EFFECTS OF NICKEL CHLORIDE ON HEMATOLOGICAL

AND DEVELOPMENTAL PARAMETERS

IN Wistar albino PREGNANT RATS

Adjroud, O.* and Mouffok, S.

Laboratory of Animal Physiology, Department of Biology Sciences, Batna University, Algeria

*Correspondence to Adjroud Ounassa

E- mail : o.adjroud@caramail.com

INTRODUCTION:

Nickel, a major environmental pollutant, was potentially produce carcinogenic, genotoxic, immunotoxic and teratogenic (Bencko et al.,1986 , Grimsrund et al., 2003; Danadevi et al., 2004;and Montanaro et al.,2005). It was extensively used in electroplating, manufacture of batteries and steel to release into the atmosphere during mining, smelting and refining operations (Caplat, 2001). Therefore, nickel had a disastrous impact on human and animal health. Also, it found to be harmful for hematopoiesis and female reproduction of laboratory rodents (Weischer et al., 1980; Dieter et al., 1988 ; and Käkelä et al., 1999). Selenium has been previously found to counteract the deleterious effects on nickel on the reproduction rats (Käkelä et al., 1999). Adequate selenium status was known to be essential for fertility in man and animal (Hensen and Deguchi, 1996). In this study, we compared the effects of NiCl₂ in drinking water and subcutaneous injection administration on day 4 of gestation, in pre-implantation period, on development and haematological parameters in pregnant Wistar rats using variations in the dose, route of administration and duration of exposure.

MATERIALS AND METHODS:

Animals:      

Adult Female albino Wistar rats (Pasteur Institute, Algiers) were kept in a lighting schedule of 12 h light: 12 h darkness at 231°C with free access for food and water. Animals were used at day 21 of pregnancy. The average length of gestation in breeding colony in our department was 22 days. Females (180-300g) were caged with males overnight and the vaginal smear examined for the presence of spermatozoa. The day on which spermatozoa were found in the smear was designated day 1 of pregnancy and animals were used on days 6 and 21. Pregnant females were housed at five rats per cage. The average of gestation in the breeding colony was 22 days (Adjroud 1995).

Chemicals:

Nickel chloride hexahydrate (NiCl₂, 6H₂O) was purchased from sigma Aldrich Laborchemikalien Gmbh; Selenium (Se) was purchased from we prolab; NaCl was purchased from panacreac Qu mica Sa, diethyl ether Ficher scientific (UK).

Experiments:

Each animal was anaesthetized with diethyl ether s.c., and was weighed before each experiment. The controled groups and treated groups were injected s.c with 0.3 ml/rat of NaCl 0.9%, or drinking distilled water.

Nickel chloride hexahydrate (NiCl₂, 6H₂O) was dissolved in sterile saline (NaCl 0.9%) and was given as a single s.c. at 25, 50 and 100 mg/kg body weight or 20 mg/100 ml in drinking distilled water. Selenium (Se) was dissolved in sterile saline and was given s.c. at 0.3 mg/kg body mass in association with the higher dose (100 mg/kg body mass) of Nickel chloride. Selenium was used to block the effects of the NiCl₂. The exposed rats or control groups were injected on day 4 of pregnancy during pre-implantation period and blood sample was collected on EDTA from jugular vein on day 6 and 21 of pregnancy for haematological study. The determination of haematological param-eters was performed by coulter Erma Inc PCE-21-ON. On day 21 of pregnancy maternal body weight was recorded, uterus were excised to evaluate the number of live foetuses and abortion; the fetuses were removed and weighed.

Statistical analysis:

Data for each group of experiments (n=6) were statistically  analysed by analysis of variance and expressed as mean S.E.M. Significant differe-nces between the treated group mean and its control group were performed by Student's "t" test. Differences were considered to be significant if P<0.05. Data were analysed with Excel for windows, version 5.1, USA.

RESULTS:

1-Effects of NiCl₂ on haematological parameters:

Subcutaneous administration of nickel chloride, 25 mg/kg b w, on day 4 during pre implantation period induced 2 days after treatment an immediate and significant decrease in the erythrocyte counts, hematocrit values and haemoglobin concentrations from day 6 to 21 of pregnancy. While NiCl_2, at50 mg/kg b w, produced a significantly reduction on day 6 of pregnancy for erythrocyte counts and hematocrit values and platelets counts but significantly increased on day 21 of pregnancy to erythrocytic counts, hematocrit values and haemoglobin concentrations. On the contrary NiCl_2, at 50 mg/kg b w, produced a significantly elevation on the platelets counts on day 21 of pregnancy. However, the highest dose of NiCl₂, 100 mg/kg b w, did not significantly affect these haematological parameters during observation period compared to control. Adding Selenium by 0.3 mg/kg b w associated with higher dose of NiCl₂, did not alter the effect of 100 mg/kg b w administered alone on the erythrocytic counts, hematocrit values, haemoglobin concentrations, and platelets counts on day 6 and 21 of pregnancy, compared to control values.

On the other hand, rats having received NiCl_2, 20 mg/100 ml in drinking water showed an immediate and significant decrease in the erythrocyte counts and hematocrit values from day 6 to day 21 of pregnancy. This dose of nickel Chloride added to drinking water had no significantly effect on haemoglobin concentrations from day 6 to day 21 of pregnancy, but slightly increased blood platelet counts on day 6 of pregnancy.

2-Effects of NiCl₂ on maternal body weight:

NiCl₂, 25, 50, and 100 mg/kg b w administered s.c., reduced progressively and significantly the maternal body weight on day 6, this fall was spectacular on day 21 of pregnancy compared to control values. Selenium (0.3 mg/kg b.w.) combined with 100 mg/kg, b.w of nickel chloride produced a slight but no significant increase in maternal body weight compared to 100 mg/kg b w of NiCl₂ alone. Rats exposed to20 mg/100 ml in drinking water showed a notable decrease in maternal body weight particularly on day 6 of pregnancy compared to control and to subcutaneous route.

3-Effects of NiCl₂ on developmental parameters:

When rats had been treated subcutaneously on day 4 of pregnancy by NiCl₂, 25, 50 and 100 mg/kg, b w the fetal body weight decreased slightly on day 21 of pregnancy. Addition of selenium to NiCl_{2 -} exposed rats did not improve, the fetal body weight on day 21 of pregnancy. Similarly, rats drinking water supplemented with 20 mg/100 ml of NiCl₂, were showed a slight but not significant reduction of fetal body weight on day 21 of pregnancy.

A-Effects of NiCl₂ on live fetuses number:

Our results showed that NiCl₂, induced on day 21 of pregnancy a progressive diminution of the number of live fetuses with 25 and 50 mg /kg, b w, s.c.; this diminution was reached to maximum with 100 mg/kg, in comparison with control. Selenium did not prevent the decrease in the number of live fetuses induced by the highest dose (100 mg/kg) of NiCl₂. However, 20 mg/100 ml of NiCl₂ addedto the drinking water had no effect on the number of live fetuses.

B-Effects of NiCl₂ on number of abortions:

25 and 50 mg/kg, b.w., of NiCl₂ slightly elevated the number of abortions; while with 100 mg/kg b w the elevation was reached to maximum in comparison with control. Selenium combined with 100 mg/kg of NiCl₂ did not block theseabortions. On the contrary, 20 mg/100 ml of NiCl₂ in the drinking water did not affected significantly on the number of aborted foeti in comparison with control.

Table (1): Effects of subcutaneous nickel chloride alone or combined with selenium on haematological parameters and maternal body weight in Wistar albino pregnant rats

Each value haematological parameters or body weight represents the mean±SEM 6 rats per group

**p<0.01, *p<0.05 compared with control value, student's t test.

Table (2): Effects of oral nickel chloride alone or combined with selenium on haematological parameters and maternal body weight in pregnant Wistar albino rats

Each value haematological parameters or body weight represents the mean±SEM 6 rats per group

***p<0.001, **p<0.01, *p<0.05 compared with control value, student's t test.

Table (3): Effects of subcutaneous nickel chloride alone or combined with selenium on fetal body weight; number of fetuses and number of abortions in pregnant Wistar albino rats (day 21)

Each value represents the mean ± sem 6 rats per group

*p<0.01 compared with control value, student's t test.

Table (4): Effects of oral nickel chloride on fetal body weight; number of fetuses and number of abortions in pregnant Wistar albino rats (day 21)

Each value represents the mean ± sem 6 rats per group

DISCUSSION:

The immediate anaemia observed with the lower dose of NiCl₂ administered subcutan-eously or with 20 mg/100 ml solution given in the drinking water was also observed in rats by Weischer et al., (1980) and Nielsen (1980). The decrease in haematological parameters seemed to be related to impaired iron absorption in rats deprived of nickel in diet (Nielsen, 1980). Furthermore, NiCl₂ also acted on metabolism and in particularly the decrease in the erythrocyte glutathione peroxydase with consequent increase in oxidative stress in human and fish (De Luca et al., 2007). On the other hand, the deficiency of nickel in diet altered the fatty acids composition of total lipids and phospholipids in rats erythrocytes (stangi and Kirehess 1997). On the contrary, the middle dose of NiCl₂ only reduced on day 6 of gestation the erythrocyte counts, hematocrit values and the platelets counts, and had no effect on haemoglobin concentrations. Inversely, on day 21 of pregnancy, the middle dose increased the erythrocyte counts, the hematocrit values, and haemoglobin concentrations. This result was also obtained in rats (Weischer et al 1980) and was consistent with increase in erythropoietin synthesis in vitro in human and fish. On the other hand, the increase in haematological parameters obtained 18 days after treatment might be due to the accumulation of nickel in blood on day 21 of gestation (Dem et al., 2005) and then increased the iron content(Cempel, 2004) which induced an increase in the haematological parameters in our pregnant rats. The highest dose had not altered haematological parameters. In addition, selenium combined to the highest dose failed to modify the effects of the highest dose.

Reports suggested that soluble nickel salts might affect development. Indeed, our result showed that nickel chloride administered s.c., or in drinking water in the early gestation during pre-implantation induced a harmful effect particularly on the number of live fetuses and embryonic resorption with the highest dose s.c. These results agrees with embryotoxicity and fetal toxicity of NiCl₂ observed in rats by Sundermann et al., (1977), Saillenfait et al., (1991) and smith et al., (1993), also, in mice by Chernoff & Kawlock (1982) and Berman &Rehnberg (1983) and in women . The toxic effect of nickel seemed to appear in mice during the passage through the oviduct (Storeng & Jonsen, 1981) and during organogenesis (Mas et al., 1985) with subsequent effect on the development after implantation inducing cytotoxicity and teratogenic effect. Injection of NiCl₂ during early gestation crossed the feto-maternal barriers and entered the foetus during late gestation (Sundermann et al., 1977). In another study by Lu et al., 1981) reported that the concentration of nickel in the maternal blood and the placenta were found maximum level at 2 hours after injection of nickel chloride in pregnant mice. In addition, the yolk sac and placenta accumulated Ni in mice (Mas et al., 1985) inducing in mid pregnancy in cultured rat embryos a significant decrease in yolk sac diameter and high incidence of poor yolk sac circulation (Saillenfait et al., 1991) but did not prevent the transportation of metal to embryo or foetus. NiCl₂, in drinking water or in subcutaneous administration had no significant effect on fetal body weight looses. This result disagrees with Storeng and Jonson, (1981); Mas et al., (1985) and Georges et al.; (1989), or in rats (Saillenfait et al., (1991). Which reported a decrease in fetal body weight obtained .

Maternal body weight was progressively and significantly decreased in dose-related manner after treatment with NiCl₂, subcutaneous or in the drinking water. This result agrees with those obtained in rats by weischer et al., (1980), Georges et al., (1985), (Seidenberg et al., 1986) and smith et al., (1993) or in mice treated by nickel sulfate in drinking water (Dieter et al., (1988). This decrease in maternal body weight was probably related to the decreased metabolic organs weights such as kidney and liver (Lu et al., 1981, Novelli et al., 1998, and Pari & Prasath, 2008). In conclusion, our results demonstrated nickel chloride in drinking water or in the lower dose administ-ered subcutaneous induced immediately anaemia. The developmental parameters were only altered by the highest dose of nickel administered subcutaneous. Selenium did not antagonise the effects of nickel.

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