Involvement of oxidative stress in the mechanism of cadmium-induced toxicity on rat uterus

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 364–373

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Involvement of oxidative stress in the mechanism of cadmium-induced toxicity on rat uterus ´ Marzenna Nasiadek ∗ , Małgorzata Skrzypinska-Gawrysiak, Adam Daragó, ´ Ewa Zwierzynska, Anna Kilanowicz Department of Toxicology, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland

a r t i c l e

i n f o

a b s t r a c t

Article history:

The study was undertaken to explore whether cadmium bioaccumulation can induce oxida-

Received 27 January 2014

tive stress in the uterus of rats. Cadmium (0.09, 0.9, 1.8 or 4.5 mgCd/kg b.w.) was administered

Received in revised form 9 July 2014

by gavage for 28 days. The animals were dissected on the first day and then after 90 days

Accepted 11 July 2014

post exposure (second group of animals). The results show that cadmium accumulates in

Available online 19 July 2014

the uterus in a dose-dependent manner. The uterine Cd concentrations were almost the

Keywords:

cadmium caused significant changes in catalase (CAT) activity and lipid peroxidation (MDA)

same in both groups, which is indicative of its long half-life in this organ. The accumulated Cadmium

levels at concentrations from 0.09 to 0.35 ␮gCd/g wet uterine tissue. In summary our results

Uterus

show that the induction of oxidative stress and lipid peroxidation in the uterus may play

Oxidative stress

important roles in the mechanism of toxicity in this organ and may have a negative impact

Repeated administration

on reproductive processes. © 2014 Elsevier B.V. All rights reserved.

Rat

1.

Introduction

Cadmium (Cd) is a major environmental toxicant. In the general population, food and drinking water are the main sources of cadmium exposure (WHO, 2000; ATSDR, 2012). Assuming that the average intake of Cd from food in most countries is in the range of 10–80 ␮gCd/day, and that an average person absorbs 5–15%, this would correspond to a Cd uptake of 0.5–12 ␮g/day. However, the bioavailability of inhaled Cd oxide in smokers is relatively high: 30–50%. It is assumed that a person who smokes one packet of cigarettes per day assimilates approximately 0.5–3.0 ␮gCd (ATSDR, 2008). More importantly, Cd has a long biological half-life, with a fast component of 75–128 days (blood) and a slow component of 10–38

∗

years (tissue) in humans, and hence accumulates in the body over considerable period, particularly in the kidneys and liver (WHO, 2000). Recent studies also point out that Cd is accumulated in the uterus (Nasiadek et al., 2005, 2011; Han et al., 2006; Höfer et al., 2009; Nakamura et al., 2012). Cadmium has been designated as an endocrine disruptor (ED), mainly due to its adverse effect on the reproductive system. This toxic metal is harmful to the reproductive process, causing retardation of growth, sterility and exerting an embryotoxic effect (Salvatori et al., 2004; Thompson and Bannigan, 2008). In recent years, increasing evidence has been accumulated suggesting that cadmium exerts an estrogenic effect (Garcia-Morales et al., 1994; Stoica et al., 2000; Johnson et al., 2003; Höfer et al., 2009). Johnson et al. (2003) have reported that cadmium exposure in ovariectomised rats resulted in

Corresponding author. Tel.: +48 42 677 91 48; fax: +48 42 677 91 48. E-mail addresses: [email protected] (M. Nasiadek), [email protected] ´ ´ (M. Skrzypinska-Gawrysiak), [email protected] (A. Daragó), [email protected] (E. Zwierzynska), [email protected] (A. Kilanowicz). http://dx.doi.org/10.1016/j.etap.2014.07.007 1382-6689/© 2014 Elsevier B.V. All rights reserved.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 364–373

uterine hyperplasia, increased growth and development of mammary glands, and induction of hormone-regulated genes. Recent epidemiological studies associate cadmium exposure to hormone-related cancers including endometrial cancer (Åkesson et al., 2008). The molecular mechanism responsible for the toxic effects of Cd has not so far been completely clarified. It is known that one of the several mechanisms of Cd toxicity operates by stimulating the formation of reactive oxygen species (ROS), thus causing oxidative damage in erythrocytes and in various tissues, which results in a loss of membrane function (Sarkar et al., 1995). It has been confirmed that long-term exposure to Cd leads to disruption of the cellular antioxidant system and/or inhibition of antioxidant enzymes. Earlier studies showed that oxidative stress appears to play a major role in Cd-induced toxicity in the liver, kidneys, brain, bone, testes and ovaries (Waalkes, 2000; Casalino et al., 2002; Tandon et al., 2003; Jurczuk et al., 2004; Yalin et al., 2006; Nemmiche et al., 2007; Samuel et al., 2011; Brzóska et al., 2011). However, no studies address the induction of Cd-induced oxidative stress in uterine tissue under conditions of acute and chronic toxicity, especially after a prolonged observation period. Previous studies have described xenoestrogenic activity of cadmium on uterine tissue (Höfer et al., 2009; Ali et al., 2010), but other biochemical aspects of toxicity have not been addressed.

1.1.

Aim

The aim of this study was to assess the bioaccumulation of cadmium in female rat uterine tissue and to evaluate its influence, from the biochemical aspect, on oxidative stress therein after a 28-day oral administration, and then after 90 days of observation following the termination of Cd exposure.

2.

Materials and methods

2.1.

Reagents

CdCl2 × 2.5 H2 O was obtained from Sigma–Aldrich (St. Louis, MO). All reagents were analytical grade. The water used was demineralized ultrapure of Milli-Q plus reagent grade.

2.2.

Animal experimental design

Eighty-four female Wistar rats (aged 9–10 weeks old) obtained from the Laboratory Animal Center of the Medical University of Lodz, showing at least three consecutive regular estrous cycles, were used in the study. All procedures conducted on rats were in accordance with the Local Animal Ethical Committee of Medical University of Lodz (protocol number: LKE 46/LB/481/2009). The animals were maintained in polypropylene steel cages under controlled conditions (12-h light/dark cycles at room temperature 22 ± 1 ◦ C and relative humidity 50–60%) for 2 weeks before the beginning of the experiment. The rats had free access to water and diet (Ssniff R/M-H low phytoestrogen). To identify regular estrous cycles, cytological analyses of vaginal smears by light microscopy were performed.

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The experiments were conducted on female rats which were divided randomly into two groups, the first receiving a 28-day exposure to Cd and the second undergoing 90 days of observation after the 28-day exposure: both groups comprising a control group (n = 10) and Cd-exposed group (n = 32). Animals from control groups received by gavage water with the same volume, which was used in Cd-exposed groups. Both the Cd exposed groups received of Cd as CdCl2 , which was administered by gavage in four different daily doses (0.09, 0.9, 1.8, 4.5 mgCd/kg b.w., which corresponded to: 1/1000; 1/100; 1/50, 1/20 LD50 ) (n = 8 each group) every day for 28 days. An average oral lethal dose (LD50 ) value for CdCl2 in rat was reported as 88 mgCd/kg b.w. (Lehman, 1951). After termination of both experiments, the estrous cycle was determined and only the female rats in estrus were classified for euthanasia, that is why the sacrifice of all the animals lasted from one to seven days to assure that all female rats were in the estrus stage on the day of euthanasia. During and following the treatment, all animals were observed carefully for mortality, body weight and gross behavioral changes.

2.2.1.

Euthanasia, tissue collection and preservation

Female rats which were found to be in estrus were weighed and underwent euthanasia after 28 days of exposure, and after 90 days of observation following a 28-day exposure. The rats were exsanguinated by heart puncture under light ether anesthesia. Subsequently, whole blood was drawn into Vacutainer tubes for metal analysis (S-Monovette, Sarstedt), the uteri were trimmed of fat and weighed. The uterus tissue was collected into acid washed tubes and then stored at −70 ◦ C until biochemical analysis. For Cd analysis, whole blood (1 ml) was kept in acid washed cryo-tubes at −70 ◦ C, while the remaining part of the blood was centrifuged at 3000 × g/10 min in a refrigerated centrifuge at 4 ◦ C, to separate the plasma. The buffy coat was removed, the plasma separated and the remaining erythrocytes (RBC) drawn from the bottom, washed three times in cold physiological saline (0.9% NaCl), and hemolyzed by the addition of an equal volume of ice-cold demineralized ultrapure water (MilliQ plus reagent grade; Millipore) to yield a 50% RBC lysate. The plasma and RBC lysate were stored at −70 ◦ C in cryo-tubes for biochemical assay. The Cd concentrations in whole blood and the uterus were determined in the collected samples, as were the following oxidative stress parameters: total antioxidant status (TAS) levels in the plasma, catalase activity (CAT) in the RBC lysate and concentration of reduced glutathione (GSH), malondialdehyde (MDA) and activity of catalase (CAT) in the rats uterus.

2.3.

Biochemical analysis

2.3.1.

Determination of TAS

The total antioxidant status (TAS) includes both antioxidant enzymes and antioxidant vitamins. The plasma TAS was measured with a Ransel NX 2332 readymade test (Randox Laboratories), according to the manufacturer’s instructions. The assay based on oxidation of ABTS (2,2 -azino-di-(33-ethylbenzthiazoline sulphonate) by H2 O2 catalyzed by peroxidase (metmyoglobin) to produce the radical cation

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ABTS+ , which has a relatively stable blue-green color. Antioxidant in the added sample cause suppression of this color production to a degree which is proportional to their concentration. The TAS level was expressed as mM/l plasma.

2.3.2.

Determination of CAT activity

CAT activity in the erythrocyte lysate was measured using a CAT 240 colorimetric assay kit for catalase activity (Applied Bioanalytical Labs.). CAT activity was expressed as U/ml RBC hemolysate. Uterus tissues were rapidly excised and homogenized in an ice bath using phosphate-buffered saline (pH 7.4) with 0.01% digitonin using a Kika Labortechnik T-25 basic homogenizer. The homogenate was centrifuged at 10000 × g for 30 min at 4 ◦ C. CAT activity in the supernatant of uterus homogenate was measured using a CAT 240 colorimetric assay kit for catalase activity (Applied Bioanalytical Labs.), according to the manufacturer’s instructions. Spectrophotometric analysis was performed using a Hitachi U-2900 spectrophotometer. CAT activity was expressed as U/mg protein. Protein concentrations in supernatants were determined according to Lowry et al. (1951). Bovine serum albumin was used as standard.

2.3.3.

Determination of GSH

GSH concentration in the uterus (10% homogenate in phosphate-buffered saline pH 8.0) was determined according to Sedlak and Lindsay (1968). After deproteinization of homogenates with 5-sulfosalicylic acid, non-protein thiol groups were determined via a reaction with Ellman’s reagent. The emerging yellow coloring was determined spectrophotometrically at 412 nm. At the same time, the calibration curve was plotted with the use of reduced GSH as standard substance. GSH concentration was expressed as ␮M/g tissue.

2.3.4.

Determination of MDA

The uterine malondialdehyde (MDA) levels, an indicator of free radicals generation which increases at the end of the lipid peroxidation (LPO), were estimated according to Uchiyama and Mihara (1978). The method is based on the spectrophotometric measurement of the color generated by reaction of thiobarbituric acid (TBA) with MDA. Homogenates, prepared in the same way as for the determination of GSH above, were heated at temperature of 100 ◦ C in the presence of 1% orthophosphoric acid with 0.6% thiobarbituric acid for 45 min. Lipid peroxidation products (especially MDA) reacted with thiobarbituric acid, and the color product of this reaction after n-butanol extraction were measured using a Hitachi U-1900 spectrophotometer at 532 nm. At the same time, a calibration curve was plotted with use of tetraepoxypropane, which under analytical conditions underwent hydrolysis, producing stoichiometric amounts of malondialdehyde. The concentration of MDA was expressed as nM/g tissue.

2.3.5.

Determination of cadmium concentration

Cd concentration was analyzed in blood and uterus tissue, using a Hitachi Z-8270 GFAAS with Zeeman-type background correction, autosampler and a pyrocoated tube. Digestion of tissue samples was performed with a MarsXpress microwave digestion system (CEM, USA) with nitric acid (Ultrex II, Baker). For determination of Cd concentrations, the samples were

prepared in duplicate. The analytical quality control was within the range of reference values: reference samples were Seronorm Whole Blood-1 (Sero, Norway) and Bovine liver 1577b (National Institute of Standards and Technology). The detection limits for Cd were 0.2 ␮g/l for blood or 0.2 ng/g for wet tissue. Cd concentrations in blood and uterine tissue were expressed as ␮g/l or ␮g/g wet tissue.

2.4.

Statistical analysis

Statistica software version 9.1 (Statsoft Polska) was used for statistical analyses. Statistical significance of differences was calculated using the Kruskal–Wallis analysis of variance with a subsequent Mann–Whiney U-test. Differences and correlations were considered statistically significant at p ≤ 0.05. All results are expressed as the mean ± SD (standard deviation).

3.

Results

Characteristics of rats subjected to different Cd doses during studies are described in Table 1. As shown, none of the administered doses of Cd resulted in significant changes in water or food consumption or in final body weight of female rats. Moreover, oral Cd exposure did not result in notable changes in absolute (g) or relative (g/100 g) wet weights of the liver, kidneys and of the uteri. However, a statistically insignificant tendency for uterine wet weight to slight elevations was observed in the groups treated with cadmium at a dose of 4.5 mgCd/kg b.w., both after 28 days of exposure and 90 days following the termination of Cd exposure, compared with controls (Table 1).

3.1.

Cadmium concentrations in the blood and uterus

The study revealed increased Cd concentrations in the blood and uterus of the female rats (Figs. 1 and 2). The blood Cd concentration (0.48 ␮g/l) in animals after administration of 0.09 mgCd/kg (28 doses), was similar to that observed in the non-exposed general population. Administration of higher doses (0.9, 1.8 and 4.5 mgCd/kg) resulted in a significant dosedependent increase in blood Cd concentration (Fig. 1). Oral administration of cadmium at doses ranging from 0.9 to 4.5 mgCd/kg contributed to a statistically significant increase in its concentration in the uterus, compared with controls (Fig. 2). Similarly to blood, the concentration of Cd in uterus tissue was dose dependent. Ninety days after exposure termination, Cd concentrations in the uterus did not decrease significantly, contrary to the Cd concentration in the blood, where the reduction was about 25-fold in comparison with group after 28 days of exposure (Figs. 1 and 2). In examined females a significant correlation between Cd concentration in the blood and uterine tissues was observed both after 28 days of exposure (rs = 0.87) (not shown) and after 90 days following termination of exposure (rs = 0.83) (not shown).

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Table 1 – Daily food, water consumption, body, liver, kidneys and uterine weights of rats exposed to CdCl2 for 28 days and then after 90 days post exposure period. Parameters

Doses (mgCd/kg b.w.) Control

0.09

0.9

1.8

4.5

28-day exposure Initial body weight (g) Final body weight (g) Liver weight (g) Liver weight (g/100 g b.w.) Kidneys weight (g) Kidneys weight (g/100 g b.w.) Uterine weight (g) Relative uterine weight (g/100 g b.w.) Food consumption (g) Water consumption (ml)

199 208 7.00 3.36 1.18 0.56 0.63 0.31 14.4 27.2

± ± ± ± ± ± ± ± ± ±

7 9 0.68 0.32 0.08 0.02 0.06 0.03 1.9 6.2

209 223 7.29 3.26 1.27 0.56 0.74 0.32 15.5 30.9

± ± ± ± ± ± ± ± ± ±

5 10 0.95 0.33 0.18 0.04 0.04 0.07 1.1 5.9

198 211 7.09 3.34 1.16 0.55 0.74 0.36 13.8 30.8

± ± ± ± ± ± ± ± ± ±

6 3 0.84 0.39 0.13 0.03 0.05 0.03 2.5 3.8

210 218 7.29 3.33 1.26 0.57 0.75 0.34 15.6 42.8

± ± ± ± ± ± ± ± ± ±

9 11 0.54 0.24 0.15 0.04 0.09 0.04 1.2 16.5

210 224 7.31 3.26 1.22 0.54 0.88 0.39 14.8 26.9

± ± ± ± ± ± ± ± ± ±

7 8 0.91 0.41 0.10 0.01 0.18 0.08 2.6 2.8

90-day post exposure Initial body weight (g) Final body weight (g) Liver weight (g) Liver weight (g/100 g b.w.) Kidneys weight (g) Kidneys weight (g/100 g b.w.) Uterine weight (g) Relative uterine weight (g/100 g b.w.) Food consumption (g) Water consumption (ml)

195 249 7.80 3.13 1.34 0.54 0.89 0.35 25.2 44.2

± ± ± ± ± ± ± ± ± ±

6 21 0.40 0.24 0.08 0.03 0.09 0.08 14.2 10.2

200 257 7.49 2.91 1.35 0.52 0.83 0.32 27.4 40.9

± ± ± ± ± ± ± ± ± ±

5 15 0.49 0.14 0.08 0.04 0.08 0.03 13.6 9.7

199 248 7.66 3.08 1.30 0.52 0.89 0.36 30.2 40.2

± ± ± ± ± ± ± ± ± ±

9 4 0.59 0.15 0.05 0.03 0.08 0.03 4.8 8.6

203 257 7.76 3.02 1.32 0.51 0.98 0.38 26.4 39.5

± ± ± ± ± ± ± ± ± ±

4 15 0.49 0.16 0.10 0.02 0.06 0.09 13.3 7.3

201 255 7.80 3.05 1.36 0.53 1.00 0.39 27.5 42.4

± ± ± ± ± ± ± ± ± ±

6 20 0.68 0.11 0.09 0.03 0.18 0.06 14.5 10.3

All values are expressed as means ± SD.

Table 2 – Changes in biochemical parameters in the uterus of rats exposed to CdCl2 for 28 days and then after 90 days post exposure. Parameters

Doses (mgCd/kg b.w.) Control

0.09

0.9

28-day exposure GSH (␮M/g uterus) CAT (U/mg protein) MDA (nM/g uterus)

1.8

4.5

1.22 ± 0.20 10.23 ± 1.53 67.96 ± 11.40

1.21 ± 0.07 11.05 ± 0.94 57.44 ± 18.16

0.91 ± 0.38 11.75 ± 1.08 75.6 ± 8.90

1.14 ± 0.18 12.68 ± 1.33a ,e 97.21 ± 15.89a ,b

1.11 ± 0.24 8.05 ± 1.24a ,b ,c, d 119.19 ± 14.08a ,b , c

90-day post exposure period GSH (␮M/g uterus) CAT (U/mg protein) MDA (nM/g uterus)

0.98 ± 0.10 9.02 ± 1.65 61.62 ± 7.67

0.96 ± 0.05 9.68 ± 1.80 74.14 ± 10.25

0.98 ± 0.08 11.10 ± 1.53 71.01 ± 14.20

0.86 ± 0.14 12.25 ± 1.04a ,b, e 84.68 ± 14.91a

0.75 ± 0.18 7.56 ± 0.93a ,b ,c, d 88.02 ± 6.67a

All values are expressed as means ± SD. Significance from groups – p ≤ 0.05 Significantly different from control animals. b Significantly different from the group of rats given Cd 0.09 mg/kg/day. c Significantly different from the group of rats given Cd 0.9 mg/kg/day. d Significantly different from the group of rats given Cd 1.8 mg/kg/day. e Significantly different from the group of rats given Cd 4.5 mg/kg/day. a

Table 3 – TAS level in the plasma and CAT activity in RBC hemolysate of rats exposed to CdCl2 for 28 days and then after 90 days post exposure. Parameters

Doses (mgCd/kg b.w.) Control

0.09

0.9

1.8

28-day exposure TAS (mM/l plasma) CAT (U/ml hemolysate)

1.08 ± 0.10 34.45 ± 6.15

1.14 ± 0.06 32.82 ± 3.76

1.12 ± 0.06 36.68 ± 4.15

1.16 ± 0.08 38.92 ± 5.65

1.18 ± 0.09 42.94 ± 4.81

90-day post exposure TAS (mM/l plasma) CAT (U/ml hemolysate)

1.08 ± 0.10 36.38 ± 3.26

0.94 ± 0.06 37.46 ± 2.15

0.98 ± 0.08 34.69 ± 5.06

0.99 ± 0.04 31.16 ± 3.00

1.04 ± 0.08 30.66 ± 3.97

All values are expressed as means ± SD.

4.5

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Fig. 1 – Cadmium concentrations in the blood (␮g/l) after 28 days administration by gavage of CdCl2 and after 90 days post exposure period. All values are expressed as means ± SD. Significance from groups – *p ≤ 0.05; **p ≤ 0.01. (a) Significantly different from control animals; (b) significantly different from the group of rats given Cd 0.09 mg/kg/day; (c) significantly different from the group of rats given Cd 0.9 mg/kg/day; (d) significantly different from the group of rats given Cd 1.8 mg/kg/day; (e) significantly different from the group of rats given Cd 4.5 mg/kg/day.

3.2.

Biochemical analysis

The effect of cadmium treatment on GSH levels, CAT activities, MDA level in uterus and plasma TAS, erythrocyte CAT activity, which may represent redox status, were examined in female rats (Tables 2 and 3). In uterine tissue, both after 28 days of Cd administration and after 90 days following termination of exposure, significant changes were detected in the specific activity of catalase, one of the enzymes constituting an antioxidative barrier, in comparison to controls. When cadmium was given at a dose of 1.8 mgCd/kg b.w., the CAT activity markedly increased in females, however, it was found to decrease after a dose of 4.5 mgCd/kg b.w. (Table 2).

Malondialdehyde (MDA) level, which is an indicator of lipid peroxidation level, and thus serves as the marker of oxidative stress, was also determined. As shown in Table 2, lipid peroxidation (expressed as MDA) was significantly increased in the uterus tissue of Cd-treated rats (1.8, 4.5 mgCd/kg b.w.) both after terminating 28 days of exposure, as well as after 90 days following the termination of Cd administration, compared with the control group. However, contrary to the uterus tissue, no significant differences were detected in the level of total antioxidative status (TAS) in plasma and in the activity of CAT in erythrocytes between the study and control groups, both after 28 days of exposure and after a 90-day observation period following the termination of administration (Table 3).

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 364–373

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Fig. 2 – Cadmium concentrations in the uterus (␮g/g wet tissue) after 28 days administration by gavage of CdCl2 and after 90 days post exposure period. All values are expressed as means ± SD. Significance from groups – *p ≤ 0.05; **p ≤ 0.01. (a) Significantly different from control animals; (b) significantly different from the group of rats given Cd 0.09 mg/kg/day; (c) significantly different from the group of rats given Cd 0.9 mg/kg/day; (d) significantly different from the group of rats given Cd 1.8 mg/kg/day; (e) significantly different from the group of rats given Cd 4.5 mg/kg/day.

4.

Discussion

Growing evidence indicates the participation of reactive oxygen species (ROS) in the initiation or development of pathological processes affecting female reproductive processes and cancerous transformation of cells (Al-Gubory et al., 2010; Waalkes, 2000). Reactive oxygen species (ROS) are often implicated in Cd toxicology (Cuypers et al., 2010). Cadmium has been ranked 7th on The Priority List of Hazardous Substances constituting security risk to health of humans and animals (ATSDR, 2012). In the general population, food is the main source of exposure to cadmium. In Europe, estimated daily dietary intakes of cadmium in nonsmoking adults are 11.5–78.4 ␮g/day. The Provisional Tolerable Weekly Intake (PWTI) stipulates that the weekly intake of this metal from food must not exceed 2.52 ␮g/kg b.w./week, which represents 25.2 ␮g Cd per day for a person who weighs 70 kg (EFSA, 2012). In our study, rats were administered cadmium at doses ranging from 0.09 to 4.5 mgCd/kg b.w., which corresponds to a range of 21–1080 ␮gCd/day/rat. The present study shows the accumulation of cadmium in the uterus to be dose dependent, in accord with other studies (Han et al., 2006; Höfer et al., 2009; Nakamura et al., 2012). As observed now, the uterine Cd concentration did not change 90 days after exposure (Fig. 2). This suggests that the biological half-life of Cd in the uterus is similar to that observed in the liver or kidneys, the organs in which Cd usually accumulates with biological half-lives ranging from 10 to 38 years (Järup et al., 1998; WHO, 2000). Although

Cd concentration was tenfold lower in the uterus than in the liver or kidneys, as demonstrated in our studies (data not shown) and in the studies carried out by other authors (Höfer et al., 2009, 2010), it is supposed that the accumulation mechanism is similar to that found in other organs (liver, kidneys or intestines) in which significant Cd accumulation and metallothioneins (MTs) induction have been reported (Cousin, 1979; Klaassen et al., 2009; Höfer et al., 2010). Moreover, Höfer et al. (2010) showed that estrogens can regulate MTs expression in the liver, kidneys and small intestine. Recently, Cd induction of MT1a expression, also in combination with steroid estrogen, in the rat uterus has been shown (Kluxen et al., 2012). Since the animals in the present study were intact females, it is likely that MTs is responsible for Cd accumulation in the uterus. However, the effect of other Cd-binding ligands cannot be excluded, especially bearing in mind that Nakamura et al. (2012) showed that Cd in the uterus was bound to MTs in 10% only, while the remaining 90% was most probably bound to other proteins or peptides. Cd concentrations in the uterine tissue of rats, which were administered cadmium for 28 days, especially at doses 0.9–1.8 mgCd/kg b.w., were comparable to the Cd concentration seen in human uteri from the general population, as shown in our previous studies (Nasiadek et al., 2005, 2011). However, Cd concentrations in the uterine tissue of women occupationally exposed to cadmium can be expected to be higher, as is the case with smokers. This may be particularly relevant in the context of epidemiological studies describing the increase in premature births and low body weight

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of infants in groups of smoking mothers (Dachanet et al., 2011). Research from recent years, particularly Johnson et al. (2003) who observed uterine hyperplasia, increased uterus weight and progesterone receptor induction after single i.p. injection of cadmium at a dose of 5 ␮g/kg b.w. to ovariectomised rats, has provided new evidence that this metal represents a new class of xenoestrogens: the metalloestrogens. Differences in changes of uterine wet weight are considered a well-established endpoint for detecting estrogen-like chemicals (Diel et al., 2002; OECD, 2007). It has been suggested that the increase in the uterine weight most probably results from increased proliferation of endometrium cells (Byrne et al., 2009). Further research performed on ovariectomised rats has demonstrated a range of estrogenic responses to Cd, which probably results from the Cd distribution depending on the route of administration (Zhang et al., 2007; Höfer et al., 2009; Liu et al., 2010). In the present study in female rats with ovaries, no changes were detected in uterine wet weight after oral cadmium administration. Our results agree with those of Ali et al. (2010) after s.c. injection and Höfer et al. (2009) after oral administration. As noted by Ali et al. (2010), not only the route of administration but other factors such as species sensitivity, time of exposure, age, and basic diet are also likely to contribute to the net effects seen in different studies. The mechanism of toxic activity of cadmium on the organism is multidirectional. However, it is now assumed that the induction of oxidative stress is a key factor. Although Cd is not able to induce free radicals directly, it may disturb the oxidative/antioxidative status and lead to the development of oxidative stress indirectly via weakening of the antioxidative defense, disturbances of the mitochondrial electron transport chain and displacement of ions of metals such as iron (Fe) and copper (Cu) from cellular proteins, capable of inducing ROS directly (Valko et al., 2006; Gobe and Crane, 2010; Brzóska et al., 2011). So far, Cd-induced oxidative stress has been confirmed in the kidneys, liver, lungs, pancreas, testicles, ovaries, and bones and it has been found to depend on dose, route of administration, and the time of exposure (Casalino et al., 2002; Tandon et al., 2003; Abdel-Moneim and Said, 2007; Järup and Åkesson, 2009; Liu et al., 2009; Brzóska et al., 2011; Samuel et al., 2011). The present study demonstrates for the first time that Cd in vivo may induce oxidative stress as well as lipid peroxidation in rat uterine tissue. Changes in the activity of CAT, an enzyme which is responsible for the detoxification of H2 O2 , can be an indicator of Cd-induced oxidative stress in the uterus. The study results indicate that administration of cadmium at a dose of 1.8 mgCd/kg b.w. significantly increased and a higher dose (4.5 mgCd/kg b.w.) significantly decreased CAT activity both after a 28-day exposure and 90 days after exposure termination. It has also been reported that in kidneys, liver and testis, low concentrations of Cd stimulate antioxidant defence, while higher levels of this metal inhibit the activity of such antioxidant mechanisms as CAT and superoxide dismutase (SOD) (Wolf and Baynes, 2007; Ognjanovic´ et al., 2008; Wang et al., 2011). It is possible that higher Cd concentrations in the uterus may inhibit the activity of antioxidative enzymes, not only CAT, but also SOD or glutathione peroxidase (GPx).

Samuel et al. (2011) demonstrated that SOD, CAT and GPx activity in the rat ovary is strongly inhibited by Cd at doses of 50 or 200 mgCd/l drinking water (approximately calculated 2.5 or 10 mg/kg b.w.). The exact mechanism of Cd-induced effects on CAT activity remains to be clarified. One hypothesis is based on the interaction between cadmium and iron, which is the catalytic ´ et at.,1999; Jurczuk et al., 2004; unit of CAT (Wronska-Nofer Casalino et al., 1997). According to our own unpublished data, no increase in Fe concentration is observed in uterine tissue following Cd administration, which might suggest that in uterus, the predominant mechanism is most probably connected with increase in the levels of H2 O2 and superoxide radicals, which contribute to the inhibition of such antioxidative enzymes as CAT. It is known that hydrogen peroxide (H2 O2 ) is a key player in the metabolism of reactive oxygen species (ROS) (Dröge, 2002) and increase in its concentration may influence uterine muscle activity (Nakai et al., 2000; Warren et al., 2005). It is interesting that 90 days after terminating the exposure, a tendency to decrease GSH level in uterine tissue was observed. Reduced glutathione (GSH) is the most abundant low-molecular-weight thiol, which plays an important role in redox reactions and is considered as the first defense of the organism against the toxic effect of many compounds including Cd (Liu et al., 2009). Although GSH depletion was observed mainly after acute exposure to Cd (Dudley and Klaassen, 1984), it was also shown in the kidneys and liver of rats after subchronic exposure at doses similar to that administered in our research (Moniuszko-Jakoniuk et al., 2005; El-Sokkary et al., 2009; Ramesh and Satakopan, 2010). It cannot be excluded that in the uterus, contrary to the liver and kidneys, the GSH pool is depleted much later as a consequence of the accumulation of this metal in uterine tissue. Additionally, the age of the exposed female rats may be an important factor influencing the observed decrease in the level of GSH, since it is known that the GSH pool diminishes with age, which might, in turn, contribute to the increased sensitivity of the uterus to cadmium. It was reported that ROS may propagate the initial attack on lipid membranes to cause lipid peroxidation (LPO). MDA (malondialdehyde) is one of the numerous compounds formed during the process of LPO. It has been previously demonstrated that although Cd induces lipid peroxidation in various tissues including the liver, kidneys and ovaries, this effect was not examined in the uterus (Liu et al., 2009; Ramesh and Satakopan, 2010; Roopha et al., 2011; Samuel et al., 2011). In the present study, the probable increase in H2 O2 production in the uterus of Cd-exposed rats (especially after the highest dose of 4.5 mgCd/kg b.w., when the observed decrease in CAT activity) may be responsible for the observed increase in MDA concentration, both after a 28-day cadmium exposure period and then after a post exposure period of 90 days, indicating lipid peroxidation in uterine tissue. MDA has been shown to exert cytotoxic, mutagenic and carcinogenic effects (Siu et al., 1983; Ott et al., 2007), which appear to be of vital importance in view of epidemiological studies (Åkesson et al., 2008). These studies demonstrated a 2.9-fold increase in the risk of endometrial carcinoma in postmenopausal women to be related to longterm consumption of cadmium with food (15 ␮gCd/day). In

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addition, other studies have shown decreased antioxidative enzyme activity and increased MDA content in myomas and adenocarcinoma tissues (Pejic´ et al., 2009). Despite the fact that the observed changes in biochemical parameters (CAT an MDA) in the uterus of rats occurred at high doses of cadmium (1.8 and 4.5 mgCd/kg b.w.), the Cd concentrations in the rat uterus tissues (0.09 and 0.35 ␮gCd/g wet tissue, respectively) were similar, particularly at lower dose, to the concentration of Cd in the uterus myometrium of environmentally exposed non-smoking women in the general population (0.075 ± 0.068 ␮gCd/g wet tissue) (Nasiadek et al., 2011). It is worth mentioning that in the endometrial cancer tissue Cd concentration is twofold higher (0.15 ± 0.08 ␮gCd/g wet tissue) (Nasiadek et al., 2005). Furthermore, the sensitivity of the uterus to oxidative stress may be related to the presence of metallothioneins, the protein which plays an important role in protecting Cd toxicity and in combating Cd-induced ROS through Cd sequestration or free radical scavenging (Dudley and Klaassen, 1984; Klaassen et al., 1999). In conclusion, it is worth emphasizing that these are the first set of results to indicate that cadmium induces oxidative stress and lipid peroxidation in the uterus, similarly to other critical organs. Our results support the hypothesis that oxidative stress may be one of the mechanisms of the toxic effect of Cd on the uterus, and might adversely affect its function. It cannot be excluded that Cd-induced lipid peroxidation in uterine tissue may also play an important role in cancer etiology. Further research on molecular levels will allow clarification of this mechanism.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

Acknowledgment This work was supported by the Polish National Science Centre (NCN), Grant No. NN 404 504938.

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