and rinsed in 0.9 % NaCl to

remove any food remains in the lumen. The mucosal

epithelium of the stomach and intestine was collected

by scraping using a glass slide and were stored in

RNAlater (Ambion, Inc., USA) at -80 �C until

further processing. The remaining tissues from the

stomach and intestine, as well as tissue sample from

the liver, were collected, weighed and stored at

-80 �C for later Fe measurements.

Real-time PCR

Total RNA extraction, DNase treatment and cDNA

synthesis were carried out as described in Kwong et al.

(2010). First-strand cDNA was used as a template to

examine the transcript expression of Nramp-b and

Nramp-c isoforms and the housekeeping gene b-actin,

using real-time quantitative polymerase chain reaction

(RT-qPCR). All RT-qPCR assays were performed on a

real-time thermocycler (IQ5; Bio-Rad, USA), using

SYBR Master Mix (Fermentas, Canada). Specific

primers were designed for Nramp-b (accession num-

ber AF048761; forward: 50-CCT CCC CTC CGG CTT

CAG AC-30, reverse: 50-CCC CAA CAA CAC CCA

CAG GAG-30) and Nramp-c (accession number

EF495162; forward: 50-ACC CGC TCC ATC GCC

ATC TT-30, reverse: 50-GAC CTG CCG CCC ATC

TCT G-30). RT-qPCR was performed in triplicate

using the following conditions: 95 �C for 10 min, 45

cycles of 95 �C for 15 s, 64 �C for 30 s and 72 �C for

25 s. The specificity of primers of PCR was checked at

the end of each amplification using a DNA melt curve

analysis with a ramping rate of 1 �C/min over a

temperature range of 60–95 �C. All expression data

were normalized to the expression of b-actin from the

Table 1 Measured iron (Fe) concentration (mg Fe/kg dry

weight of food) in the diet and the associated daily dose (mg

Fe/kg fish wet weight/day) to rainbow trout

Fe level Daily dose

Control 136 ± 11 2.7

High iron 1,256 ± 25 25.1

Values are mean ± SEM, n = 5 for Fe level in the diets

Fish Physiol Biochem (2013) 39:363–372 365

123

same tissue. Relative expression was calculated based

on the method described by Pfaffl (2001), where the

expression levels of each gene from the high-dietary-

Fe-treated fish were calculated relative to the control

on the same day.

Iron measurement

The experimental diets and tissue samples were

digested in 5 volumes of 1 N HNO3 at 60 �C for

48 h and then centrifuged at 15,000 g for 4 min. The

supernatant was collected, diluted appropriately with

0.2 % HNO3 and analyzed for Fe concentrations using

a flame atomic absorption spectrometry (AA800,

PerkinElmer, USA). The quality control and assurance

of Fe analysis were maintained using appropriate

method blanks and a certified standard for Fe (Fisher

Scientific, Canada) and were validated with certified

reference materials (DOLT-4; National Research

Council, Canada).

Plasma iron status

Total plasma Fe concentrations and unsaturated

Fe-binding capacity (UIBC) were measured using a

commercial kit (Pointe Scientific, Inc., USA). The

protocol was adapted for measurement in a 96-well

plate using a microplate reader (Varioskan Flash,

Thermo Scientific, USA) at 560 nm. The total plasma

Fe concentrations and UIBC values were used to

determine total Fe-binding capacity (TIBC) and

transferrin saturation (%) according to the manufac-

turer’s instructions.

Calculations and statistical analysis

Condition factor [K = 100 9 weight (g)/length (cm)3],

gastro-somatic index (GSI), intestinal somatic index

(ISI) and hepato-somatic index (HSI) [GSI, ISI or HSI

(%) = weight of each tissue 9 100/total body weight]

were calculated from individual fish sampled on days 0

and 14 (n = 6 per treatment).

Statistical analysis was performed using Sigma-

Plot� (version 11.0; Systat Software, Inc., Point

Richmond, CA, USA). Data were either analyzed

using one-way or two-way ANOVA (with exposure

time and dietary Fe as two independent variables),

followed by a post hoc Holm–Sidak test. Data were

either log-transformed or square-root-transformed

when the data did not meet the assumptions of equal

variance or normal distribution. Data are reported as

the mean ± SEM, and differences were considered

significant at p \ 0.05.

Results

Physiological status

The weight, condition factor and GSI values of fish

were not significantly different between the two

treatment groups during the exposure (Table 2). On

days 7 and 14, the HSI values of fish fed with a high-Fe

diet were significantly higher than day 0 (p \ 0.05).

Similarly, a significant increase in HSI (p \ 0.05) was

also recorded on day 7 in fish exposed to elevated

dietary Fe when compared with fish fed with normal

Fe diet (control) although the difference did not persist

on day 14. A significant increase in the ISI value was

also observed in fish exposed to the high-Fe diet

Table 2 Physiological status of rainbow trout exposed to

control and high-Fe diet

Days Control High Fe

Weight (g) 0 195.0 ± 17.8A

7 183.4 ± 17.5Aa 192.9 ± 25.5Aa

14 191.0 ± 28.9Aa 222.1 ± 33.6Aa

Condition factor 0 1.22 ± 0.04A

7 1.25 ± 0.04Aa 1.26 ± 0.04Aa

14 1.22 ± 0.06Aa 1.19 ± 0.04Aa

HSI (%) 0 0.95 ± 0.06A

7 1.09 ± 0.05Aa 1.32 ± 0.08Bb

14 1.10 ± 0.10Aa 1.18 ± 0.06ABa

GSI (%) 0 1.89 ± 0.11A

7 1.96 ± 0.18Aa 2.16 ± 0.16Aa

14 1.40 ± 0.12Ba 1.69 ± 0.09Aa

ISI (%) 0 5.25 ± 0.23A

7 5.31 ± 0.10Aa 6.59 ± 0.37Bb

14 4.73 ± 0.22Aa 5.62 ± 0.48ABb

HSI Hepato-somatic index, GSI Gastro-somatic index, ISIIntestinal somatic index. Values labeled with different letters

indicate statistical difference (two-way ANOVA followed by a

post hoc Holm–Sidak test, p \ 0.05). Upper case: among days

in the same diet treatment (including comparison with day 0 of

the control for the high-Fe diet treatment); lower case: between

two treatments on the same day. Values are mean ± SEM,

n = 6

366 Fish Physiol Biochem (2013) 39:363–372

123

compared with that in control fish on days 7 and 14

(p \ 0.05).

Nramp mRNA expression

In both the stomach and intestine, the mRNA expres-

sion level of Nramp-c was about three- to fourfold

higher than that of Nramp-b on day 0 (p \ 0.05)

(Fig. 1a). The higher expression level of Nramp-c than

Nramp-b was also recorded on days 7 and 14 (data not

shown). In addition, the expression level of Nramp-c

was significantly higher in the intestine relative to that

in the stomach (p \ 0.001), whereas the expression

level of Nramp-b was comparable between the two

tissues (Fig. 1b). Similar spatial expression pattern of

Nramp-b and Nramp-c in the stomach and intestine

was also observed on days 7 and 14 (data not shown).

In the stomach, elevated dietary Fe did not signif-

icantly affect the expression of Nramp-b (Fig. 2a).

However, fish fed with a high-Fe diet exhibited a

significant increase in Nramp-c expression in the

stomach on day 7 (p \ 0.05), followed by a decrease

to the level comparable with that in control fish on day

14 (Fig. 2b). In the intestine, both Nramp-b and

Nramp-c mRNA expression levels were significantly

increased in fish fed with a high-Fe diet on day 7

(p \ 0.001–0.01), followed by a decrease to the level

similar to that in control fish on day 14 (Fig. 2c and d).

A two-way ANOVA revealed no interactive effects

between exposure time and dietary Fe treatments.

Tissue iron concentration

Exposure to elevated dietary Fe resulted in a significant

increase (p\0.001) in Fe concentration of the gastroin-

testinal tissue of fish during the entire exposure period

except in stomach at day 14 (Fig. 3a and b). However, Fe

concentration in the liver remained unchanged between

the two treatment groups over the entire exposure period

(Fig. 3c). A two-way ANOVA revealed a significant

interaction between exposure time and dietary Fe treat-

ments for both the stomach and intestine (p\0.001).

Plasma iron status

The plasma Fe concentration, TIBC and transferrin

saturation (%) were significantly increased in fish

exposed to high-Fe diet (p \ 0.001-0.05) during the

entire exposure period (Fig. 4a, c and d). However, no

significant change was observed in UIBC between the

two treatment groups on either day 7 or day 14

(Fig. 4b). A two-way ANOVA revealed no significant

interaction between exposure time and dietary Fe

treatments in any of the above measurements.

Discussion

To the best of our knowledge, this is the first study

to report the linkage between the expression of

(a)F

old

diffe

renc

e

0

1

2

3

4

5Nramp-βNramp-γ

(b)

Nramp isoforms

Stomach Intestine

Beta Gamma

Fol

d di

ffere

nce

0

1

2

3

4

5

20

40

60

80StomachIntestine

a

a

a

b

b

b

a a

Fig. 1 a Relative difference in Nramp-b and Nramp-c mRNA

expression level in the stomach and intestine of rainbow trout.

Data were normalized with b-actin from the same tissue and

were expressed relative to the Nramp-b expression in the same

tissue. b Relative difference in Nramp-b and Nramp-c mRNA

expression level between the stomach and intestine of rainbow

trout. Data were normalized with b-actin from the same tissue

and were expressed relative to the expression of each respective

gene in the stomach. Bars labeled with different letters are

statistically different from each other (one-way ANOVA

followed by a post hoc Holm–Sidak test, p \ 0.05). Values

are mean ± SEM, n = 6

Fish Physiol Biochem (2013) 39:363–372 367

123

iron-transporting Nramp genes and systemic Fe reg-

ulation in teleost fish. Our study showed that the

expression of Nramp transcripts in the gastrointestinal

tract is modulated by dietary Fe level and subsequently

affects Fe accumulation in fish. In addition, we

demonstrated that regulation of Fe-binding pool in

the plasma is probably a key mechanism for regulating

Fe homeostasis in fish during exposure to elevated

dietary iron.

The Fe level in the control diet (136 mg Fe/kg dry

weight) used in this study was well within the range of

the dietary Fe level (100–250 mg Fe/kg dry weight)

suggested for normal physiological functioning in

salmonid fish (Andersen et al. 1996). The Fe concen-

tration in natural fish diet has been found to vary

between 160 and 12,500 mg/kg dry weight (Wint-

erbourn et al. 2000), and thus, the Fe level (1,256 mg

Fe/kg dry weight) in the Fe-enriched diet used in this

study was environmentally relevant. It should also be

noted here that no apparent toxicity (e.g., oxidative

stress) was reported in rainbow trout following

4 weeks of treatment with a diet containing a much

higher Fe level (1,975 mg Fe/kg dry weight) relative

to the Fe-enriched diet of the present study (Carri-

quiriborde et al. 2004). The present study indicated

that the exposure to the elevated dietary Fe does not

affect the condition factor, but significantly increases

the HSI value in rainbow trout. Kwong et al. (2011)

also reported similar findings in rainbow trout exposed

to elevated dietary Fe (1,108 mg Fe/kg dry weight) for

4 weeks. In contrast, Carriquiriborde et al. (2004) did

not observe any change in HSI value in rainbow trout

chronically treated with a high-Fe diet (high Fe:

1,975 mg Fe/kg dry weight). Nevertheless, they

recorded an enlargement of liver cells and suggested

that the metabolic activity in the liver was increased in

Nramp-β

Time (day)

Fol

d di

ffere

nce

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 ControlHigh Fe

Nramp-γ

Time (day)

Fol

d di

ffere

nce

0

2

4

6

8

10

Nramp-β

Time (day)

Fol

d di

ffere

nce

0

1

2

3

4

5

6

7 ControlHigh Fe

Nramp-γ

Time (day)

7 14 7 14

7 14 7 14

Fol

d di

ffere

nce

0

2

4

6

8

10

12

Stomach

Intestine

b

aa

ab

a

b

a

ab

a

b

aab

(a) (b)

(c) (d)

a

a a

a

Fig. 2 Alterations in the mRNA expression level of Nramp-band Nramp-c in the stomach (a, b) and intestine (c, d) of rainbow

trout exposed to normal (control) and high-Fe diets. Data were

normalized with b-actin from the same tissue and were

expressed relative to the control at the same exposure time.

Bars labeled with different letters are statistically different from

each other (two-way ANOVA followed by a post hoc Holm–

Sidak test, p \ 0.05). Values are mean ± SEM, n = 6

368 Fish Physiol Biochem (2013) 39:363–372

123

fish fed with the high-Fe diet (Carriquiriborde et al.

2004).

In mammals, the Nramp2 is suggested to be

important in dietary Fe absorption (Gunshin et al.

1997; Mackenzie and Garrick 2005). In rainbow trout,

two different isoforms of Nramp protein (Nramp-band Nramp-c) are shown to mediate apical Fe uptake

when expressed in Xenopus oocytes (Cooper et al.

2007). The mRNA expression of both of these Nramp

isoforms has been recorded in the gastrointestinal tract

of rainbow trout, and the in vitro examination of Fe

uptake indicated their involvement in Fe transport in

trout intestine (Kwong et al. 2010; Kwong and Niyogi

2008). Interestingly, a considerably higher expression

of Nramp-c relative to Nramp-b was observed in both

stomach and intestine during the entire exposure

period. This finding suggested that Nramp-c may play

a more prominent role in Fe transport in trout. This

was also supported by our observation that the

increase in the expression of Nramp-c was much

higher in magnitude relative to that of Nramp-b in the

gastrointestinal tract of rainbow trout, following

7 days of treatment to elevated dietary iron. In

addition, the increased expression of Nramp-c gene

corresponded with the significant elevation of stomach

Fe level (discussed later) following exposure to high-

Fe diet, indicating that the stomach is an important site

of Fe absorption in trout. This is in line with the

previous studies that also reported stomach is an

important site for Cu absorption in trout (Nadella et al.

2011).

In mammals, it has been shown that exposure to an

elevated Fe diet led to the down-regulation of Nramp2

gene expression in the intestine, thereby reducing Fe

absorption (Frazer et al. 2003; Gunshin et al. 1997).

Interestingly, we observed that high dietary Fe expo-

sure increased the transcript expression of Nramp in

the gastrointestinal tract of fish on day 7, which was

followed by a decrease to the level similar to that in the

control fish (fed with a normal Fe diet) on day 14. The

induction of Nramp gene expression was also accom-

panied by an increase in gastrointestinal Fe accumu-

lation in the fish (discussed later). These findings

suggested an up-regulation of dietary Fe absorption

during the early phase of exposure to elevated dietary

Fe, while a reduction in Nramp gene expression during

the later phase might have been an adaptive response

to ameliorate systemic Fe burden. At present, it is not

clear why the exposure to elevated dietary Fe causes

(a) Stomach

0

5

10

15

20

25 Control

High Fe

(b) Intestine

0

10

20

30

40

50

(c) Liver

Time (day)

0 7 14

0 7 14

0 7 140

20

40

60

80

100

Fe

conc

entr

atio

ns (

μ g/g

wet

wt.)

120

a

a a

b

ab

a a a

b

b

a

a

aa

a

Fig. 3 Iron (Fe) concentration in the a stomach, b intestine and

c liver of rainbow trout exposed to normal (control) and high-Fe

diets. Bars labeled with different letters are statistically different

from each other (two-way ANOVA followed by a post hoc

Holm–Sidak test, p \ 0.05). Values are mean ± SEM, n = 6

Fish Physiol Biochem (2013) 39:363–372 369

123

an initial increase in the gastrointestinal expression of

Nramp genes in fish. We were not able to examine

whether the increased expression of Nramp genes

leads to corresponding increase in Nramp protein

levels, because of the lack of commercially available

antibodies specific to different Nramp proteins. It is

apparent that the regulation of Nramp proteins, not

only in the gut but in other major epithelia as well (e.g.,

gill, kidney), in relation to altered dietary Fe exposure

in fish requires further investigations. Overall, our

observations suggested that body Fe status probably

regulates the gastrointestinal expression of Nramp

genes in fish, at least in part.

The present study showed that Fe accumulation in the

stomach and intestine of fish was significantly increased

following exposure to high dietary Fe. However, Fe

level in the stomach appeared to reduce the control level

on day 14, which occurred likely due to the down-

regulation of the Nramp genes on day 14. In addition, we

found that fish exposed to the high-Fe diet had an

elevated plasma Fe level—a consequence of increased

dietary Fe absorption. The increase in the gastrointe-

stinal and plasma Fe levels correlated with the increase

in gastrointestinal Nramp transcript levels. In order to

handle elevated Fe level in the plasma, fish appeared to

up-regulate the plasma Fe-binding capacity by increas-

ing the plasma transferrin level (equivalent to TIBC), as

well as by increasing the percentage of Fe saturation in

the transferrin. These responses are physiologically

imperative since excess unbound Fe in the plasma may

cause oxidative damage to the vital cells/organs in the

body (Yamaji et al. 2004b). Transferrin is mainly

synthesized in the liver and regulated by the liver Fe

concentration (Anderson and Frazer 2005). Interest-

ingly though, we did not observe any apparent change in

the hepatic Fe level in fish exposed to elevated dietary

Fe. Since the first hepatic Fe measurement was

conducted on day 7 of the exposure, it is possible that

fish exposed to an elevated Fe diet exhibited a transient

increase in the hepatic Fe level, which was not recorded

in our study. In general, our results suggested that the

hepatic Fe status is tightly regulated in fish.

It is important to note that, in addition to Nramp and

transferrin, there are other proteins that are known to

Time (day)

Pla

sma

Fe

(μM

)

0

10

20

30

40

50

60ControlHigh Fe

Time (day)

UIB

C (

μ M)

0

20

40

60

80

100

Time (day)

TIB

C (

μ M)

0

25

50

75

100

125

150

175

Time (day)

7 14 7 14

7 14 7 14

Tran

sfer

rin s

atur

atio

n (%

)0

10

20

30

40

a

b

a

b

a a a a

a

b

ab

a

b

a

b

(b)(a)

(c) (d)

Fig. 4 a Plasma iron (Fe) level, b unsaturated Fe-binding

capacity (UIBC), c total Fe-binding capacity (TIBC), and

d transferrin saturation (%), in rainbow trout exposed to normal

(control) and high-Fe diets. Bars labeled with different letters

are statistically different from each other (two-way ANOVA

followed by a post hoc Holm–Sidak test, p \ 0.05). Values are

mean ± SEM, n = 6

370 Fish Physiol Biochem (2013) 39:363–372

123

play an important role in the regulation of Fe

homeostasis in vertebrates. For example, it has been

demonstrated that high-Fe diet decreases the intestinal

expression of IREG1, thereby reducing Fe extrusion

into the blood circulation (Chen et al. 2003). In

zebrafish, however, exposure to an elevated dietary Fe

was found to have no effect on the IREG1 levels in the

gut (Craig et al. 2009). On the other hand, hepcidin has

been suggested to play a critical role in the regulation

of systemic Fe balance in mammals (Ganz 2005).

Specifically, exposure to a high-Fe diet in mammals

has been shown to increase the level of hepcidin,

which mediates the degradation of Nramp2 (Brasse-

Lagnel et al. 2011) and IREG1 (Ganz 2005). Similarly,

Rodrigues et al. (2006) reported increased hepatic

mRNA expression of hepcidin in fish during Fe

overload. However, there is currently a debate about

whether hepcidin regulates systemic Fe level by acting

primarily on apical or basolateral Fe transport (Chung

et al. 2009; Yamaji et al. 2004a). Further studies are

required to understand the role and physiological

importance of IREG1 and hepcidin in systemic Fe

regulation in fish.

In conclusion, the present study demonstrated that

exposure to elevated dietary Fe induces the gastroin-

testinal expression of Nramp genes in fish, resulting in

increased Fe level in the gastrointestinal tissue and

plasma. Nevertheless, Nramp genes probably play an

important role in homeostatic regulation of Fe uptake,

particularly at the later phase of the exposure as the

systemic Fe level begins to increase. In addition, it

appears that fish maintain Fe homeostasis during

exposure to elevated dietary Fe, to a large degree, by

up-regulating the transferrin level and Fe-binding pool

in the plasma. Overall, our study provides fundamen-

tal insights into the homeostatic regulation of dietary

Fe absorption and systemic Fe handling in fish.

Acknowledgments We thank Drs Jose Andres and Ken

Wilson (University of Saskatchewan) for allowing us to use

their laboratory equipment. A Discovery Grant from the Natural

Sciences and Engineering Research Council of Canada

(NSERC) to S.N. supported this research. This work was also

supported (in part) by the Alexander Graham Bell Canada

Graduate Scholarship, as well as the Society of Environmental

Toxicology and Chemistry (SETAC)/ICA Chris Lee Award for

Metals Research, sponsored by the International Copper

Association, to R.W.M.K. This work was approved by the

University of Saskatchewan’s Animal Research Ethics Board

and adhered to the Canadian Council on Animal Care guidelines

for humane animal use.

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