Toxicology 209 (2005) 289–301
Sex-specific effects of neonatal exposures to low levels of
cadmium through maternal milk on development and
immune functions of juvenile and adult rats
St´ ephane Pilleta,b,1, Andrew A. Rooneya,1,2, Jean-Marie Bouquegneaub,
Daniel G. Cyra, Michel Fourniera,∗
aINRS-Institut Armand-Frappier, Universit´ e du Qu´ ebec, 245 Hymus Boulevard, Pointe-Claire, Que., Canada H9R 1G6
bDepartment of Oceanology, University of Li` ege, B6 Sart-Tilman, B-4000 Li` ege, Belgium
Received 23 September 2004; accepted 12 December 2004
Available online 19 February 2005
Cadmium (Cd) is a major environmental contaminant. Although immunotoxic effects have been associated with Cd exposure,
the inconsistency of experimental results underlines the need of an experimental approach more closely related to environmental
conditions. We investigated the effects of exposing neonatal Sprague–Dawley rats to environmentally relevant doses of Cd
through maternal milk. Dams received 10 parts per billion (ppb) or 5 parts per million (ppm) Cd chloride (CdCl2) in drinking
water from parturition until the weaning of the pups. Half of the offspring was sampled at weaning time. The remaining
juvenile rats received water without addition of Cd until adulthood. Cd accumulation in kidneys of juvenile rats fed from dams
exposed to Cd indicated the transfer of the metal from mother to pups through maternal milk. This neonatal exposure resulted
in decreased body, kidney and spleen weights of just weaned females but not of males. This effect was more pronounced in
the less exposed females fed from dams exposed to 10ppb Cd, which also displayed lower hepatic metallothionein-1 (MT-1)
mRNA levels. The effect of Cd exposure on body and organ weights did not persist to adulthood. In contrast, we observed
gender-specific effects of neonatal Cd exposure on the cytotoxic activity of splenic NK-cells of both juvenile and adult rats.
Cd also strongly inhibited the proliferative response of Con A-stimulated thymocytes in both male and female adult rats 5
weeks after the cessation of Cd exposure. These immunotoxic effects were observed at doses much lower than those reported
to produce similar effects when exposure occurred during adulthood. In conclusion, neonatal exposures to environmentally
∗Corresponding author. Tel.: +1 514 630 8824; fax: +1 514 630 8850.
E-mailaddress:Michel.Fournier@inrs-IAF.uquebec.ca (M. Fournier).
1Both authors contributed equally to this study.
2Present address: U.S. Environmental Protection Agency, National Center for Environmental Assessment, Integrated Risk Information
System Staff, Research Triangle Park, NC 27711, USA.
0300-483X/$ – see front matter © 2005 Published by Elsevier Ireland Ltd.
290S. Pillet et al. / Toxicology 209 (2005) 289–301
© 2005 Published by Elsevier Ireland Ltd.
Keywords: Cadmium; Maternal milk; Gender-specific effects; Adults; Juveniles; Immune functions
Cadmium (Cd) is a ubiquitous environmental con-
largest potential sources of Cd exposure for humans
are food and cigarette smoke (Goyer, 1996; Nordberg,
1996; Koller, 1998). Cd concentrations in food nor-
mally range from 2 to 100 parts per billion (ppb), al-
though Cd can reach levels higher than 1 part per mil-
1–3ppm. Oral exposure is usually in the form of solu-
ble salts, such as cadmium chloride (CdCl2) (Elinder,
1985; Koller, 1998).
The primary sites of Cd bioaccumulation in mam-
mals are the liver and kidneys. During long-term ex-
posures, between 30 and 50% of the total Cd body
burden accumulates in the kidneys (Goyer, 1996;
Nordberg, 1996). An important proportion of the Cd in
these organs is associated with low, cysteine-rich pro-
teins, the metallothioneins (Nordberg, 1996). Four iso-
3 and MT-4 are highly tissue-specific, MT-1 and MT-2
have been detected in multiple tissues and cell types,
although these isoforms are mainly produced in the
liver and kidneys (Moffatt and Denizeau, 1997; Miles
et al., 2000; Cyr et al., 2001). These two isoforms are
very similar and appear to be coordinately regulated in
covery and widespread tissue distribution, most of the
research on metallothioneins has been focused on MT-
1 and MT-2. Considering the similarity between these
isoforms, all subsequent mention of these proteins in
this paper will refer to both MT-1 and MT-2 (MT, in-
cations, such as Cd or zinc (Zn), and exposure to these
metals strongly increases MT synthesis. As a result of
these properties, MT are important regulators of the
homeostasis of essential cations and MT sequestration
of toxic heavy metals represents a major detoxifica-
tion process in the cells. In fact, resistance to the toxic
tracellular MT and transgenic mice that do not express
MT are highly sensitive to Cd, thus, demonstrating the
protective role of these proteins in metal toxicity (Liu
et al., 1995, 2000; Klaassen et al., 1999).
Nephrotoxicity is usually associated with chronic
exposure to Cd, while acute exposure results primar-
ily in hepatotoxicity (Nordberg, 1996). Toxic effects
of Cd have also been demonstrated on bone forma-
tion, male reproductive functions and immune system
(Nordberg, 1996; Koller, 1998; Waalkes et al., 1999).
Toxic effects of Cd on immune organs and differen-
tiation of immune cells, as well as on the specific
and non-specific immune responses, indicate an im-
pact on the immune system as a whole, which can lead
to a significant decrease in host-resistance. However,
the inconsistency of experimental results has revealed
that the immunotoxicity of Cd depends on the speci-
ation of the metal employed, the route, dose and du-
ration of exposure, as well as the physiological sta-
tus of the animal (Descotes, 1988, 1992; Payette et
al., 1995; Koller, 1998). Therefore, experimental ap-
proaches closely related to environmental conditions
are needed to better assess the risk of Cd exposure
for humans and wildlife. Moreover, the developmen-
tal stage, during which exposure takes place, has been
shown as a critical factor for risk assessment of chem-
ical exposure (Miller, 1983; Goyer, 1996). A higher
sensitivity to immunotoxic effects when exposure oc-
curs during foetal or early neonatal periods has been
reported. Chemical exposure at these stages results in
alterations in the establishment of the immune system
and can lead to a persistent impairment of immune
competence (Gehrs et al., 1997; Miller et al., 1998;
Holladay and Smialowicz, 2000; Bunn et al., 2001;
Rooney et al., 2003). However, no information is cur-
response to Cd exposure during early stages of devel-
S. Pillet et al. / Toxicology 209 (2005) 289–301291
Although relatively low, the transfer of Cd through
exposure when rodents are exposed during both ges-
tational and lactational periods (Petersson Graw´ e and
Oskarsson, 2000). Moreover, Cd levels in maternal
milk are correlated with the degree of maternal ex-
posure (Petersson Graw´ e and Oskarsson, 2000). The
objective of the present study was to investigate the
short- and long-term immunotoxic effects of neona-
tal exposure to environmentally relevant levels of Cd
through maternal milk. A concentration of 10ppb Cd
in dam’s drinking water was selected as representative
of levels found in food and water. A higher level of ex-
posure of 5ppm was selected as representative of lev-
els found in cigarette smoke or in some contaminated
2. Materials and methods
2.1. Animals and tissue collection
Pregnant female Sprague–Dawley rats, purchased
1 week prior to parturition (Charles River Canada
Inc., St. Constant, Que., Canada), were housed in a
pathogen-free animal facility, and cared for in compli-
ance with the Canadian Council for the Animal Care.
Within 12h of birth (day 1), pups were removed from
their mothers, sexed and randomly re-assigned to lac-
tating females so as to provide same-sex pups per fe-
male. These lactating females received either 0ppb
(control), 10ppb or 5ppm Cd as CdCl2(Sigma Chem-
icals, Oakville, Ont., Canada) in their drinking water.
Cd addition was provided.
On days 28 (juveniles) and 63 (adults), rats were
weighed and euthanized by CO2 asphyxiation. The
spleen, thymus, liver and kidneys were aseptically
removed and weighed. The kidneys and liver were
immediately frozen in liquid nitrogen and stored at
−80◦C. Cell suspensions from the spleen and thy-
mus were obtained by passing the tissues through a
stainless-steel mesh in HBSS medium (pH 7.3; Gibco,
HEPES (Gibco) and 100IU penicillin and 100?g/ml
streptomycin (Bio Media, Mississauga, Ont., Canada).
Debris was removed by passing the cell suspension
and viability was determined by trypan blue exclu-
sion. Cells were then resuspended in RPMI medium
(Gibco) supplemented with 25mM HEPES (Gibco),
10% heat inactivated foetal bovine serum (Bio Media),
Media) (complete RPMI) at a final concentration of
2.2. Cadmium and zinc analysis
Kidneys were freeze-dried for 36h and digested
with a mixed solution of hydrochloric and nitric acid
(1:3, v/v) slowly heated to 100◦C, until the digestion
was complete. Atomic absorption spectrophotometry
(ARL 3510) was used to determine Zn and Cd concen-
trations. A set of certified material samples (DORM-2,
surement Standards, Ont., Canada) spiked with grade
concentrations of Cd was analysed in parallel to en-
sure the accuracy of the method. Recoveries ranged
from 73.3±4.6% and 88.5±1.5% of the certified val-
ues for Zn and Cd, respectively. Detection limits were
0.15?g/g dry weight (DW) for Zn, and 0.14?g/gDW
2.3. MT-1 mRNA analysis
Relative levels of hepatic MT-1 mRNA were mea-
sured by Northern blot analysis according to the previ-
ously published protocol (Cyr et al., 2001). The cDNA
probe was labelled by random priming with32P dCTP
(Oligonucleotide Labelling Kit, Amersham Pharma-
cia Biotech, Baie d’Urf´ e, Que., Canada). Hybridiza-
tion of the cDNA probe was performed according to
the method outlined by Cyr et al. (2001). Autora-
diography was subsequently performed using Phos-
phor Screens (Molecular Dynamics, Amersham Phar-
macia Biotech). Following autoradiography, the mem-
branes were stripped according to manufacturer’s in-
cleotide probe, which was used to standardize RNA
loading. The resulting unsaturated phosphorimages
were analysed by densitometry using a PhosphorIm-
ager and ImageQuant software (Molecular Dynam-
ics). The integrated area under the curve for each sig-
nal was standardized against the signal for the 18S
rRNA in order to determine relative levels of the MT-1
292S. Pillet et al. / Toxicology 209 (2005) 289–301
2.4. Cytotoxic activity of NK-cells
The cytotoxic activity of splenic NK-cells was as-
sessed by measuring their ability to kill mice tumor
cell lines (YAC-1; American Type Culture Collection,
Manassas, VA, USA) as described by Brousseau et al.
(1999). Briefly, YAC-1 cells were stained with 3,3?-
dioctadecyloxacarbocyanine perchlorate (DiO; Sigma
1 were combined at four different ratios 100/1, 50/1,
25/1, 10/1 in complete RPMI. Mixed cell suspensions
were then centrifuged at 300×g for 5min at 20◦C.
After 1h incubation at 37◦C, a solution of propidium
iodide in RPMI (PI; Sigma Chemicals) was added in
order to obtain a final concentration of 50?g/ml, and
were then analysed using a FACScan flow cytometer
(Becton Dickinson, San Jose, CA, USA). CellQuest
Pro software (Becton Dickinson) allowed us to dis-
criminate between DiO positive (DiO+) YAC-1 cells
and DiO negative (DiO−) splenocytes. Based on this
distinction, the acquisition consisted of a minimum of
5000 DiO+ gated events. PI enters all cells but is ac-
tively excreted by living cells. PI positive (PI+) cells,
thus, represent dead cells. Therefore, double positive
PI+ and DiO+ cells are dead YAC-1 cells. The pro-
portion of dead YAC-1 cells represents the cytotoxic
activity of NK-cells.
2.5. Lymphoproliferative response
An aliquot of 5×105splenocytes was cultured
in triplicate in polystyrene flat-bottom 96-well cul-
ture plates (Costar, Corning, NY, USA) in complete
RPMI supplemented with 10?g/ml dextran (Sigma
Chemicals) alone or with one of three mitogens: con-
canavalin A (5?g/ml) (Con A; Sigma Chemicals),
phytohaemagglutinin (20?g/ml) (PHA; Sigma Chem-
icals), or lipopolysaccharide (25?g/ml) (LPS; Sigma
Chemicals). Similarly, thymocytes were incubated in
either medium alone or with one of the two mito-
gens: Con A (2.5?g/ml) or PHA (20?g/ml). Con A
and PHA stimulate the proliferation of T-lymphocytes,
while LPS stimulates B-lymphocytes. After 72h incu-
bation at 37◦C in a humidified chamber with 5% CO2,
0.5?Ci of [3H] TdR (ICN Biomedicals, Costa Mesa,
CA, USA) was added to each well, and cells were in-
cubated for an additional 18h. The DNA of the cells
incorporated radioactivity was measured with a liquid
scintillation counter. Mean disintegration per minute
(DPM) for the triplicate cultures was assessed. The
data were then expressed as a stimulation index, i.e.
the ratio of the mean DPM of mitogen-stimulated cells
to the mean DPM of unstimulated cells.
2.6. Statistical analysis
Statistical analyses were performed using STATIS-
TICA (1998 edition, Statsoft, Tulsa, OK, USA) and
SigmaStat (Version 2.0, Jandel Corporation, Chicago,
IL, USA) softwares. Data were first tested for normal-
ity and homogeneity of variance and then compared
using a two-way analysis of variance (ANOVA) to as-
sess the effects of gender and treatment, as well as the
occurrence of specific effects of treatment on each sex.
When significant effects were observed, a Tukey test
a posteriori was used to determine significant differ-
ences between groups. Correlations between variables
having a normal distribution and homogenous vari-
When parametric tests could not be applied, a Spear-
man rank order correlation was used. Significance was
established at p<0.05.
3.1. Cd and Zn concentrations in juvenile rat
Renal Cd concentrations in juvenile rats fed from
control dams were below the limit of detection. Cad-
mium concentrations in kidneys of juvenile rats fed
from dams exposed to 10ppb were also below the de-
tection limit except for one female, which had Cd con-
centration between the detection limit (0.14?g/gDW)
and the quantification limit (0.70?g/gDW). In con-
trast, all juvenile rats fed from dams exposed to 5ppm
Cd had quantifiable levels of Cd in their kidneys.
There were no differences between sexes with re-
spect to Cd concentrations. However, we observed
a significant sex-specific effect of treatment on Zn
concentrations in kidneys. Females fed from control
S. Pillet et al. / Toxicology 209 (2005) 289–301293
dams had higher Zn concentrations in kidneys than
males, but no significant differences were observed
between sexes in rats fed from dams exposed to Cd
or between treatment groups within males or females
3.2. Effect of Cd exposure on body and organ
In juvenile rats, an effect of gender on body, kid-
ney and spleen weights, as well as an effect of treat-
ment on body, kidney and liver weights was observed.
Moreover, the effects of treatment on body, kidney
and spleen weights were gender-dependent (Fig. 1).
The body weight of juvenile females, but not males,
fed from dams exposed to Cd was reduced as com-
pared to gender-matched control. This effect was sig-
nificantly more pronounced in females fed from low
were observed for kidney and spleen weights, with the
exception that the spleen weights of females fed from
dams exposed to 5ppm Cd were not significantly dif-
C). The most important reduction in body, kidney and
spleen weights observed in females fed from dams ex-
posed to 10ppb Cd and the absence of effect on males
led to a significant difference between the sexes within
this treatment group (Fig. 1A–C). Liver weights were
also affected by the treatment, but there was no gen-
der effect. Only juvenile rats fed from dams exposed
Fig. 1. Weights of body (A), kidneys (B), spleen (C) and liver (D) of female and male juvenile rats fed from dams exposed to 0ppb (control),
10ppb Cd or 5ppm Cd in drinking water. Data are expressed as the mean±S.E. (n=5–6). The effects of treatment were dependent on the
(symbols). The (*) indicates a significant difference between sexes within a group. Shared Greek letters indicate a lack of statistical difference
between groups when effect did not depend on the gender. Significance was established at p<0.05.
294S. Pillet et al. / Toxicology 209 (2005) 289–301
Zn and Cd concentrations in kidneys of juvenile rats fed from dams
exposed to 0ppb (control), 10ppb Cd or 5ppm Cd in drinking water
Control 10ppb 5ppm
Results are expressed as ?g/g dry weight (DW); mean±S.E.<LD
indicate levels below the detection limit. The (*) indicates signifi-
cant differences between males and females fed from control dams
aCd concentration in kidneys from one female rat fed from a
dam exposed to 10ppb Cd was between the limit of detection
(0.14?g/gDW) and the limit of quantification (0.70?g/gDW).
to 10ppb Cd had lower liver weight when compared to
animals fed from control dams (Fig. 1D). There were
no effects of treatment on organ weights normalized to
body weight (data not shown).
In adults, Cd exposure via maternal milk had no ef-
fect on organ or body weights (data not shown). Adult
rats displayed the expected strong sexual dimorphism
mus weights did not differ between genders and were
not affected by treatment in juvenile or adult rats (data
3.3. Hepatic MT-1 mRNA levels in juvenile rats
hepatic MT-1 mRNA levels of each sex. Females fed
males fed from dams exposed to 5ppm Cd exhibited
two other experimental groups of females (Fig. 2). By
contrast, there was no effect of treatment on males, re-
sulting in a gender difference within rat fed from dams
exposed to 10ppb Cd (Fig. 2).
We observed a significant correlation between renal
Cd concentrations and hepatic MT-1 mRNA levels in
rats fed from dams exposed to 5ppm Cd (Fig. 3A).
Hepatic MT-1 mRNA levels of all the rats were also
correlated with renal Zn concentrations (Fig. 3B).
Fig. 2. Hepatic MT-1 mRNA levels in female and male juvenile rats
fed from dams exposed to 0ppb (control), 10ppb Cd or 5ppm Cd
in drinking water. MT-1 mRNA levels were quantified by Northern
blot analysis and levels were standardized for loading with the 18S
rRNA. Data are expressed as the mean±S.E. (n=5–6). The effect
of treatment was dependent on the gender. Shared superscripts indi-
cate a lack of statistical difference between groups within females
(numbers) or males (symbols). The (*) indicates a significant differ-
ence between sexes within a group. Significance was established at
3.4. Cytotoxic activity of splenic NK-cells
The percent of YAC-1 cells killed by splenic NK-
cells decreased as the ratio of splenocytes to YAC-1
cells decreased in both control and Cd-exposed ani-
mals, indicating a normal response pattern (data not
shown) (Brousseau et al., 1999). A single ratio of 50/1
of gender and treatment. This ratio was selected based
on its use in the literature (Chowdhury and Chandra,
1989; Cifone et al., 1990), and because it allowed for
potential increases and decreases in cytotoxic activity
as it did not represent maximum or minimum response
among the tested ratios.
Treatment had significant differential effects on
each sex in both juvenile and adult rats. Only males
were significantly affected regarding the cytotoxic ac-
tivity of splenic NK-cells, but the effects depended on
Juvenile males fed from dams exposed to 5ppm Cd
tivity as compared to gender-matched control, while
the responses of males fed from less exposed dams
were similar to control. Although this immune param-
eter was unaffected in females fed from dams exposed
S. Pillet et al. / Toxicology 209 (2005) 289–301295
Fig. 3. Correlation between the relative hepatic MT-1 mRNA levels
of juvenile rats and the Cd (A) or Zn (B) concentrations in kidneys
(?g/g dry weight). Cd levels could only be quantified in juvenile rats
fed from dams exposed to 5ppm Cd, Cd concentrations in the other
groups being below the quantification limit.
to Cd as compared to gender-matched control, we ob-
served a significant difference between the responses
of the two groups of exposed females. Splenic cells
of females fed from dams exposed to 10ppb Cd dis-
played lower NK cytotoxic activity than splenic cells
of females fed from dams exposed to 5ppm Cd. NK
cell cytotoxic activity of males and females fed from
ferences in rats fed from dams exposed to Cd. Males
fed from dams exposed to 10ppb Cd had a greater re-
sponse than females belonging to the same treatment
of rats fed from dams exposed to 5ppm Cd (Fig. 4A).
Adult males fed from dams exposed to 10ppb Cd
had significantly higher splenic NK cell cytotoxic ac-
tivity as compared to gender-matched control. How-
ever, males fed from dams exposed to 5ppm Cd dis-
Fig. 4. Cytotoxic activity of splenic NK-cells in juvenile (A) and
adult (B) rats fed from dams exposed to 0ppb (control), 10ppb Cd
or 5ppm Cd in drinking water. The cytotoxic activity is expressed as
the mean percent of dead target cells (YAC-1)±S.E. (n=5–6). The
effects of treatment were dependent on the gender for either juve-
nile and adult rats. Shared superscripts indicate a lack of statistical
difference between groups within females (numbers) or males (sym-
bols). The (*) indicates a significant difference between sexes within
a group. Significance was established at p<0.05.
played a similar response to gender-matched control
(Fig. 4B). There were no gender differences between
and dams exposed to 5ppm Cd. However, males fed
from dams exposed to 10ppb had a higher NK cyto-
toxic activity than females belonging to the same treat-
ment group (Fig. 4B).
3.5. Proliferative response of splenic and thymic
lymphocytes to mitogens
In juvenile rats, the proliferation of Con A-
stimulated splenocytes from rats fed from dams ex-
296 S. Pillet et al. / Toxicology 209 (2005) 289–301
Fig. 5. Proliferative response of lymphocytes to Con A-stimulated splenocytes (A and B) and thymocytes (C and D) in juvenile (A and C)
and adult (B and D) rats fed from dams exposed to 0ppb (control), 10ppb Cd or 5ppm Cd in drinking water. The proliferative response of
lymphocytes is expressed as the mean stimulation index±S.E. (n=5–6). The effect of treatment was not dependent on the gender. Shared
Greek letters indicate a lack of statistical difference between groups. The (*) indicates a significant difference between gender. Significance was
established at p<0.05.
posed to Cd did not differ from control, but the stim-
ulation index in rats fed from dams exposed to 10ppb
Cd was significantly higher than those in rats fed from
dams exposed to 5ppm Cd (Fig. 5A). No effect was
found for Con A-stimulated thymocytes (Fig. 5C).
In adult rats, treatment did not affect the prolifera-
erative response of Con A-stimulated thymocytes was
suppressed in both males and females fed from dams
exposed to 10ppb and 5ppm Cd more than 5 weeks
after the end of the Cd exposure through maternal milk
We did not observe any effect in the proliferative
response of splenocytes and thymocytes to LPS and
The presence of Cd in the kidneys of juvenile rats
fed from dams exposed to 5ppm Cd indicates a trans-
fer of Cd to the offspring via the maternal milk. The
rats fed from dams exposed to 10ppb Cd probably re-
sults from levels being below the detection limit rather
than from an absence of Cd transfer to the offspring.
S. Pillet et al. / Toxicology 209 (2005) 289–301297
Petersson Graw´ e and Oskarsson (2000) reported Cd
transfer through maternal milk of dams exposed via a
mini-osmotic pump connected to the jugular vein. Our
results confirmed that Cd transfer occurs using a more
environmentally relevant route and doses of exposure.
The concentration of Cd measured in the kidneys of
juvenile rats were lower than those found in several
human populations living in industrialized areas, and
much lower than the concentration of 200?g/g fresh
(Curry and Knott, 1970; Lauwerys et al., 1984; WHO,
1992; Friis et al., 1998; Benedetti et al., 1999).
Although very low, the neonatal Cd exposure lev-
els in this study significantly delayed the early devel-
opment of females but not males. The absence of ef-
fects on the weights of kidneys, spleen and liver when
normalized to body weight indicates that the reduction
of organ weights likely results from a general devel-
opmental effect. Hormones can modulate Cd toxicity
in several tissues or organs. It has been reported that
90% of patients with Itai–Itai disease (the most severe
form of chronic Cd intoxication in humans) were post-
may play an important role in the development of the
pathology (Jarup et al., 1998). Ovariectomy also en-
hanced nephrotoxicity and hepatotoxicity of acute ex-
posure to Cd in Sprague–Dawley rats (Katsuta et al.,
thought to be mediated through the modulation of MT
synthesis (Shimada et al., 1997). MT are coordinately
regulated, and their synthesis is primarily controlled at
the transcriptional level (Andrews, 1990; Moffatt and
Denizeau, 1997; Miles et al., 2000). Interestingly, lev-
els of MT-1 mRNA were significantly reduced in the
liver of the juvenile females fed from dams exposed
to 10ppb Cd. MT synthesis following exposure to Cd
has been shown to be modulated by hormones, but this
modulation appeared to be complex and depended on
the hormone, the route of exposure, the gender and the
tissue (Blazka and Shaikh, 1991; Shimada et al., 1997;
Sogawa et al., 2001). Sogawa et al. (2001) showed that
male mice have lower basal levels of hepatic MT-1
mRNA than females; however, a subcutaneous injec-
tion of Cd induced higher MT-1 mRNA levels in male
liver as compared to female liver. Although the higher
level of exposure in our experiment was too low to
induce a significant increase in hepatic MT-1 mRNA,
we observed a similar pattern when comparing males
and females from control and exposed groups. At this
point, we cannot explain the decrease of MT-1 mRNA
levels in the liver of females fed from dams exposed to
10ppb Cd. However, considering the protective role of
MT against Cd toxicity, the low levels of MT-1 mRNA
may be related to the more marked effect of Cd expo-
sure on global development observed in this group.
The correlation between MT-1 mRNA levels in the
liver and Zn or Cd concentrations in kidneys illus-
trates the relationship between MT and Zn homeosta-
sis or Cd detoxification. An important component of
Cd toxicity involves the disturbance of Zn homeosta-
receptor (ER). Cano-Gauci and Sarkar (1996) demon-
strated the occurrence of a reversible exchange of Zn
between ER and MT. Our results also show that Cd
exposure through maternal milk has significant sex-
and females observed in control animals. Therefore,
gender effects of Cd exposure could be related to sex-
specific alterations of MT synthesis and Zn homeosta-
Immunotoxicity has previously been associated in
laboratory models with exposure to high levels of Cd
that are unlikely to occur in the environment (Stacey et
al., 1988; Cifone et al., 1989a; Dan et al., 2000). In the
present experiment, we demonstrate that exposure to
development of the immune system affects the cyto-
toxic activity of splenic NK-cells in both juvenile and
adult male rats, and strongly decreases the prolifera-
tive response to Con A of thymocytes in adults of both
and/or longer exposure periods to Cd were necessary
to affect NK cell cytotoxic activity and the lympho-
during adulthood (Thomas et al., 1985; Stacey et al.,
1988; Cifone et al., 1989a). The early stages of devel-
opment are critical for the establishment of major un-
derpinning processes of several systems, including the
immune system, making foetal and early postnatal pe-
result in persistent effects (Holladay and Smialowicz,
298S. Pillet et al. / Toxicology 209 (2005) 289–301
2000). For example, exposure to 250ppm lead (Pb)
acetate in drinking water during gestation resulted in
decreased total leukocyte counts, elevated production
of tumor necrosis factor-? by LPS-stimulated spleno-
cytes and decreased delayed-type hypersensitivity in
female offspring at adulthood, while none of these ef-
fects were observed in dams directly exposed to Pb
(Miller et al., 1998). In the current study, the cyto-
toxic activity of splenic NK-cells and the prolifera-
tive response of thymic lymphocytes to Con A were
affected in adulthood, 5 weeks after the end the expo-
sure through maternal milk. Immunotoxic effects, and
especially the strong inhibition of lymphoproliferative
response of thymocytes to Con A, that we observed in
adults after neonatal exposure to Cd suggest that Cd
may act on progenitor cells and thereby alter the de-
velopment of the related immune function of mature
cells. Exposure to Cd is known to cause morphologi-
cal changes at different sites of maturation of immune
cells including bone marrow, spleen and thymus. Al-
tered hematopoietic processes, distribution and rela-
tive proportion of lymphocyte subpopulations in the
blood and spleen of rodents have been reported fol-
lowing Cd exposure (Yamada et al., 1981; Ohsawa et
al., 1983; Yamano et al., 1998). Some of these effects
appear to be more pronounced, when exposures oc-
curred in older adult rats, which illustrate the impor-
tance of age at the time of exposure (Yamano et al.,
1998). Ultimately, all lymphoid cell types are derived
from pluripotent hematopoietic stem cells. These cells
display a sequential rather than simultaneous loss of
other developmental potential en route to becoming
a particular type of mature immune cell (Marquez et
al., 1998; LeBien, 2000). T-cells and NK-cells share
a common stem cell, while B-cells stem earlier in the
differentiation pathway from lymphoid cells. Interest-
and NK-cells when exposure occurs in an early stage
of development, while the proliferative response of B-
cells remains unaffected. At the cellular level, Cd has
been shown to disturb major endpoints of the differen-
tiation pathway of effective immune cells. Cd induced
apoptosis in thymocytes and the different subsets of
thymocytes have different susceptibilities to the apop-
totic effects of Cd (El Azzouzi et al., 1994; Dong et
al., 2001; Shen et al., 2001). The complex cellular pro-
cesses leading to mature immune cells also require the
presence of appropriate cytokines, and Cd has been
demonstrated to alter cytokine production (Theocharis
et al., 1991; Funkhouser et al., 1994; Kayama et al.,
1995) as well as the expression of cytokine receptors
(Cifone et al., 1989b; Payette et al., 1995).
Regulation of immune functions by the endocrine
system has been extensively described (Grossman,
1985; Schuurs and Verheul, 1990; Sorachi et al., 1993;
Nilsson and Carlsten, 1994). Krzych et al. (1981) re-
ported that proliferation of splenocytes stimulated by
mitogens was higher in female mice than in males. We
observed similar results in the current study with Con
A-stimulated splenocytes from adult rats. We did not
observe any significant effect of treatment on splenic
NK cell cytotoxic activity in either juvenile or adult
females, while this immune function was altered in
ing in utero exposure to Pb (Bunn et al., 2001). Sex
steroid hormones could be partly involved in the ex-
pression of these gender differences. Several studies
have demonstrated endocrine disrupting effects of Cd.
testosterone levels (Zeng et al., 2002, 2003). Cd can
also bind and activate ER-?, at least in vitro (Stoica et
strated the estrogenic activity of Cd following in vivo
exposure in ovarectomized female rats. Although the
activity but the mechanisms of this modulation appear
to be complex and depend on the hormone, the dose
and route of exposure (Screpanti et al., 1987; Schuurs
and Verheul, 1990; Sorachi et al., 1993; Curran et
al., 2001; Lang et al., 2003). Sex steroid hormones
also play an important role in immune organ devel-
opment as well as in the maturation and differentia-
tion of immune cells (Erlandsson et al., 2001, 2003;
Pejcic-Karapetrovic et al., 2001). Therefore, the tran-
sitory and persistent gender-specific effects of Cd on
NK cell cytotoxic activity could be partly mediated by
the potential endocrine disrupting effects of Cd.
Luster et al. (1993) showed a high concordance
between decrease of host-resistance and concomi-
tant alterations of T-lymphocyte proliferation and NK
cytotoxic activity. Immunotoxicity of chemicals was
also found to be well correlated to carcinogenicity
(Luster et al., 1992). Therefore, the effects observed
on these two immune biomarkers resulting from expo-
S. Pillet et al. / Toxicology 209 (2005) 289–301299
In conclusion, neonatal exposure to environmen-
tally relevant levels of Cd via maternal milk resulted
in gender-specific delays in the development of fe-
considering the limited number of lactating dams im-
posed by logistical constraints. Moreover, exposure to
persistent alterations of immune functions. Some of
surable Cd accumulation in the kidneys. The immuno-
toxic effects were dependent on the type of immune
cytotoxic activity of NK-cells were only observed in
males. To our knowledge, this is the first report of a
gender-specific difference in the immunotoxic effects
of Cd. The transitory and persistent immunotoxic ef-
fects were observed at levels of exposure much lower
than those reported in the literature as affecting the
immune functions when exposure occurs during adult-
hood. Considering that most of the previous studies
have been performed on adult rodents as a model for
human exposure, levels reported to be immunotoxic
and the potential effects on human health could be un-
R.Biondo(Universit´ edeLi` ege),M.Fortier,M.Gre-
are thanked for their assistance. This study was sup-
ported by a grant from the Canadian Toxic Substances
Research Initiative and the Canada Research Chair
in Environmental Immunotoxicity (MF). AR was a
recipient of an INRS post-doctoral fellowship and
SP was supported by the Fonds pour la Formation
` a la Recherche dans l’Industrie et dans l’Agriculture
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