Protective effects of oestradiol against cadmium-induced changes in blood parameters and oxidative damage in rats

Summary The aim of this study was to investigate the protective effects of oestradiol (E2, 4 mg kg-1 b.w. i.p.) against cadmium-induced (Cd, 2 mg kg-1 b.w. i.p.) blood changes in rats. Cadmium induced a significant decline in haemoglobin, haematocrit, and total erythrocyte, lymphocyte, and thrombocyte count, whereas total leukocytes and granulocytes increased. A significant increase was also observed in serum cholesterol, triglycerides, glucose, AST, and ALT activities, whereas total protein and albumin levels dropped significantly. Administration of E2 in combination with Cd alleviated most of these adverse effects. In terms of oxidative stress, Cd significantly increased oxygen-free radicals (O2 •- and H2O2) in neutrophils and lipid peroxidation in erythrocytes, whereas E2 treatment reversed these changes to control values. Acute Cd poisoning significantly lowered antioxidant enzyme (SOD and CAT) activity and the level of non-enzymatic antioxidants (GSH and vitamin E), while increasing in GSSG. Treatments with E2 reversed Cd-induced effects on the antioxidant defences and significantly lowered Cd-induced oxidative damage in erythrocytes. This study suggests that exogenous E2 effectively restores redox balance in rat erythrocytes and counters adverse haematological and biochemical effects of Cd poisoning. It also improves the antioxidant capacity of erythrocytes, acting in synergy with endogenous antioxidants. Sažetak Cilj ovog istraživanja bio je ispitati moguće zaštitne učinke estradiola (E2, 4 mg kg-1 tjelesne težine i.p.) protiv kadmijem izazvanih (Cd, 2 mg kg-1 tjelesne težine i.p.) promjena u krvi štakora. Kadmij je značajno smanjio vrijednosti hemoglobina, hematokrita, eritrocita, limfocita i trombocita te povećao vrijednosti ukupnih leukocita i granulocita. Također je uočen značajan rast razina AST i ALT, serumskog kolesterola, triglicerida i glukoze te pad razina ukupnih proteina i albumina. Primjena E2 u kombinaciji s Cd ublažila je većinu tih štetnih učinaka Cd. U pogledu oksidacijskog stresa, Cd je značajno potaknuo ne samo nastanak slobodnih kisikovih radikala (O2 •- i H2O2) u neutrofilima nego i lipidnu peroksidaciju u eritrocitima, a primjena E2 te je promjene svela na kontrolne vrijednosti. Akutno otrovanje kadmijem osjetno je smanjilo aktivnost antioksidativnih enzima SOD i CAT te razinu neenzimskih antioksidansa GSH i vitamina E, a povećalo razinu GSSG. Primjena E2 preokrenula je promjene u sustavu antioksidacijske obrane i značajno spriječila oksidacijska oštećenja u eritrocitima izazvana kadmijem. Prema rezultatima ovog istraživanja, E2 se učinkovito bori s disbalansom redoks statusa eritrocita i negativnim hematološkim i biokemijskim učincima nastalima nakon akutnog trovanja štakora kadmijem. Egzogeno primijenjen E2 pridonosi poboljšanju antioksidativnog kapaciteta eritrocita, djelujući sinergistički s endogenim antioksidansima.

Exposure to Cd can results in toxic effects on a variety of tissues, but the first to be affected is blood, as Cd binds to the membrane of erythrocytes and plasma albumin and is then transported to the liver (1), causing haematological and biochemical changes (2) and anaemia (3)(4)(5).Furthermore, erythrocytes are the most common markers of oxidative stress due to the sensitivity of their cell membranes and antioxidant enzymes to free radicals (6,7).
The mechanism of acute Cd toxicity involves depletion of glutathione, which results in increased production of reactive oxygen species (ROS) such as superoxide anion (O 2 •-), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (•OH) (8,9).This in turn leads to increased lipid peroxidation, DNA damage, protein oxidation, and eventually to cell dysfunction and necrosis (10).
Female sex hormone oestradiol (17β-oestradiol, E2) inhibits lipid peroxidation caused by free radicals (11) and improves intracellular SOD and CAT activity (12).It can act directly, by scavenging free radicals and by chelating redox-active metal ions or indirectly, by regulating antioxidant enzyme expression (13).
A number of studies have already established its protective role in the cardiovascular and nervous system (14,15), but little is known about its effects against Cd toxicity in blood in vivo.
The aim of this study was to address this gap by investigating how E2 counters the effects of Cd on haematological, biochemical, and oxidative stress parameters, including antioxidant defences in the blood of acutely exposed rats.We started with the assumption that the protective mechanism of E2 against Cd-induced toxicity is based on its antioxidative action and therefore investigated how it counters Cdinduced oxidative burst in neutrophils and redox imbalance in erythrocytes as markers of oxidative stress.Given the complexity of E2 action and difficulties in distinguishing its genomic from nongenomic effects, this study aimed to better characterise its beneficial effects in vivo.We hope that our findings will have practical implications for E2 therapy in conditions of metal-induced oxidative stress.

Chemicals
Chemicals for this study were obtained from Sigma Chemical Co.(St.Louis, MO, USA) and Merck (Darmstadt, Germany).All reagents and chemicals were of analytical grade or higher purity.

Animals
The study included 32 male, eight-week-old Wistar albino rats weighing 200-220 g.As we wanted to investigate non-genomic effects of oestradiol, we used only male rats to avoid interaction between exogenous oestradiol and oestrogen receptors as well as hormonal changes during the oestrous cycle (16).The animals were maintained in plastic cages under standard laboratory conditions (temperature 22±2 °C; 12-hour light/dark cycle).The rats had free access to drinking water and standard rodent laboratory diet.All animal experiments were approved by the institutional ethics committee.

Experimental design
Animals were randomly divided into four groups of eight.Group 1 served as control and received saline [0.1 mL kg -1 body weight (b.w.)], whereas the remaining three experimental groups received single intraperitoneal (i.p.) injections as follows: group 2 -2 mg kg -1 (b.w.) of CdCl 2 in 0.1 mL saline; group 3 -4 mg kg -1 (b.w.) of 17β-oestradiol; and group 4 -17β-oestradiol 24 h after CdCl 2 in the above doses.The animals were anaesthetised with ether and decapitated 24 h after treatment.
On the day of sacrifice blood samples were collected in K-EDTA tubes for haematological analysis or in tubes without anticoagulants for other analyses.Haematological and biochemical parameters were measured on the day of sacrifice.
Blood biochemistry included serum concentrations of total protein, albumin, total cholesterol, triglycerides, glucose, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), measured on an autoanalyser (Architect C8000, Wiesbaden, Germany) using standard diagnostic kits (Abbott Laboratories, Abbott Park, IL, USA).The rest of the samples were stored at -20 °C until analysis but for no longer than seven days.
To measure oxidative stress parameters, blood samples were centrifuged at 1000 g (+4 °C) for 10 min and serum was removed.Erythrocytes were washed three times with an equal volume of cold saline (0.9 %, v/v), and 1 mL of washed erythrocytes was lysed on ice in 3 mL of dH 2 O (0 °C) for 30 min.To determine the levels of superoxide anion (O 2 •-) and hydrogen peroxide (H 2 O 2 ), we extracted erythrocyte lysates by adding 0.25 mL of 3 mol L -1 perchloric acid and 1 mL of 0.02 mol L -1 EDTA to 0.5 mL of lysate.After extraction on ice and centrifugation at 1000 g for 10 min, lysate extracts were neutralised with 2 mol L -1 K 2 CO 3 .
The determination of O 2 •-was based on the reduction of nitroblue tetrazolium (NBT) in the presence of O 2 •- (17).The determination of H 2 O 2 was based on the oxidation of phenol red (PR) in the presence of horseradish peroxidase (HRPO) as catalyst (18).Both measurements were done on a JENWAY 6105 UV-Vis spectrophotometer (Bibby Scientific Ltd., Staffordshire, UK) and concentrations are expressed as µmol L -1 of erythrocytes.
Lipid peroxidation was determined according to method of Ohkawa et al. (19), based on the reaction of lipid peroxidation products malondialdehyde (MDA) with thiobarbituric acid (TBA).Briefly, haemolysate samples were extracted by adding 0.4 mL of 28 % trichloroacetic acid (TCA) to 0.8 mL of lysate and centrifuged at 1000 g for 10 min.Colour reaction was obtained out by adding 0.2 mL of 1 % TBA and incubating the samples in a bath at 90 °C for 15 min.These results are expressed in µmol L -1 of erythrocytes using a molar extinction coefficient for MDA.
Superoxide dismutase (SOD, EC 1.15.1.1)activity was determined using the method of Marklund and Marklund (20), based on pyrogallol oxidation by O 2 •and its dismutation by SOD.Enzyme activity is expressed as units per milligram (U mg -1 ) of haemoglobin.
Catalase (CAT, EC 1.11.1.6)activity was measured with the JENWAY 6105 UV-Vis spectrophotometer at 230 nm (21) as the rate of H 2 O 2 degradation by CAT and is expressed in μmol min -1 g -1 of haemoglobin.
Reduced glutathione (GSH) was determined using the method of Beutler (22), based on GSH oxidation with 5,5'-dithiobis(2-nitrobenzoic acid) and its concentrations expressed as mmol L -1 of erythrocytes.The concentrations of oxidised glutathione (GSSG) were determined after enzymatic reaction with glutathione reductase (23) after inhibition of GSH oxidation by N-ethylmaleimide, and expressed in µmol L -1 of erythrocytes.In both cases, haemolysate samples were extracted by adding 0.4 mL of 0.1 % EDTA and 0.75 mL of precipitation solution (containing 25 % HPO 3 , 0.005 mol L -1 EDTA and 0.1 mol L -1 phosphate buffer) to 0.1 mL of lysate and centrifuging the mix at 1000 g for 10 min.
Vitamin E was determined in haemolysate extracts (obtained by adding 0.05 mL of 1 % EDTA to 0. Neutrophils were isolated according to the method of Russo-Carbolante et al. (25).Aliquots of blood were collected into plastic tubes containing K-EDTA and Histopaque reagent (1077 mg mL -1 ) was added.The tubes were centrifuged at 400 g for 45 min and the upper phase removed.The lower phase was added 6 % dextran solution (in 0.15 mol L -1 NaCl) and PBS and incubated at 37 °C for 20 min.To lyse erythrocytes, the pellet was resuspended in 0.83 % NH 4 Cl and after 5 min centrifuged at 480 g for 10 min.After washing with PBS, the cells were resuspended in 1 mL of PBS in the concentration of 10 6 cells mL -1 .The obtained neutrophils were used to measure O 2 •-and H 2 O 2 levels using a modified colorimetric NBT test (26) and an ELISA reader (Optic Ivymen System, Model 2100C, Biotech SL, Madrid, Spain).The results are expressed as optical density (OD) of 10 6 cells mL -1 .

Statistical analysis
All data were evaluated using the SPSS for Windows software, version 13 (SPSS Inc., Chicago, IL, USA).The results are expressed as mean±standard error of the mean (SEM).Comparisons were made using either factorial analysis of variance (ANOVA) with a post-hoc Bonferroni/Dunnett's multiple analysis or Kruskal-Wallis test (for comparison across several groups) and Mann-Whitney U-test (for comparison between two groups).Differences at p<0.05 were considered statistically significant.

Haematological and biochemical findings
Acute poisoning of rats with Cd significantly lowered Hb, Hct, TEC, lymphocyte, and PLT counts and increased TLC and granulocytes.Administration of E2 in combination with Cd alleviated the harmful effects of Cd on most haematological parameters (Table 1).
Table 2 shows significant increase in serum cholesterol, triglycerides, glucose, AST, and ALT, while total protein and albumin levels significantly dropped in the Cd group compared to control.Treatment with Cd+E2 significantly reversed these effects to nearly normal values compared to the Cd group.

Oxidative stress parameters
After treatment with Cd, H 2 O 2 concentration in erythrocytes dropped significantly compared to control, whereas O 2 •-increased, but not significantly.E2 treatment did not affect O 2 •-and H 2 O 2 in erythrocytes (Figure 1).In contrast, neutrophil concentrations of O 2 •-and H 2 O 2 significantly increased after Cd   treatment, while E2 lowered them significantly (Figure 2).Treatment with E2 reversed Cd-induced changes in these oxidative stress parameters.
Cadmium significantly increased LPO in erythrocytes, whereas treatment with E2 restored it to control levels (Figure 3).
Antioxidant defence enzymes SOD and CAT significantly dropped in Cd-treated rats, whereas, again, E2 treatment restored these to control values (Figure 4).
Table 3 shows changes in GSH, GSSG, and vitamin E. As with other parameters, E2 significantly reversed Cd-induced changes.

DISCUSSION
Our findings agree with earlier reports (3,5,(27)(28)(29) and confirm the ability of Cd to induce oxidative stress in rat erythrocytes.ROS production and lipid peroxidation induced by acute Cd treatment resulted in erythrocyte haemolysis and anaemia, in the activation of immune system, and in disorders of carbohydrate and lipid metabolism in hepatocytes.
Oestradiol seems to efficiently counter the adverse haematological and biochemical effects of Cd in rats and may substantially reinforce endogenous protection against oxidative stress.
The most important findings of this study are briefly discussed below.Consistent with other studies (30,31), E2 showed beneficial effects on carbohydrate metabolism as it lowered high blood glucose in Cdtreated rats.The observed drop in AST and ALT may indicate protective activity of E2 against hepatocyte damage.In addition, E2 alleviated inflammation.According to previous reports (32,33), this was probably mediated by reduced spontaneous secretion of inflammatory cytokines and migration of leukocytes into inflamed areas.
Our results show that Cd increased granulocyte count and induced neutrophil oxidative burst.Some of the molecular mechanisms of Cd toxicity, as reported in a review by Waisberg et al. (1), include release of a large amount of O 2 •-and H 2 O 2 from activated neutrophils into circulation, which leads to the influx of ROS in erythrocytes.In our study, acute  Cd treatment resulted in significantly lower H 2 O 2 concentration and increased LPO concentration in erythrocytes.These cells are capable of producing and storing nitric oxide (NO) (34).Being faster than SOD, NO scavenges O 2 •-to form cytotoxic peroxynitrite (ONOO -), which in turn mediates protein nitration and lipid peroxidation (6,7).Since increased LPO disrupts the normal function or destroys the membrane of erythrocytes, the final outcome of these events is a decrease in haematocrit level, and eventually anaemia, which was also observed in our study.
We found that treatment with E2 significantly reduced the concentration of both O 2 •-and H 2 O 2 in neutrophils, suggesting that E2 effectively counteracted oxidative burst induced by Cd.This is in line with the study by Priyanka et al. (35), who have reported that this hormone inhibits production and release of large amounts of O 2 •-and H 2 O 2 from neutrophils that can pass erythrocyte membranes and induce oxidative stress.
Oestrogens protect cells against oxidative stress in direct or oestrogen receptor-independent manner by decreasing ROS production and preventing intracellular H 2 O 2 accumulation (15,36,37).By transferring the hydrogen atom from its phenol-hydroxyl ring to O 2 •-, E2 scavenges oxyradicals and blocks their intracellular accumulation, thus preventing the formation of ONOO -and lipid peroxidation (11,13).
Our results have confirmed that E2 counters Cd toxicity by inhibiting LPO in erythrocytes and by preventing haemolysis.Previous studies (11,13) suggest that this is mediated by the phenol hydroxyl group of E2, which donates hydrogen atoms to a lipidderived radical.The resulting oestrogen phenoxyl radical is stabilised by delocalisation of unpaired electrons in the aromatic ring (13,14).Oestrogens thus prevent LPO by sacrificing themselves to oxidation, turning into quinol, which can be formed directly from E2 and •OH without the participation of metabolic enzymes, and by converting back to E2 using NADPH as the reducing agent without the production of ROS (13,38).In the erythrocytes of Cd-exposed rats we found lower SOD and CAT activity and a significant depletion of non-enzymatic antioxidants (GSH and vitamin E).Since Cd is able to substitute divalent metals in metalloenzymes (1), the lowering effect of Cd on SOD activity is the consequence of interactions between Cd and Zn or Cu in the SOD molecule.Lower SOD activity reflects on lower H 2 O 2 concentration, which finally results in lower CAT activity, whose substrate is H 2 O 2 (6,7).
The degradation of H 2 O 2 is also potentiated by vitamin E and GSH (6,7).Our results confirm the protective effects of E2 against changes in both enzymatic and non-enzymatic antioxidants in rat erythrocytes.These findings are in line with Hambden et al. (30), who found that E2 enhanced SOD and CAT activities in the liver of diabetic rats.Similar protective effects were observed in cultured rat hepatocytes (39), where E2 inhibited ROS generation, lipid peroxidation, as well as the loss of SOD and GSH-Px activities.The results of our study suggest that E2 improves erythrocyte antioxidant capacity due to either direct scavenging of ROS or induction of antioxidant enzymes.
To prevent LPO and cell damage, exogenous antioxidants may act in synergy with endogenous antioxidants (5,27,40,41).Here we investigated two endogenous antioxidants -GSH and vitamin E -and found that E2 treatment increased their concentrations.Molecules rich in -SH groups, such as GSH, have a key role in binding Cd and reducing its toxicity.Dlugosz et al. (38) have suggested that E2 can increase the levels of -SH groups.

CONCLUSION
The results of this study show that exogenous E2 effectively coped with the oxidative overload in rat neutrophils, erythrocyte redox imbalance, and adverse haematological and biochemical effects of acute Cd poisoning.E2 improved erythrocyte antioxidant capacity by acting synergistically with endogenous antioxidants.
We hope that our results will shed more light on direct, nongenomic effects of E2 in vivo and provide important information about the protective mechanism of E2 in Cd-induced toxicity.They may also have broader implications for the therapeutic use of E2, especially in postmenopausal women.
5 mL of lysate) by measuring the reduction of ferric into ferrous ions in the presence of tocopherol and p r o d u c t i o n o f c o l o u r e d c o m p l e x w i t h bathophenanthroline (24).The absorbance of the p r o d u c e d c o m p l e x w a s m e a s u r e d spectrophotometrically (JENWAY 6105 UV-Vis spectrophotometer) at 535 nm.Vitamin E concentrations are expressed in mg L -1 of erythrocytes.