Human and Experimental Toxicology

30(9) 1233–1245
Nephrotoxicity induced by chromium ª The Author(s) 2010
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(VI) in adult rats and their progeny DOI: 10.1177/0960327110387454
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Nejla Soudani1, Mediha Sefi1,*, Hanen Bouaziz1,*,

Yassine Chtourou1, Tahia Boudawara2 and
Najiba Zeghal1

Abstract
To assess kidney damages in pregnant and lactating rats and in their suckling pups, Wistar female rats were
given, through drinking water, 700 parts per million (ppm) of K2Cr2O7 from the 14th day of pregnancy until
day 14 after delivery. Toxicity was objectified by a significant increase in malondialdehyde (MDA), glutathione
(GSH) and nitric oxide (NO) levels in kidney of chromium-treated mothers and their suckling pups. Moreover,
lactate dehydrogenase (LDH) was increased in kidney and decreased in plasma of K2Cr2O7-treated rats.
Activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were increased
in dams and decreased in their pups. Interestingly, these biochemical modifications were accompanied by
higher plasma and lower urinary levels of creatinine, a specific indicator of glomerular function, and of urea
than those of controls. Significant increase in creatinine clearance was also found in treated mothers and in
their progeny. Histological studies showed an infiltration of mononuclear cells, necrosis and vascular congestion
in kidney of pups and dams. Based on the present findings, K2Cr2O7 administrated to female rats during late
pregnancy and early postnatal periods provoked kidney damages in dams and their offspring.

Keywords
potassium dichromate, suckling rats, nephrotoxicity, histopathological studies

Introduction to Cr(VI). This metal could also traverse the placental

Chromium (Cr) in the environment occurs predomi-
nantly in two valence states: hexavalent [Cr(VI)] and
trivalent chromium [Cr (III)].1,2 Due to its widespread
use in leather tanning, textile and pigment electroplating
industries, Cr(VI) becomes one of the most abundant
pollutants in aquatic and terrestrial ecosystems.3 The
deposition of Cr(VI) wastes in landfills and waterways
by chromate industries affects millions of people drink-
ing Cr-containing water, residing in the vicinity of dan-
gerously polluted sites.4 The wide environmental
distribution in Cr leads to an increased interest of its
toxicity and biological effects.5 In reproductive toxicol-
ogy, Cr is highly toxic and affects growth of fetuses
and suckling pups in humans and animals. Indeed,
women working in Cr industries and living around
Cr-contaminated areas present gynaecological illness,6
postnatal hemorrhage and birth complications with high
levels of Cr in blood and urine.7,8 Interestingly, it has
been reported by Barceloux,9 that Cr was transported
to offspring through milk of lactating women exposed
barrier in rodents.9,10 Previous findings demonstrated
that Cr, administrated through drinking water for
20 days before gestation state, revealed abnormal
gestational changes in mice and rats, with a decreased
number of implantation sites and of viable fetuses.11-13
According to Junaid et al.,14 K2Cr2O7 produces embry-
otoxic and fetotoxic effects.
The exact mechanism of Cr(VI)-induced toxicity
has not been elucidated yet; however, reduction of
Cr(VI) and related free radical reactions have been
1
Animal Physiology Laboratory, Sfax Faculty of Sciences, Sfax,
Tunisia
2
Anatomopathology Laboratory, CHU Habib Bourguiba, Sfax,
Tunisia
* Authors contributed equally to this work
Corresponding author:
Najiba Zeghal, Animal Physiology Laboratory, Life Sciences
Department, Sfax Faculty of Sciences, BP1171, 3000 Sfax, Tunisia
Email: najiba.zeghal@tunet.tn
1234
Table 1. Body and relative kidney weights of suckling and adult rats, controls and K2Cr2O7 treated with 700 parts per
million (ppm) administered in their drinking water from the 14th day of pregnancy until day 14 after deliverya
Parameters and treatment Controls
Final body weight (g; n ¼ 6) 191.83 +
Relative kidney weight (mg/g bw; n ¼ 6) 0.41 +
Average food intake (g/day/mother; n ¼ 14) 31.59 +
Water consumption (mL/day/rat; n ¼ 14) 41.03 +
Quantities of K2Cr2O7 ingested
(mg/day/mother; n ¼ 14)
a Food intake and quantities of K2Cr2O7 ingested by lactating rats. The number of determinations is indicated in parenthesis. Data are
shown as means + SD.
b
K2Cr2O7 treated vs controls: p < 0.05.
c
K2Cr2O7 treated vs controls: p < 0.001.
d
K2Cr2O7 treated vs controls: p < 0.01.
postulated as possible mechanisms mediating the
toxicity of Cr(VI) compounds. In fact, free radicals
produce a number of toxic effects including DNA
damage and lipid peroxidation; therefore, the toxic
effects of Cr(VI) may be, at least in part, associated
with the production of reactive species via Fenton
or Haber-Weiss-type reactions.15 Since the kidney is
a main target organ of K2Cr2O7, this metal causes
acute renal failure (ARF) in mammals.16,17 Previous
findings showed that chromate affected selectively
the convoluted section of the proximal tubules17-19
and induced acute necrosis of renal tubules.19,20
To our knowledge, no studies have been conducted
on kidney of suckling rats whose mothers were treated
by K2Cr2O7. Therefore, the present experiment was
undertaken to evaluate biochemical parameters, lipid
peroxidation, oxidative stress and histopathological
changes in kidney of female rats and their progeny
following subchronic exposure to K2Cr2O7 during
late pregnancy and lactating periods.
Materials and methods
Chemicals
Potassium dichromate (K2Cr2O7), was obtained
from Merck (Darmstadt, Germany). Sodium selenite
(Na2SeO3) was purchased from Sigma (St. Louis,
Missouri, USA). All other chemicals of analytical
grade were purchased from standard commercial
suppliers.
Animals and treatments
Experiments were performed on female Wistar rats
weighing 170 + 10 g and purchased from SIPHAT
Human and Experimental Toxicology 30(9)
Mothers Offspring (n ¼ 30)
K2Cr2O7 treated Controls K2Cr2O7 treated
5.19 184.33 + 4.08b 23.57 + 1.51 17.51 + 2,13c
0.01 0.41 + 0.02 0.49 + 0.04 0.55 + 0.07d
1.64 28.81 + 3.71c
4.81 37.81 + 4.57d
26.46 + 3.81
(Tunisia). After 1 week of adaptation period in a room
with controlled temperature (22 + 2 C) and lighting
(12 h-light/12 h dark), female rats were mated with
males. A sperm-positive vaginal smear was taken to
indicate the first day of pregnancy. They were housed
individually in stainless steel cages and were
provided daily with standard pellet diet (SICO, Tuni-
sia) and water ad libitum. Twelve pregnant female
rats were randomly divided into two groups of six
each: group I served as controls and group II received
700 parts per million (ppm) of K2Cr2O7 (equivalent to
67 mg/kg) through the drinking water from the 14th
day of pregnancy until day 14 after delivery. Since the
major route of Cr for the general population is the oral
way,11 the present study is designed to investigate the
toxicity of K2Cr2O7 given to rats via the oral route.
The dose of K2Cr2O7 and the period of treatment were
selected on the basis of previous studies.11,14,21-23
The levels of K2Cr2O7, used in the present study, are
not usually found in the environment but may be
encountered in the workplace or in the vicinity of
industrial establishments.11 The day of parturition
was considered as day 0 of lactation. Pups were
counted, weighed and each litter was reduced to eight
pups (four males and four females, if possible) as it
has been shown that this procedure maximized lacta-
tion performance.24 Daily K2Cr2O7 intake by lactat-
ing rats was determined after measuring drinking
water consumption. So each lactating rat, treated with
K2Cr2O7, ingested daily 26.46 + 3.81 mg of K2Cr2O7
(Table 1). All animals were observed for signs of
treatment-related effects.
The experimental procedures were carried out
according to the general guidelines on the use of liv-
ing animals in scientific investigations25 and approved
Soudani N et al.
by the Ethical Committee of Sciences Faculty of Sfax.
The amount of ingested diet was calculated as the
difference between the weight of feed that remained
in the food bin (Da) and the amount placed 1 day before
(Db). These data were then used to calculate the daily
average feed intake, according to the formula:
Average feed intake ¼ ðDb  DaÞ
Quantities of K2Cr2O7 ingested by each rat were
calculated from daily water consumption (Table 1).
Before sacrifice, urinary samples were obtained
from each animal housed in a specially designed
metabolic cage, where fecal contamination was
avoided. They were collected into bottles within 24-h
cycles. Urinary volume of 14-day-old rats was calcu-
lated by taking away the 24-h urine volume of mothers
kept with their pups and those kept alone in metabolic
cages. The volume of each urine sample was recorded
and centrifuged at 3000 g for 5 min.
On postnatal day 14, 12 dams and 96 pups were
anesthetized with chloral hydrate by intra-abdominal
route. After sacrifice, blood samples were collected
into heparin tubes by aortic puncture in dams and
brachial artery in pups. Plasma samples were drawn
from blood after centrifugation at 2200 g for 15 min.
They were kept at 80 C until analysis.
Kidneys were removed, cleaned and weighed.
Some samples were rinsed, homogenized (10% w/v)
in phosphate buffer (pH 7.4) and centrifuged. The
resulting supernatants were used for biochemical
assays. Others were immediately fixed in 10% formalin
solution for histological studies.
Biochemical determinations
Estimation of urea, uric acid, creatinine and creatinine
clearance. The levels of urea, uric acid and creatinine
in plasma and urine were estimated spectrophotome-
trically using commercial diagnostic kits (ref 20151,
20143, 20091, respectively) purchased from Bioma-
greb (Ariana, Tunisia). Creatinine clearance, an index
of glomerular filtration rate, was calculated by UV/P
equation26 where U is the urinary creatinine level,
V the volume of urine sample collected within 24 h
and P the plasma creatinine concentration.
Lipid peroxidation. Concentration of MDA in tissues, an
index of lipid peroxidation, was determined spectropho-
tometrically according to Draper and Hadley method.27
A 0.5-mL aliquot of kidney extract supernatant was
mixed with 1 mL of trichloroacetic acid solution and
1235
centrifuged at 2500 g for 10 min. One millilitre of a solu-
tion containing 0.67% thiobarbituric acid (TBA) and 0.5
mL of supernatant were incubated for 15 min at 90 C
and cooled. Absorbance of TBA-MDA complex was
determined at 532 nm using spectrophotometer. Lipid
peroxidation was expressed as nmol of thiobarbituric
acid reactive substances (TBARS), using 1,1,3,3-tetra-
ethoxypropane as standard.
Estimation of kidney nitric oxide level. Production of
nitric oxide (NO) was determined based on the Griess
reaction.28 Briefly 100 mL of deproteinized sample
was incubated with 100 mL of Greiss reagent at room
temperature for 10 min. Absorbance was measured at
550 nm, using a microplate reader. Nitrite concentration
was measured using sodium nitrate as standard and was
expressed as nmol/mg protein of tissue.
Non-protein thiols content in kidney. Kidney non-protein
thiols (NPSH) levels were determined by the method
of Ellman.29 A 500 mL aliquot of supernatant was
mixed with 10% trichloroacetic acid (1V/1V). After
centrifugation, the protein pellet was discarded and
free–SH groups were determined in a clear superna-
tant. A 100 mL aliquot of supernatant was added to
850 mL of 1 M potassium phosphate buffer (pH ¼ 7.4)
and to 50 mL of 5,5-dithiobis-2 nitro benzoic acid
(DTNB; 10 mM). The absorbance of colorimetric
reaction was measured at 412 nm.
Estimation of lactate dehydrogenase (LDH) activities.
Lactate dehydrogenase activities in plasma and
kidney, used as biochemical markers for renal
damage, were determined by enzymatic method using
commercial reagent kit (Biomaghreb, Ariana, Tunisia.
Ref 20012).
Protein analysis in kidney. Protein content in kidney was
determined according to Lowry et al30 using Bovine
serum albumin as standard.
Antioxidant defense system assays. The catalase (CAT)
activity was determined according to the method of
Aebi.31 The H2O2 decomposition rate was followed
by monitoring absorption at 240 nm. One unit of CAT
activity is defined as the amount of enzymes required
to decompose 1 mmol of hydrogen peroxide in 1 min.
The enzyme activity was expressed as mmol H2O2
consumed/min/mg protein.
Glutathione peroxidase (GPx) activity was measured
according to Flohe and Gunzler.32 The enzyme activity
1236
was expressed as nmol of GSH oxidized/min/mg
protein.
Superoxide dismutase (SOD) activity was estimated
according to Beauchamp and Fridovich.33 The reac-
tion mixture contained 50 mM of tissue homogenates
in potassium phosphate buffer (pH 7.4), 0.1 mM
L-methionine, 2mM riboflavine and 75 mM Nitro Blue
Tetrazolium (NBT). The developed blue color reaction
was measured at 560 nm. Units of SOD activity were
expressed as the amount of enzyme required inhibiting
the reduction of NBT by 50% and the activity was
expressed as U/mg protein.
Reduced glutathione (GSH) levels were determined
by the method of Ellman29 modified by Jollow et al34
based on the development of a yellow color when
DTNB was added to compounds containing sulfhydryl
groups. Five hundred milliliters of tissue homogenate
were added to 3 mL of 4% sulfosalicylic acid. The
mixture was centrifuged at 1600 g for 15 min. Five
hundred milliliters of supernatant were taken and
added to Ellman’s reagent. The absorbance was
measured at 412 nm after 10 min. Total GSH content
was expressed as mg/g tissue.
Histopathological examination
Kidney histological sections of 5 mm were placed on
slides and stained with hematoxylin-eosin for histo-
pathological studies. Six slides were prepared from
each kidney. All sections were evaluated for the
degree of tubular and glomerular injury and necrosis.
Each kidney slide was examined and assigned for
severity of changes using scores on a scale of none
(), mild (þ), moderate (þþ) and severe (þþþ)
damages.
Statistical analysis
Comparison of mean values followed by SD between
treated and control rats was made using Student’s
t-test.35 The 0.05 level was selected as the point of
minimal statistical significance.
Results
Evaluation of body, absolute and relative kidney
weights
During the experimental period, there are no clinical
signs and no death in K2Cr2O7-treated group. Food
and water consumption was reduced by 9% and 8%,
respectively, in K2Cr2O7-treated dams when compared
to controls (Table 1). The body weights of Cr-treated
Human and Experimental Toxicology 30(9)
rats were decreased by 4% in mothers and 26% in their
offspring as compared to controls. Relative kidney
weights of treated dams were similar to those of control
rats, while a marked increase in the relative kidney
weight was observed in pups (þ12%) when compared
to corresponding controls.
Urinary volume, creatinine, urea and uric acid
levels in plasma and urine
K2Cr2O7-intoxicated rats showed a constellation of
disorders in renal function witnessed by increased
urine output and changes in creatinine, urea and uric
acid levels. In fact, the 24-h urine volume in treated
mothers and in their pups was higher than in the con-
trols (þ52% and þ56%, respectively; Figure 1A).
Creatinine levels in K2Cr2O7-treated mothers and
their pups were higher in plasma (þ87%; þ85%) and
lower in urine (10; 28%) than those of correspond-
ing controls (Table 2). So we have found, after
K2Cr2O7 treatment, a reduction in creatinine clear-
ance, an indicator of glomerular dysfunction in adult
rats (32%) and in their pups (37%; Figure 1B).
Urea levels in treated mothers and in their suckling
pups were higher in plasma (þ86%; þ40%) and lower
in urine (57%; 32%), respectively, than those of
controls (Table 2). While uric acid levels were lower
in plasma of K2Cr2O7-treated mothers (44%) and of
their offspring (37%) than those of corresponding
controls. Urinary excretion of uric acid, a nitrogenous
waste product, was increased in K2Cr2O7-treated
mothers (þ37%) and their pups (þ59%) compared
to controls (Table 2).
Kidney malondialdehyde content
Subacute toxicity of K2Cr2O7 provoked a significant
increase in kidney malondialdehyde (MDA) levels
in dams (77%) and in their offspring (1.5-fold). The
rate of lipid peroxidation in kidney was more pro-
nounced in pups than in their mothers (Table 3).
Lactate dehydrogenase activities in kidney and
plasma
In K2Cr2O7 group, lactate dehydrogenase activity, a
marker of kidney cellular damage, was increased in
plasma by 53% and 57% and decreased in kidney
extracts by 61% and 47%, respectively, in adult rats
and in their suckling pups (Figure 2).
Soudani N et al. 1237

their suckling pups (Table 3), while, NPSH levels

A Mothers Offspring decreased significantly by 47% in mothers and by
62% in their offspring (Table 3).
14 ***
(6)
Effects of K2Cr2O7 on kidney antioxidant enzyme
activities
10 K2Cr2O7 treatment led to a significant increase in
Urinary volume (ml/24 h)

(6)
CAT, SOD and GPx activities in kidney homogenates
by 86%, 65% and 22%, in mothers and a decrease of
6 *** these parameters by 50%, 47% and 48% in their pups,
(8)
2 respectively, when compared to those of control
group (Figure 3).
(8)

1 Histopathological studies
Abnormalities in kidney histological pictures,
detected in glomeruli and in convoluted tubules, were
0
CT K2Cr2O7 CT K2Cr2O7 shown in mothers (Figure 4B1, B2, B3 and B4) as
well as in their pups (Figure 5D1, D2, D3 and D4)
compared to those of corresponding controls
B Mothers Offspring (Figures 4A and 5C). The severity of changes was
270 assessed for each slide by scoring using a scale of
no (), mild (þ), moderate (þþ) and severe (þþþ)
220 (6) damage. The kidney of K2Cr2O7-treated mothers
exhibited vascular congestion inside glomeruli and
Creatinine clearance (µl/min)

170 *** between tubules and narrowed Bowman’s space.

(6)
Furthermore, convoluted tubules were dilated (Figure
120 B1, B2). Tubular cells showed vacuoles formation,
which indicates a necrosis step (Figure 4B2). Infiltra-
70
40 (8)
tion of lymphocytes and polynuclear cells particularly
between tubules was also observed (Figure 4B3, B4).
30 **
(8) Moreover, in their pups, we have observed tubular
20 dilatation and cell necrosis (Figure 5D2, D4). Nonethe-
less, these effects are more pronounced in mothers.
10
The histopathological changes were graded and sum-
0 marized in Table 4.
CT K2Cr2O7 CT K2Cr2O7

Figure 1. Urinary volume (A) and creatinine clearance (B)

in adult rats and their pups: controls and K2Cr2O7 treated
with 700 parts per million (ppm) administered in the drink-
ing water from the 14th day of pregnancy until day 14 after
delivery. K2Cr2O7 treated versus controls: **p < 0.01;
***
p < 0.001. Data are shown as means + SD. The number
of determinations is indicated above columns of each
diagram.
Kidney NO, GSH and NPSH levels
In K2Cr2O7 group, NO and GSH levels in kidney
homogenates were increased by 64% and 27%,
respectively, in adult rats and by 65% and 26% in
Discussion
Throughout the course of gestation to parturition,
females’ biological systems were deeply modified in
order to ensure embryo-fetal development and
well-being.36 These disorders threatened both mothers
and their fetuses. Impairment of gestation and compli-
cations during pregnancy and childbirth have been
reported in female workers exposed to Cr at the work-
place.7,8 The same disorder was also observed in mice
exposed to Cr(VI) during gestation period.11,14,21,22,37
In the present study, we have focussed our attention
on K2Cr2O7 potential causing alterations in kidney
1238 Human and Experimental Toxicology 30(9)

Table 2. Plasma and urinary levels of creatinine, urea and uric acid in adult rats and their pups, controls and K2Cr2O7–
treated from the 14th day of pregnancy until day 14 after deliverya
Mothers Offspring
Parameters and treatments Controls (n ¼ 6) K2Cr2O7 (n ¼ 6) Controls (n ¼ 8) K2Cr2O7 (n ¼ 8)
Creatinine (mmol/L)
Plasma 126.05 + 16.92 235.73 + 26.91b 102.15 + 8.28 188.59 + 14.49b
Urine 4184.27 + 344.71 3752.09 + 212.82c 4769.67 + 240.78 3446.61 + 213.47b
Urea (mmol/L)
Plasma 5.42 + 0.46 10.65 + 0.93b 6.14 + 0.98 8.59 + 0.91d
Urine 3.42 + 0.42 1.45 + 0.27b 3.96 + 0.77 2.67 + 0.3b
Uric acid (mmol/L)
Plasma 252.24 + 21.72 142.45 + 21.15b 218.24 + 20.88 138.11 + 28.95b
Urine 1111.82 + 73.09 1520.82 + 116.91d 1592.82 + 116.91 2540.69 + 219.35b
a
The number of determinations is indicated in parenthesis. Data are shown as means + SD.
b
K2Cr2O7 treated vs controls: p < 0.001.
c
K2Cr2O7 treated vs controls: p < 0.05.
d
K2Cr2O7 treated vs controls: p < 0.01.

Table 3. MDA, NO, GSH and NPSH contents in kidneys of adult rats and their pups controls and K2Cr2O7 -treated with
700 parts per million (ppm) administered in their drinking water from the 14th day of pregnancy until day 14 after deliverya
Parameters and treatments MDAb NOc GSHd NPSHe
Mothers
Controls 42.23 + 4.39 10.31 + 2.07 74.56 + 4.77 1.99 + 0.14
K2Cr2O7 74.94 + 8.81f 16.93 + 2.77f 94.79 + 8.13f 0.76 + 0.08f
Offspring
Controls 77.91 + 12.93 11.78 + 2.25 80.56 + 9.76 2.16 + 0.23
K2Cr2O7 192.71 + 20.32f 19.42 + 3.21f 101.43 + 6.98f 1.43 + 0.16f
Abbreviations: GSH: glutathione, MDA: malondialdehyde, NO: nitric oxide, NPSH: non-protein thiols.
a
The number of determinations is indicated between parenthesis. Data are shown as means + SD.
b
MDA ¼ nmoles/g tissue.
c
Nitric oxide ¼ nmol/mg protein.
d
Glutathione-glutathione ¼ mg/g tissue.
e
Non-protein thiols ¼ mmol/g tissue.
f
K2Cr2O7 treated vs controls: p < 0.001.

function of female rats and their pups during the
suckling period.
The results, presented in this work, clearly showed
several modifications, induced by K2Cr2O7 during
late pregnancy and early postnatal periods, resulted
in nutritional status and metabolism. In fact, ingestion
of Cr(VI), through drinking water, induced a signifi-
cant decrease in food consumption and in water intake
by female rats. So, body and kidney weights of their
offspring were altered. These findings might be attrib-
uted to a Cr transfer through both placenta and milk as
demonstrated either by Barceloux,9 or/and by cell
growth inhibition of the cell cycle.38 Similar results
were observed by Gilbert et al.39 in humans exposed
to chromium picolinate.
In the present study, K2Cr2O7 strongly affected
plasma and urinary parameters such as the 24-h urine
volume, creatinine levels and creatinine clearance.
In the current study, oral administration of K2Cr2O7
to lactating rats induced an increase in 24-h urine out-
put both in mothers and in their offspring. Polyuria
might be explained by the inhibition of antidiuretic
hormone secretion, which normally provoked water
reabsorption across the collecting ducts after exposure
to metals.40 In the present investigation, a reduction of
the 24-h creatinine excretion and of creatinine clear-
ance indicated kidney impairment since these para-
meters were used as qualitative and quantitative
indexes of the alteration in glomerular filtration rate
(GFR).
Soudani N et al. 1239

and/or with the conversion of ammonia to urea as a

A Mothers Offspring result of increased synthesis of arginase enzyme
involved in urea production. Another biochemical
*** *** marker, used in this study to evaluate kidney function,
400 (6) (8)
was uric acid levels in plasma and urine. In fact, many
drugs could affect plasma uric acid levels by influencing
the net reabsorption of uric acid in the proximal tubules
300
of the nephron.41 Under our experimental conditions,
Plasma LDH activity (IU/l)

(8) K2Cr2O7 poisoning in adult and young rats resulted in

(6) a decrease in plasma uric acid levels and an increase
200 in its urinary excretion. Kidney dysfunction could
probably occur via kidney oxidative damage. In fact,
uric acid is a potent scavenger of peroxynitrite.42
100 Once inside the cell, Cr(VI) is rapidly reduced in
order to generate ROS,43,44 so reactive Cr intermedi-
ates can directly interact with cellular constituents.
Indeed, Cr V, Cr IV and the more stable Cr III are all able
0
CT K2Cr2O7 CT K2Cr2O7
to generate free radicals such as superoxide, nitrogen
species like peroxynitrite, NO and hydroxyl causing
damage consistent with oxidative stress.45 Free radicals
B Mothers Offspring can damage virtually all types of macromolecules
including lipids, causing an increase of kidney MDA
125 (6)
content,5 the major product of lipid peroxidation. In fact,
in our study, we have found that Cr(VI) treatment
(8) increased the amount of MDA levels in kidney tissue
100 of mothers and their pups, suggesting oxidative stress
Kidney LDH activity (U/g tissue)

in kidney and nephrotoxicity. Our results corroborated

75
***
50 (6)
25
0
CT K2Cr2O7
Figure 2. Lactate dehydrogenase (LDH) activities in plasma
(A) and in kidney (B) of adult rats and their pups: controls
and K2Cr2O7 treated with 700 parts per million (ppm)
administered in their drinking water from the 14th day of
pregnancy until day 14 after delivery. K2Cr2O7 treated versus
controls: ***p < 0.001. Data are shown as means + SD. The
number of determinations is indicated above columns of each
diagram.
In addition, urea, a major nitrogen-containing
metabolic product of protein metabolism, was increased
in blood of K2Cr2O7-treated rats. These results corre-
lated with an increased protein catabolism in mammals
with previous findings cited above. Additionally, lipid
peroxidation levels were found to be higher in young
*** rats than in mothers, this may be due to deleterious
(8)
changes as transfer of K2Cr2O7 from the mothers to their
fetuses through placenta and to their newborns through
milk.9 Our results confirmed an intoxication of pups
during a lactating period which was necessary for their
development.
Furthermore, a previous study of Fujihara et al.46
CT K2Cr2O7
showed that a basal amount of NO might be important
to maintain normal renal function. However, increasing
evidence suggested that excessive production of NO
played a major role in oxidative stress and tissue damage
in the pathophysiology of acute renal failure.47 Under
our experimental conditions, kidney NO levels
increased significantly in K2Cr2O7-treated mothers and
in their offspring when compared with those of the
control group. Thus indicated that Cr might lead to the
induction of inducible nitric oxide synthase, resulting
in an increased production of NO, leading to the
formation of toxic peroxynitrite.48 An enhancement
of NO was reported by Bagchi et al.49 in peritoneal
exudates cells, mainly the macrophages from sodium
dichromate-treated rats.
1240 Human and Experimental Toxicology 30(9)

The increased oxidative stress presented the opposite

A Mothers offspring picture to the changes in antioxidants status. In fact, the
impairment of the antioxidant defense system was con-
100
CAT( µmol H2O2degraded/min/mg protein)

*** sidered as a critical event in Cr-induced nephrotoxi-

(6)
city.50 Exposure of Cr was characterized by the
75 depletion of tissue and circulating non-enzymatic anti-
(8)
oxidants including GSH and NPSH.51 Indeed, GSH was
(6)
considered as the essential compound which maintained
50
cell integrity because of its reducing properties and its
***
(8)
participation in the cell metabolism. Current data
25 revealed that K2Cr2O7 caused a significant increase in
GSH level in mothers and their progeny. This finding
might well be a compensatory response to protect tubu-
0 lar cells from the ravaging effects of lipid peroxidation
CT K2Cr2O7 CT K2Cr2O7
by increasing the levels of endogenous GSH. Our results
corroborates with previous study of Cupo and Wetter-
B Mothers Offspring
hahn52 who reported that Cr(VI)-induced an increase
1000 of GSH level in chicken embryo hepatocytes. Further-
*** more, It has been suggested that low level of GSH
(6)
750 (8)
enhanced Cr(VI) toxicity by promoting the formation
SOD (U/mg protein)

of Cr(V), while high concentrations of glutathione and

(6)
ascorbate would reduce rapidly Cr(VI) to the final prod-
500
*** uct Cr(III), thus yielding lower steady state concentra-
(8)
tion of Cr(V) and scavenge Cr(VI) as Cr(III)-GSH
250 complex.53
In addition, another product NPSH, considered as
0 one of the important primary defenses that counteract
CT K2Cr2O7 CT K2Cr2O7 oxidative stress, decreased significantly in kidney of
treated mothers and of their offspring when compared
C Mothers Offspring to the control group. Depletion of NPSH following
140
Cr(VI) exposure might be due to either induction of
** (8) oxidative stress and to consumption of GSH in the
(6)
GPx (nmol GSH/min/mg protein)

120 Cr(VI) reduction process54 or to the excretion of

(6)
100
80
60
40
20
0 effects on the cellular resistance. Our findings showed
CT
Figure 3. Antioxidant enzyme activities CAT (A), SOD (B)
and GPx (C) in kidney of adult rats and their pups: controls
and K2Cr2O7 treated with 700 parts per million (ppm)
administered in their drinking water from the 14th day of
pregnancy until day 14 after delivery. A: catalase (CAT),
B: superoxide dismutase (SOD), C: glutathione peroxidase
(GPx). K2Cr2O7 treated versus controls: *** p  0.001.
Data are shown as means + SD. The number of determi-
nations is indicated above columns of each diagram.
Cr(III) in the urine as a complex with GSH.55
**
Antioxidant enzymes are frequently used as mar-
(8) kers of oxidative stress.56 Among the first line of cel-
lular defense, SOD, CAT and GPx are important in
the preservation of homeostasis for normal cell func-
tion. According to Avti et al.,57 deviations in the phy-
siological concentrations of these enzymes has drastic
K2Cr2O7 CT K2Cr2O7 a significant increase in the activities of SOD, GPx
and CAT in kidneys of mothers and a decrease in
those of pups. As these enzymes have a protective role
against oxygen free radical-induced damage, their
induction can be understood as an adaptative response
to oxidative stress. Sengupta et al.58 observed also an
increase of antioxidant enzyme activities in adult rats
exposed to hexavalent Cr. Similar results, concerning
a decrease of the enzymes cited above, have been
found by others in suckling mice whose mothers
Soudani N et al. 1241

A X 200

B1 X 200 B2 X 400

B3 X 200 B4 X 400

Figure 4. Kidney histological sections of adult rats: controls (A) and treated (B1, B2, B3 and B4) who have received 700 parts
per million (ppm) K2Cr2O7 in their drinking water from the 14th day of pregnancy until day 14 after parturition. Optic micro-
scopy: HE (200) A, B1 and B3; HE (400) B2 and B4. Arrows indicate: intra-glomeruli hemorrhage, tubular dilation,
glomeruli showing large Bowman’s space, markedly lobulated glomeruli, infiltration of mononuclear cells, necrosis,
vacuolation, vascular congestion.
1242 Human and Experimental Toxicology 30(9)

C X 200

D1 X 200 D2 X 400

D3 X 200 D4 X 400

Figure 5. Kidney histological sections of 14-day-old rats: controls (C) and whose mothers have received K2Cr2O7 (D1,
D2 D3 and B4) in their drinking water from the 14th day of pregnancy until day 14 after parturition. Optic microscopy HE
(200) C1, D1 and D3; HE (400) D2 and D4. Arrows indicate: hemorrhage, tubular dilation, glomeruli showing
large Bowman’s space, necrosis, vascular congestion.
Soudani N et al.
Table 4. Grading of the histopathological changes in the kidney sectionsa
Hemorrhage Tubular dilatation
Groups Mothers Offspring Mothers Offspring Mothers Offspring
Controls    
K2Cr2O7 þþþ þþþ þþþ þþ
a
Scoring was done as follows: none (), mild (þ), moderate (þþ) and severe (þþþ).
were treated by heavy metals such as fluoride.59 The
inhibition of most antioxidant enzymes, induced by
Cr, may be due either to the direct binding of the
metal to their active site or to their increased usage
in scavenging free radicals induced by the metal, thus
causing irreversible inhibition of their activities. The
difference between deviation in concentration of these
enzymes between mothers and pups could be also
explained by the fact that K2Cr2O7 has different
mechanisms depending on the age of animals, route
and period of administration.
These biochemical modifications corresponded
histologically. In fact, histological changes, seen in
the kidney of mothers and their pups treated with
K2Cr2O7, were characterized by necrosis, hemorrhage
and tubular dilation. Besides, in mothers, extensive
vacuolization and intra-glomeruli hemorrhage were
observed. Changes in the histoarchitecture were also
observed by Pedraza-Chaverri et al.50 after K2Cr2O7
treatment. This might be due to the increased amount
of ROS product which might attack membranes, lipid
and proteins and affect kidney histoarchitecture.60
In conclusion, we found that a high concentration
of K2Cr2O7 in drinking water, ingested by pregnant
and lactating rats, induced an oxidative stress in renal
tissue of both adult rats and their pups, leading to lipid
peroxidation. Balance between oxidative and antioxi-
dant systems was disturbed in K2Cr2O7-treated rats.
High K2Cr2O7 intake by lactating rats appeared to
have pronounced toxic effects in their suckling pups.
These evidences, presented in our experimental data,
show for the first time the occurrence of nephrotoxi-
city after exposure of rats to K2Cr2O7 during the late
pregnant and lactating period, make clear the need for
attention of pregnant women on K2Cr2O7 exposure
not only during pregnancy but also during lactation
period.
Acknowledgments
The authors are indebted to Miss Dalenda Kchaou for her
assistance in histolological techniques.
1243
Necrosis Enlarged glomeruli space Vacuolization
Mothers Offspring Mothers Offspring
     
þþþ þþ þþþ þ þþ þ
Funding
This work was supported by the DGRST grants (Appui à la
Recherche Universitaire de Base ARUB 99/UR/08-73),
Tunisia.
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