Chemosphere 73 (2008) 10901095

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Effects of a selenium-enriched diet on antioxidant response in adult craysh

(Procambarus clarkii)
Ambrosius Josef Martin Drr a, Nicole Pacini a, Maria Cesarina Abete b, Marino Prearo b,c,
Antonia Concetta Elia a,*
a

Laboratory of Ecotoxicology, Department of Cellular and Environmental Biology, University of Perugia, 06123 Perugia, Italy
C.Re.A.A. National Reference Centre for the Surveillance and Monitoring of Animal Feed, State Veterinary Institute, Torino, Italy
c
Laboratory of Fish Pathology and Aquaculture, State Veterinary Institute, Torino, Italy
b

a r t i c l e

i n f o

Article history:
Received 5 April 2008
Received in revised form 13 July 2008
Accepted 21 July 2008
Available online 9 September 2008
Keywords:
Procambarus clarkii
Antioxidant enzymes
Glutathione
Selenium
Diet

a b s t r a c t
Many diets employed in aquaculture are enriched with selenium to improve the diet quality and its conservation. The aim of the study was to investigate the effects of a diet enriched in selenium
(1.21 mg kg1) on the antioxidant response of Procambarus clarkii (Girard, 1852). Craysh fed a diet with
lower selenium content (0.30 mg kg1) were the control. Selenium accumulation, enzymatic activities,
and total glutathione were evaluated in hepatopancreas of adults of both sexes fed with both diets for
50 days at two experimental times (T30, T50 days). Treated females exhibited the highest selenium bioaccumulation during both experimental times, while treated males displayed the highest selenium concentration after 30 days, compared to control craysh. A sex-related difference was found for the response of
the analyzed enzymes in the selenium diet-treated specimens. In fact, superoxide dismutase, glutathione
peroxidase and glyoxalase I activities in males were more sensitive compared with females, showing
depleted activities in both experimental times. Catalase activity was induced in females (T50), while glutathione S-transferase activity was the highest in treated females and the lowest in treated males, compared with own controls. Only glutathione reductase activity and glutathione content showed the same
trend in both sexes, which were both lowered in treated specimens, when compared with control craysh. This result might be due to the effect of selenium toxicity on this freshwater species. Males and
females of P. clarkii showed a different susceptibility to the prooxidant effects in a Se-enriched diet.
2008 Elsevier Ltd. All rights reserved.

1. Introduction
Selenium is an essential trace element for animals. It belongs to
group VI of the periodic table, and is similar to the homologous sulphur. In the environment, selenium is present as selenate SeO2
4
and selenite SeO2
3 . Among its inorganic forms, the reduced selenium is present as Se2; in organic forms it can be substituted in
amino acids as cystein-Se, cystin-Se and methyonin-Se. CysteinSe (one selenium atom substitutes the sulphur), is a component
of seleniumproteins, with important enzymatic functions in
organism (Barceloux, 1999).
Many of them are important Se-dependent enzymes with Secystein in their active site. The chemical difference between cystein and Se-cystein is that the rst shows the sulphidrilic protonated group in physiologic pH, while the analogous group of the
other one is dissociated, to permit the catalytic role of selenium
in Se-proteins (Combs and Combs, 1984). Selenium is essential
* Corresponding author. Tel.: +39 0755855717/18; fax: +39 0755855733.
E-mail address: elia@unipg.it (A.C. Elia).
0045-6535/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2008.07.054

for growth of sh (Diplock and Hoekstra, 1976; Reddy and

Massaro, 1983; Sunde, 1984). However, there is a ne partition line
between deciency and toxicity. In fact, in high cellular concentration selenium can be utilized instead of sulphur, causing errors in
protein synthesis, compromising the functionality of proteins.
High selenium concentration may cause bioaccumulation in the
trophic chain, representing a risk for organisms (Sorensen, 1991;
Hamilton, 2004), but its toxicity depends essentially on its chemical structure. In vitro studies (Seko et al., 1989) demonstrated that
the reduction of selenium facilitates the reactions with the
sulphidrilic groups of molecules containing thiol, like glutathione
(GSH), producing some intermediate metabolites, such as Se-diglutathione (GSSeSG), glutathioneselenol (GSSeH), hydrogen
selenide (HSe), elemental selenium (Se0) with hydrogen peroxide
and radical anion superoxide O
2 .
Many diets employed in aquaculture are enriched with selenium to improve the diet quality and its conservation. The maximum selenium concentration allowed in diets employed in
aquaculture is 0.5 mg kg1 (Directive 70/524/ECC). Recently, during a monitoring program for the control of diet quality, carried

A.J.M. Drr et al. / Chemosphere 73 (2008) 10901095

out by C.Re.A.A. National Reference Centre for the Surveillance

and Monitoring of Animal feed (Italy) was found a diet with an
higher selenium concentration (1.21 mg kg1).
Selenium accumulation and enzymatic activities of superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (Se
GPx), glutathione reductase (GR), glutathione S-transferase (GST),
glyoxalase I (GI) and total glutathione content were investigated
in hepatopancreas in both sexes of adult Procambarus clarkii (Girard, 1852) fed for 50 days either a diet enriched in selenium
(1.21 mg kg1) and a selenium-control diet (0.30 mg kg1). Since
works are scanty on sex-specic antioxidant response against Setoxicity in craysh (Elia et al., 2007), we studied antioxidant response in male and female P. clarkii in order to investigate their
detoxicant ability. Furthermore, P. clarkii is the predominant craysh species in aquaculture all over the world.

2. Materials and methods

2.1. Experimental design
Adult P. clarkii were collected from Lake Trasimeno, reparted for
sex, and acclimatised without food subministration for 15 days in
four ow-through tanks of 2 m2 each at the Fish Breeding Center of
Lake Trasimeno (S. Arcangelo, Umbria, Italy). In each tank were collocated three multi-hole bricks (24 holes/brick) used as shelter by
craysh. After the acclimatisation period, craysh were fed ad libitum either a commercial control diet (0.30 mg kg1) and a commercial enriched Se-diet (1.21 mg kg1) for 50 days. At each
experimental time (T30T50) craysh were weighed and counted
in order to get the total and mean weight of all specimens for each
tank. Both diets had the same composition, i.e., pellet dimension:
1.51.6 mm; protides: 47%; lipids: 20%; and shmeal: 45%. The
quantity offered to craysh was 1.40% body weight/day from the
start of the experiment until T50 and both diets were fed during
the morning of every experimental day.
Specimens were observed daily in order to record mortality and
moults (Table 1). The water of all experimental tanks was monitored daily for chemical and physical parameters to keep them
constant and similar in all tanks (pH 7.9 0.5; T C 23.0 3.5;
O2 mg L1 7.9 0.3; Cond. lS cm1 1383.9 11.5). After 30 and
50 days of treatment, samples of 10 individuals from each of the
four tanks were removed and transferred to the laboratory. Further, vesix specimens from each experimental group of 10 were
selected and the chemical and biochemical analyses were conducted separately on each animal. The morphometric parameters
were recorded for all samples (Table 1), and the hepatopancreas

Table 1
Morphometric parameters of P. clarkii, moults and mortality for each experimental
group
Number
of
specimens

Cephalothorax
length (mm)

Weight
(g)

Number
of
moults

Mortality
after
moult

Total
mortality

T30
CF
F
CM
M

21
24
25
26

52.4 2.7
58.3 4.6
54.0 3.1
55.4 1.5

31.2 5.3
40.4 6.9
37.4 1.6
36.2 5.9

2
2
2
2

0
2
2
1

1
2
2
3

T50
CF
F
CM
M

10
12
13
13

54.9 1.4
55.6 1.7
52.8 1.3
51.2 1.7

35.2 3.5
35.3 2.8
33.4 2.5
28.9 1.1

0
1
0
1

0
1
0
0

0
1
2
0

C = control; F = females; M = males.

1091

of each individual was removed and stored at 80 C until

analysed.
2.2. Selenium determination
Tissue samples (max 1 g) were mineralized in microwave oven
with 8 ml of pure nitric acid 65% and hydrogen peroxide 30%
(1.5 ml), then ltered after the digestion through ashless lter paper (diameter 90 mm, retention 7 lm, SchleicherSchuell) into a
25 ml ask and carried to volume with ultrapure water (Aleixo
and Nbrega, 2003). The PerkinElmer AAnalyst 800 atomic
absorption spectrophotometer equipped with transversely heated
graphite atomizer (THGA) system, with Zeeman-effect for background correction and integrated autosampler was used. End cap
graphite tubes with a pyrolytic graphite coating and platforms
made of a pyrolytic graphite were used throughout the work. Argon of 99.998% purity was used as inert gas. Operative parameters
were: wavelength 196.0 nm, slit width 0.7 nm, a mixture of Pd
(NO3)2 and Mg (NO3)2, in nitric acid (0.2%) as matrix modier.
The determination of selenium was carried out by method of standard additions; the standard concentrations were 25
50 ppb (lg L1). The correlation coefcient (R) of the calibration
curves was >0.990 and the quantication limit (LOQ) was
0.01 ppm (mg kg1).
To determine the concentration of selenium in the samples the
following formula was used.
Selenium (mg kg1) = (lg L1 in sample solution) (nal volume
25 ml)/(g sample weight g) 1000.
2.3. Enzymes and glutathione determination
The biochemical analyses were conducted on the cellular cytosolic fractions. Each hepatopancreas analyzed for the enzyme
activities (0.5 g) was homogenized in 10 volumes of 100 lmM TRIS
buffer, pH 7.8, containing 100 lM phenylmethylsulphonyl uoride
(PMSF) and bacitracin 0.1 mg mL1 as reported in Elia et al. (2006).
Superoxide dismutase (SOD) activity was determined by measuring the reduction of cytochrome c by the xanthine/hypoxanthine
system (McCord and Fridovich, 1969) at 550 nm. One unit of SOD
is dened as the amount of enzyme that inhibits by 50% the reduction of cytochrome c. Different volumes of sample were utilized to
determine the 50% inhibition of reaction rate. The amount of SOD
activity was expressed as U mg1 protein.
CAT activity was measured following the decrease in absorbance at 240 nm due to H2O2 consumption (e = 0.04 mM1 cm1),
following the method of Greenwald (1985). SeGPx activity toward
H2O2 as substrate was determined and the oxidation of NADPH
was followed at 340 nm (e = 6.22 mM1 cm1) according to Lawrence and Burk (1976). GR activity was assayed following the decrease in absorbance at 340 nm (e = 6.22 mM1 cm1) due to
the oxidation of NADPH according to Chung et al. (1991). GST
activity was measured according to Habig et al. (1974) using the
substrate 1-chloro-2,4-dinitrobenzene (CDNB). The assay of the
activity with CDNB measured the formation of the conjugate with
GSH at 340 nm (e = 9.6 mM1 cm1). GI activity was determined
according to Oray and Norton (1977) at 240 nm (e = 3.37
mM1 cm1) using 1.5 mM GSH/methylglyoxal hemithioacetal as
substrate. Enzymatic activities were analyzed at 25 C. Protein concentration was determined according to the method reported in
Lowry et al. (1951).
Weighted tissues (0.5 g) were used for the determination of
cytosolic total glutathione analysis as previously reported by Elia
et al. (2006). Total glutathione content (GSH + 2GSSG) of hepatopancreas of both sexes was assayed by the GR recycling assay at
412 nm according to Akerboom and Sies (1981). The reaction

1092

A.J.M. Drr et al. / Chemosphere 73 (2008) 10901095

was monitored at 412 nm and the amount of total glutathione was

expressed as nmol/g tissue.
2.4. Statistical analysis
MannWhitney U test was used to test differences between
samples. Biochemical and chemical data were conducted individually in vesix specimens of each experimental group and run by
triplicate. Results are given as medians, minimum and maximum.
Signicance was tested at the 5% level (P < 0.05).
3. Results
The number of moults was low at both experimental times and
the low mortality was essentially due to moults for both treated
and untreated specimens (Table 1). Females fed with Se-enriched
diet (1.21 mg kg1) evidenced an increased selenium content of
about 60% at T30 and of about 80% at T50, compared to own control
(0.3 mg kg1), while treated males exhibited a slight increase only
at T30, compared to their control (Fig. 1). Higher SOD activity was
recorded in Se-treated females, but did not reach the statistical signicance; on the contrary, activity was markedly reduced in males
for about 80100%, during all investigated times. Controls of both
sexes at T50 showed also higher activity of about 50% compared
with controls of the rst experimental time (Fig. 2). CAT activity

of Se-treated females showed an increased level (+130%) at T50;

on the contrary, activity in control males was higher at the second
experimental time (90%) and similar between treated and untreated specimens (Fig. 2). The hepatic SeGPx activity in Se-treated males was reduced (statistically signicant) for about 4090%,
during all investigated times, while no important variation was recorded for Se-treated females, compared with their controls
(Fig. 3). GR activity measured in Se-treated samples was lowered
for females at T30 (60%) and for males at T50 (80%) compared
with own controls. Enzyme activity in control males was higher
at the second experimental time (60%) (Fig. 3). The specic activity
of GST was higher in Se-treated females compared with the own
control at T30 (+160%) and T50 (55%). Moreover, Se-treated females
at T50, showed a higher enzyme level than at T30 (160%). GST activity of Se-treated males evidenced a reduced activity at T30 (70%)
and at T50 (80%) (Fig. 4) and their control was remarkable higher
at T50, compared with the same control at T30 (100%). GI activity of
Se-treated females, showed a tendency to an increased activity,
and the statistical signicant difference was not reached. Adult
Se-treated males showed a lowered GI activity at T30 (85%) and
T50 (45%), compared to control samples (Fig. 4). Total glutathione
measured in Se-treated females was reduced (statistically signicant) for about 40%, during the second experimental time, while
males showed a lower content of about 30% at T30, compared to
control samples (Fig. 5).

Selenium content in hepatopancreas of females and males of Procambarus clarkii

0.75

*
*

*
*

0.50

mg/kg

*
*

0.25

0.00
CFT30

FT30

CFT50

FT50

F= Se-treated females

CF= control females

CMT30

MT30

CMT50

CM= control males

MT50

M= Se-treated males

Fig. 1. Selenium accumulation (medians, minimum and maximum ranges) in hepatopancreas for both sexes of Procambarus clarkii fed with a selenium-control diet
(0.30 mg kg1) and with a selenium-enriched diet (1.21 mg kg1). The statistical signicant differences between treated and untreated samples are reported by the symbol (*)
(P < 0.05).

Superoxide dismutase (SOD) and catalase (CAT) activities in hepatopancreas of females and males of Procambarus clarkii
100

100

80

a
60

80

* b
*

40

60

b
*

40

a
b

20

20

CAT mol/min/mg prot

SOD U/mg prot

*a

CFT30 FT30 CFT50 FT50 CMT 30 MT30 CMT 50 MT50 CFT30 FT30 CFT50 FT50 CMT 30 MT30 CMT 50 MT50
CF= control females

F= Se-treated females

CM= control males

M= Se-treated males

Fig. 2. SOD and CAT activities (medians, minimum and maximum ranges) in hepatopancreas for both sexes of Procambarus clarkii fed with a selenium-control diet
(0.30 mg kg1) and with a selenium-enriched diet (1.21 mg kg1). The statistical signicant differences between treated and untreated samples are reported by the symbol
(*), and between controls by different letters (a, b) (P < 0.05).

1093

A.J.M. Drr et al. / Chemosphere 73 (2008) 10901095

Glutathione peroxidase (Se-GPx) and glutathione reductase (GR) activities in hepatopancreas of females and males of Procambarus clarkii

150

120

120

*
*

90

90
a *
*

60

60

30

GR nmol/min/mg prot

Se-GPx nmol/min/mg prot

150

30

0
CFT30 FT30 CFT50 FT50 CMT 30 MT30 CMT 50 MT50 CFT30 FT30 CFT50 FT50 CMT 30 MT30 CMT 50 MT50
CF= control females

F= Se-treated females

CM= control males

M= Se-treated males

Fig. 3. SeGPx and GR activities (medians, minimum and maximum ranges) in hepatopancreas for both sexes of Procambarus clarkii fed with a selenium-control diet
(0.30 mg kg1) and with a selenium-enriched diet (1.21 mg kg1). The statistical signicant differences between treated and untreated samples are reported by the symbol
(*), and between controls by different letters (a, b) (P < 0.05).

Glutathione S-transferase (GST) and glyoxalase I (GI) activities in hepatopancreas of females and males of Procambarus clarkii
2000

1500

a *
1500

a*
1000

1000

b*
b*

500

500

GI nmol/min/mg prot

GST nmol/min/mg prot

2000

CFT30 FT30 CFT50 FT50 CMT30 MT30 CMT50 MT50 CFT30 FT30 CFT50 FT50 CMT30 MT30 CMT50 MT50
CF= control females

F= Se-treated females

CM= control males

M= Se-treated males

Fig. 4. GST and GI activities (medians, minimum and maximum ranges) in hepatopancreas for both sexes of Procambarus clarkii fed with a selenium-control diet
(0.30 mg kg1) and with a selenium-enriched diet (1.21 mg kg1). The statistical signicant differences between treated and untreated samples are reported by the symbol
(*), and between controls by different letters (a, b) (P < 0.05).

Total glutathione content in hepatopancreas of females and males of Procambarus clarkii

nmol/g wet tissue

1000
800

600

400
200
0

CFT30

FT30

CF= control females

CFT50

FT50

CMT30

F= Se-treated females

MT30

CM= control males

CMT50

MT50

M= Se-treated males

Fig. 5. Total glutathione (medians, minimum and maximum ranges) in hepatopancreas for both sexes of Procambarus clarkii fed with a selenium-control diet (0.30 mg kg1)
and with a selenium-enriched diet (1.21 mg kg1). The statistical signicant differences between treated and untreated samples are reported by the symbol (*) (P < 0.05).

4. Discussion and conclusion

In the present study, the highest selenium concentration given
with a diet enriched in selenium (1.21 mg kg1), did not cause

mortality for both sexes of P. clarkii. In fact, the low mortality recorded for all samples, fed with control diet or with selenium diet,
was due mainly to the natural event of moult. The number of
moults for craysh was low at both experimental times. As already

1094

A.J.M. Drr et al. / Chemosphere 73 (2008) 10901095

reported by Drr et al. (2006) adults of Procambarus clarkii from

Lake Trasimeno are subjected to moult only one or two times a
year.
Selenium can represent high risk for the exposed organisms
(Hamilton et al., 2002; Hamilton, 2004; Wang et al., 2006; Hoang
and Klaine, 2008).
The evaluation of selenium bioaccumulation in hepatopancreas
of P. clarkii was performed on specimens fed with both diets, measuring the total selenium concentration (organic and inorganic
form). Se-treated females exhibited the highest selenium accumulation throughout all experimental times, while Se-treated males
displayed different selenium level only at T30 compared with
own controls. For both Se-treated sexes, a lower selenium accumulation level was found for craysh sampled after 50 days, compared with those of the rst experimental time.
The effects of Se on the antioxidant enzymes are reported for
living organisms; carp fed a Se yeast-supplemented diet showed
an efcient antioxidant system (evaluated from GPx, CAT, SOD,
GR, GST in erythrocytes and liver) compared to those fed a diet
with sodium selenate (Jovanovic et al., 1997). However, little information is available on the prooxidant effects of Se on the antioxidant response of P. clarkii fed with an enriched selenium diet
(Elia et al., 2007). Our previous results evidenced higher Se accumulation in specimens fed a Se-enriched diet for 15 days. A sex-related difference was found for the response of the analyzed
enzymes in the treated specimens. Catalase and glutathione peroxidase activities were low in both selenium treated females and
males, respectively, whereas glutathione reductase activity
showed signicant reductions in both sexes (Elia et al., 2007).
Our present results conrm those previously reported; P. clarkii
fed with both diets showed different enzymatic trends, related to
sex. SOD enzyme detoxify from superoxide radical by dismutation
and H2O2 formation, while CAT and SeGPx are two enzymes involved in the cellular detoxication of H2O2. Treated males of P.
clarkii evidenced a lower SOD activity in all experimental times,
which might indicate a reduced ability to protect them against the
toxic effect of superoxide anion radical. Males fed with a Se-enriched
diet showed a major susceptibility to oxidative stress, compared
with females, to the prooxidant effect of selenium; SeGPx activity,
which was very similar between control and treated females, was
depleted in treated males. Particularly, the induction of CAT activity
in females fed with a Se-enriched diet, after a longer exposure time
to selenium, represents a valuable defence response of these specimens against the prooxidant effect of reactive oxygen species (ROS).
Selenium in elevated concentration might show toxic effects
with the impairment of enzymatic function. For example, the results of studies conducted on embryos and juvenile trout treated
with Se evidenced an oxidative stress status (Palace et al., 2004)
with a decrease of the GSH-GSSG ratio (Schlenk et al., 2003). The
toxicity of nitrite increased also in prawn Penaeus vannamei when
fed with a high selenium concentration diet. In this case, the
authors evidenced that the reduction of SOD, GPx and CAT activities was more pronounced in prawn fed the higher selenium diet,
than in those of the basal diet (Wang et al., 2006).
The highest selenium content in diet induced a lowering of GR
activity, in both sexes in almost all experimental times, compromising the ability to regenerate GSH, as the main scavenger of oxyradicals, from GSSG. Only few data about the thiol content in this
craysh species and the differences linked to sexes are reported
in the literature (Almar et al., 1987; Elia et al., 2006). Particularly,
the authors evidenced slight differences in glutathione levels between sexes. In the present study, the highest selenium concentration, affected total glutathione of both treated sexes and this result
might be related to the lower glutathione reductase activity found
in these specimens. The prooxidant effect of selenium might induce both a lowering of GR activity and the production of some

toxic intermediate metabolites, which caused a reduced thiol level.

In vitro studies (Seko et al., 1989) demonstrated that selenium produces intermediate metabolites such as Sediglutathione (GSSe
SG), glutathioneselenol (GSSeH) and the hydrogen selenide
(HSe) which might cause damages to membranes and DNA.
Se-treated males showed a lower GST activity, while treated females showed higher activity throughout the experimental times.
Unexpected was the high GST activity, mainly, in control males
after 50 days, and this result can suggest that even the low selenium concentration might affect enzyme activity. GST activity
seems to act as good marker to detect the prooxidant effect, induced by selenium diet.
GI activity is involved in the detoxication of toxic metabolites,
produced during lipid peroxidation. A depletion of GI activity indicates a compromised detoxicant ability against the peroxidative
metabolites. Since the highest selenium concentration affected
only treated males, it seems that males are more susceptible than
females to this prooxidant factor.
In conclusion, the enriched selenium diet caused a different bioaccumulation in P. clarkii for both sexes; females showed an increased selenium concentration throughout all experimental
times, while males displayed the highest Se content only during
the rst period. Selenium concentration affected in different way
the antioxidant response of both sexes of P. clarkii. Males and females show different detoxication ability versus selenium, probably mediated by hormonal control. Further studies carried on
juveniles of P. clarkii fed a Se-enriched diet are ongoing in our laboratory to investigate the effect of selenium on these antioxidant
enzymes and metabolites in this freshwater species.
Acknowledgements
We thank Dr. M. Natali of the Fish Breeding Centre of Lake Trasimeno (S. Arcangelo, Umbria, Italy) for his kindly assistance. This research was supported by Ministero della Salute (Ricerca Corrente,
Italy).
References
Akerboom, T.P.M., Sies, H., 1981. Assay of glutathione disulde and glutathione
mixed disulde in biological samples. Methods Enzymol. 71, 373382.
Aleixo, P.C., Nbrega, J.A., 2003. Direct determination of iron and selenium in bovine
milk by graphite furnace atomic absorption spectrometry. Food Chem. 83, 457
462.
Almar, M.M., Diaz-Mayans, J., Romero, F.J., 1987. Glutathione content and
glutathione S-transferase activity in midgut gland of Procambarus clarkii sex
differences, the effect of fasting, and their implications in cadmium toxicity.
Comp. Biochem. Phys. C 87, 433435.
Barceloux, D.G., 1999. Selenium. J. Toxicol-Clin. Toxic. 37, 145172.
Chung, P.M., Cappel, R.E., Gilbert, H.F., 1991. Inhibition of glutathione disulde
reductase by glutathione. Arch. Biochem. Biophys. 288, 4853.
Combs, G.F., Combs Jr., S.B., 1984. The nutritional biochemistry of selenium. Annu.
Rev. Nutr. 4, 5984.
Diplock, A.T., Hoekstra, W.G., 1976. Metabolic aspects of selenium action and
toxicity. CRC Crit. Rev. Toxicol. 5, 271329.
Drr, A.J.M., La Porta, G., Pedicillo, G., Lorenzoni, M., 2006. Biology of Procambarus
clarkii (Girard, 1852) in Lake Trasimeno. B. Fr. Peche. Piscic. 380381, 1155
1168.
Elia, A.C., Drr, A.J.M., Mastrangelo, C., Prearo, M., Abete, M.C., 2006. Glutathione and
antioxidant enzymes in the hepatopancreas of craysh Procambarus clarkii
(Girard, 1852) of Lake Trasimeno (Italy). B. Fr. Peche. Piscic. 380381, 1351
1361.
Elia, A.C., Drr, A.J.M., Taticchi, M.I., Prearo, M., Abete, M.C., 2007. Detoxication
enzymes of freshwater craysh Procambarus clarkii fed a diet enriched in
selenium: preliminary results. Mar. Freshwater Behav. Phy. 40, 15.
Greenwald, R.A., 1985. Handbook of Methods for Oxygen Radicals Research. CRC
Press, Boca Raton, FL.
Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The rst
enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 71307139.
Hamilton, S.J., 2004. Review of selenium toxicity in the aquatic food chain. Sci. Total
Environ. 326, 121.
Hamilton, S.J., Holley, M., Buhl, K.J., Bullard, F.A., Weston, L.K., McDonald, S.F., 2002.
Toxicity of selenium and other elements in food organisms to razorback sucker
larvae. Aquat. Toxicol. 59, 253281.

A.J.M. Drr et al. / Chemosphere 73 (2008) 10901095

Hoang, T.C., Klaine, S.J., 2008. Characterizing the toxicity of pulsed selenium
exposure to Daphnia magna. Chemosphere 71, 429438.
Jovanovic, A., Grubor-Lajsic, G., Djukic, N., Gardinovacki, G., Matic, A., Spasic, M.,
1997. The effect of selenium on antioxidant system in erythrocytes and liver of
the carp (Cyprinus carpioL.). Crit. Rev. Food. Sci. 37, 443448.
Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in seleniumdecient rat liver. Biochem. Biophys. Res. Commun. 71, 592598.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement
with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
McCord, J.M., Fridovich, I., 1969. Superoxide dismutase: an enzymatic function for
erythrocuprein (hemocuprein). J. Biol. Chem. 244, 60496055.
Oray, B., Norton, S.J., 1977. Two step purication of mouse liver glyoxalase I and
evidence of its dimeric constitution. Biochim. Biophys. Acta 483, 203209.
Palace, V.P., Spallholz, J.E., Holm, J., Wautier, K., Evans, R.E., Baron, C.L., 2004.
Metabolism of selenomethionine by rainbow trout (Oncorhynchus mykiss)
embryos can generate oxidative stress. Ecotoxicol. Environ. Saf. 58, 1721.

1095

Reddy, C.C., Massaro, E.J., 1983. Biochemistry of selenium: an overview. Fundam.

Appl. Toxicol. 3, 431436.
Schlenk, D., Zubcov, N., Zubcov, E., 2003. Effects of salinity on the uptake,
biotransformation, and toxicity of dietary seleno-L-methionine to rainbow
trout. Toxicol. Sci. 75, 309313.
Seko, J., Sarto, Y., Kitahara, J., Imura, N., 1989. Active oxygen generation by the
reaction of selenite with reduced glutathione in vitro. In: Wendel, A. (Ed.),
Selenium in Biology and Medicine. Berlin, Springer-Verlag, pp. 7073.
Sorensen, E.M.B., 1991. Metal Poisoning in Fish. CRC Press, Boca Raton, FL.
Sunde, R.A., 1984. The biochemistry of selenoproteins. J. Am. Oil Chem. Soc. 61,
18911900.
Wang, W., Wang, A., Zhang, Y., 2006. Effect of dietary higher level of selenium and
nitrite concentration on the cellular defense response of Penaeus vannamei.
Aquaculture 256, 558563.