The effects of air pollution and smoking on placental cadmium, zinc concentration and metallothionein expression

Toxicology 238 (2007) 15–22

The effects of air pollution and smoking on placental cadmium, zinc concentration and metallothionein expression Hulya Cetin Sorkun a,∗ , Ferda Bir b , Metin Akbulut b , Umit Divrikli c , Gulten Erken d , Huriye Demirhan a , Ender Duzcan b , Latif Elci c , Ismail Celik e , Unsal Yozgatlı f a

Health Services, Vocational School of Pamukkale University, Denizli, Turkey Department of Pathology, Pamukkale University, School of Medicine, Denizli, Turkey Department of Chemistry, Pamukkale University, Faculty of Arts and Science, 20020 Denizli, Turkey d Department of Physiology, Pamukkale University, School of Medicine, Denizli, Turkey e Department of Biochemistry, State Hospital, Denizli, Turkey f Department of Gynecology and Obstetrics, Denizli State Hospital, Turkey b

c

Received 6 April 2007; received in revised form 4 May 2007; accepted 14 May 2007 Available online 26 May 2007

Abstract This study is designed to determine the placental zinc (Zn) and cadmium (Cd) levels in mothers who were smokers, mothers who were thought to be exposed to air pollution, and mothers who were non-smokers and to investigate the relationship between the expression of placental metallothionein (MT) binding these metals and blood progesterone level. Placental Zn and Cd levels were measured by atomic absorption spectrometry. Presence of placental MT was determined immunohistochemically. Placental changes were examined by light microscope after H&E and PAS staining. Immunohistochemical MT staining of syncytiotrophoblastic and villous interstitial cells were scored as positive or negative. Among the 92 mothers included in the study, 33 were smokers (Group I), 29 had been exposed to air pollution (Group II) and 30 were non-smoker rural residents who had never been exposed to air pollution (Group III). Mean off-spring birth weight of 3198.62 ± 380.01 g and mean placenta weight of 561.38 ± 111.55 g of Group II were lower when compared with those of other two groups. In Group I, mean placental Cd and Zn were 0.063 ± 0.022 ␮g/g and 39.84 ± 15.5 ␮g/g, respectively, being higher than in other groups. In Group II, mean placental Cd and Zn levels were higher than those of Group III. Blood progesterone levels of subjects in Group I (121 ng/ml) were the lowest of all groups. While the mean count of villi was the highest in Group III; the highest mean count of syncytial knots was in Group II. Thickening of vasculo-syncytial membrane was most prominent in Group I. Similarly, MT staining was positive and very dense in 72.7% (24/33) of cases in Group I (p ≤ 0.05). MT staining was positive in 69.0% (29/20) and denser in Group II cases compared to 36% (11/30) in Group III (p ≤ 0.05). This study showed that smoking increased Cd levels in placenta and accompanied an increase in placental MT expression immunohistochemically. The effects of exposure to air pollution are equally harmful as smoking related effects. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Placenta; Cadmium; Zinc; Metallothionein; Smoking; Air pollution

1. Introduction ∗

Corresponding author. Tel.: +90 2582637206; fax: +90 2582637206. E-mail address: [email protected] (H.C. Sorkun).

Placenta provides a connection between maternal and fetal circulations, and all the necessary nutrients for fetus. It permits the passage of trace elements and

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2007.05.020

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minerals such as calcium, copper, zinc and iron that are necessary for growth and development. On the other hand, prevention of transport of toxic materials such as cadmium can be achieved by the binding of these metals by mediator molecules, like MT (Galicia-Garcia et al., 1997; Goyer and Cherian, 1992; Lafond et al., 1991; WHO, 1996). Studies conducted in various parts of the world have regarded human placenta as an indicator organ when exposed to metals (Bush et al., 2000; Kantola et al., 2000; Lagerkvist et al., 1996; Osman et al., 2000; Reichrtova et al., 1998; Tabacova et al., 1994). Smoking is the major source of cadmium. Each cigarette contains 1–2 ␮g Cd. Daily Cd intake of smokers can be twice as much as non-smokers (ATSDR, 1999). Depending on the number of cigarettes smoked a day, birth weight of the babies and the weight of the placenta are lower in smokers than in non-smokers (Abel, 1980; Demir et al., 1994). Exposure of humans to Cd in industrial work environments is mainly due to inhalation of this metal at small doses (50 ␮g/m3 ) (Piscator, 1976). Exposure to Cd results in fetotoxic and embryotoxic effects (Eisenmann and Miller, 1994; Hazelhoff Roelfzema et al., 1987; Levin et al., 1987). Cadmium accumulates in the placenta of mothers exposed to Cd during pregnancy (Goyer and Cherian, 1992; Kuhnert et al., 1982; Wier et al., 1990). Binding of Cd is related to MT synthesis and partially protects the fetus from the detrimental effects of the metal (Boadi et al., 1991; Goyer, 1991). At the cellular level, one of the most important bivalent metal (Zn, Cd and Cu) binding proteins is metallothionein. This lowmolecular weight (6000–7000 Da) cysteine-rich protein is important for the regulation of Zn homeostasis and metabolism (Richards, 1989; K¨agi, 1991). It has been proposed that MT maintains Zn homeostasis at the cellular level by increasing Zn and regulating the distribution, excretion and short-term storage of Zn (Richards, 1989; Klaassen et al., 1999). Presence of Cd and Zn are potent factors for the MT synthesis (Harford and Sarkar, 1991; K¨agi and Schaffer, 1988). MT binds to Cd with greater affinity than Zn. Therefore Zn is easily released from MT and transported while Cd strongly binds MT (Klaassen et al., 1994; Lau et al., 1998; Min et al., 1992). Presence of MT has been shown in human placenta and fetal membranes (Itoh et al., 1996; Kantola et al., 2000; Klaassen et al., 1999; Milnerowicz, 1993). Zn is a trace element necessary for the growth of the fetus (Wellinghausen, 2001). Insufficient transport of Zn from the placenta to the fetus may be held responsible for low birth weight in babies of smoker women. Increase in placental Cd concentration as well as increase in Zn has been reported in

smokers (Kuhnert et al., 1987a, 1987b, 1988; Osman et al., 2000; Ronco et al., 2005; Sorell and Graziano, 1990). Placenta plays an important role in the production of sex steroid hormones necessary to maintain pregnancy (Solomon, 1988; Strauss et al., 1996). It is the primary source of progesterone during pregnancy (Baron et al., 1990; Bernstein et al., 1989). Bernstein et al. (1989) argued that smoking may cause spontaneous abortions by decreasing progesterone concentration. With the present study, our aim was to explore the effects of smoking and air pollution on the quantity of placental Cd and Zn. We also investigated the relationship between immunohistochemical MT protein expression in the placentas of these pregnant women and placental Cd and Zn levels, as well as their effects on blood progesterone levels. 2. Materials and methods Placentas of 92 women who were in their 2nd and 3rd trimesters in winter months were obtained from normal term, uncomplicated pregnancies, excluding any intercurrent disease or other complication after spontaneous vaginal delivery or caesarean section. Placentas were collected immediately after deliveries at the Department of Gynecology and Obstetrics of Denizli State Hospital. Denizli is located in the inner part of Aegean region of T¨urkiye, a major industrial centre producing textiles, manufacturing machinery, electrical components, food, and chemicals and has air pollution. Mean values in winter months including December, January, February were 216 ± 121.0 ␮g/m3 for SO2 , and 246 ± 98.2 ␮g/m3 for PM10 (Particular materials). There were no major outdoor or indoor metal exposures at home or work, and dietary habits of the parturients included predominantly mixed diet types with no alcohol consumption. Recruited subjects were divided into three groups: Group I, smokers (n = 33), consisted of women who smoked throughout their pregnancy and stopped smoking less than 12 months before the onset of their last pregnancy; Group II, urban residents of Denizli (n = 29), were non-smokers who were thought to have been exposed to air pollution; and Group III, nonsmokers (n = 30), never smoked and lived in a rural area. Blood for progesterone concentration (Abbott Architect i 2000 SR - Microparticle Immunoassay) and hemoglobin (Hgb) and hematocrite (Hct) levels were obtained at the time of delivery. After deliveries, all placentas were weighed and placed in plastic bags. Each bag was marked with the subject’s identification code. The fetal weight and gender were obtained from the clinical case-sheets. 2.1. Metal analyses For metal analyses, one sample was taken from each placenta and was kept frozen (−80 ◦ C) until analyzed. Placenta samples were prepared using the technique described

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by Brown et al. (1986) and Manca et al. (1992). Briefly, placenta samples were weighed and put into tubes and then they were added 2 ml of nitric acid (Merck). The samples were left at 100 ◦ C until their volume diminished as much as half of their total volume. Following the addition of 2 ml perchloric acid (Merck) the samples were left at the same condition as mentioned above. Deionised distilled water was added for dilution to reach 5 ml of the final volume. For the analyses of elements in blood and tissues standard solutions were prepared by using Cd and Zn standards (Merck). Deionised distilled water was used as blank. Blank and standard solutions were used for calibration of atomic absorption spectrophotometer (Brown et al., 1986; Manca et al., 1992). Calculations of concentrations were based on tissue weight. The concentrations of Zn were measured with flame atomic absorption spectrophotometer (Perkin Elmer AAS-700 Ueberlingen, Germany). Tissue concentrations of Cd were determined by atomic absorption spectrophotometer with an HGA graphite furnace (Perkin Elmer AAS-700 Ueberlingen, Germany). 2.2. Histopathological examination In separate marked bags, placentas went through macroscopic examination. They were sliced and preserved in 10% formalin for 24 h. The samples were taken from between the central region and the periphery of the maternal surface and fetal surface. Sections obtained were embedded in paraffin following routine histopathologic examination. After embedding in paraffin, sections were cut at 4–4.5 ␮m and stained with haematoxylin & eosin, periodic acid Schiff (PAS) and then examined using Nikon Eclipse E200 light microscope. The whole of randomized sections in 10 different areas were examined at 400× magnification and villus & knots were counted. Vasculo-syncytial membrane thickness was assessed based on presence of trophoblast and endothelial basal membrane thickness areas in PAS stained preparations. 2.3. Immunohistochemistry Sections expressing the most representative blocks were chosen for further immunohistochemical analysis. Fourmicron sections of formalin-fixed, paraffin-embedded tissue sections were prepared. Briefly, all sections were then deparaffinized in xylene, rehydrated through a graded series of alcohol and washed in distilled water. Distilled water was used for all subsequent washes and dilution of the antibody. Tissue sections were heated twice in a microwave oven for 10 min each at 700 W in citrate buffer pH 6 and then processed with the Standard streptavidin–biotin–immunoperoxidase method automatically with Ventana-Nexes immunostainer (Ventana Medical Systems, Tucson, AZ). Monoclonal anti-MT (Novacastra Laboratories, Ltd., Newcastle-upon-Tyne, United Kingdom, Fig. 1) at a 1/50 dilution were used. Diaminobenzidine (Ventana Medical Systems, Tucson, AZ) was used as the final chromogen, and Mayer’s haematoxylin as the nuclear counterstain.

Fig. 1. Immunohistochemical expression of MT in (A) Group I (200×, MT, original magnification), (B) Group II (400×, MT, original magnification), and (C) Group III (200×, MT, original magnification).

Immunohistochemical staining for MT was scored as (+) or (−) by determination of cytoplasmic staining of syncytial trophoblastic cells and villous interstitial cells. 2.4. Statistics Data were analyzed using SPSS 10.0 software. Means, standard deviations and percentage values were computed. Specific baby and mother characteristics were assessed by nonpara-

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Table 1 Data related to mother, mother bloods progesterone, Hgb, Hct levels and newborn birth weights

Number Age Parity Pregnancy duration/day Number of abortions Birth weight Placental weight Mother blood progesterone (ng/ml) Median Mother blood Hgb Mother blood Hct

Smokers (Group I)

Residents of Denizli (Group II)

Rural residents (Group III)

Groups I and III, p value

Groups II and III, p value

n = 33 24.88 ± 4.48 1.88 ± 1.02 277.15 ± 10.95 6/33 3276.36 ± 469.01 575.15 ± 74.01 121

n = 29 24.69 ± 2.97 1.48 ± 0.69 277.14 ± 9.77 4/29 3198.62 ± 380.01 561.38 ± 111.55 129

n = 30 25.53 ± 4.39 1.97 ± 1.03 276.63 ± 10.51 2/30 3410.00 ± 562.42 609.33 ± 111.26 168

0.472 0.432 0.659 0.190 0.315 0.208

0.542 0.359 0.982 0.368 0.150 0.052

11.44 ± 1.38 35.80 ± 3.61

12.30 ± 0.87 38.06 ± 2.99

11.80 ± 1.41 37.55 ± 4.69

0.978 0.316

0.243 0.697

Data are expressed as mean ± S.D., Student’s t-test, chi-square tests and significance at p < 0.05.

metric chi-square test. Parametric tests used were one-way ANOVA, Mann–Whitney U, Kruskall–Wallis, and Independent samples t-tests. Statistical significance was considered at p < 0.05.

3. Results The average age of 92 subjects included in the study was 25.03 ± 4.00 (max = 36, min = 17). Those subjects who stopped smoking 12 months prior to pregnancy were not included in the Group I (n = 33) which consisted of smokers. Of those who were smoking before pregnancy, 11 smoked 5 or fewer cigarettes a day, 9 smoked 6–9 cigarettes, 7 smoked 10–14, 4 smoked 15–19, and 2 smoked more than 20 cigarettes. Twenty people in the group had continued smoking 1–14 cigarettes a day throughout their pregnancies. Thirteen subjects who had stopped smoking had done so 6 months prior to or 2 months after their pregnancies. Twenty-four of smokers lived in the urban city and nine lived in rural areas. Distribution of maternal data including demographic features, blood progesterone levels and hematological

data as well as placental and birth weights according to groups is presented in Table 1. Forty-six of the babies were girls and 46 were boys. Mean birth weight was 3291.09 ± 441.35 g for boy babies and 3299.78 ± 519.96 g for girl babies. There were no twin births among our subjects. Taking into account the age of mother, average placental and baby weights, duration of pregnancies, number of births and number of abortions no statistically significant difference was observed between Group I and Group III, and between Group II and Group III (p > 0.05). However, although it was statistically insignificant, the average birth and placental weights of Group II were less than those of Group I (Table 1). Mean placental Cd and Zn levels were highest in Group I (Cd = 0.063 ± 0.022, Zn = 39.84 ± 15.5), and lowest in Group III (Cd = 0.038 ± 0.012, Zn = 32.70 ± 16.3). A statistically significant positive relationship was found between placental Cd amounts of Group I and Group III (p ≤ 0.05). Assessment of placental Cd amounts between Group II and Group III has also yielded a statistically significant positive relationship although not as prominent as that

Table 2 The relationship of the placental Cd and Zn amounts with the presence of MT Placental Cd (␮g/g tissue)

Placental Zn (␮g/g tissue)

Smokers (Group I) Residents of Denizli (Group II) Rural residents (Group III)

0.063 ± 0.022 0.048 ± 0.014 0.038 ± 0.012

39.84 ± 15.5 38.15 ± 15.3 32.70 ± 16.3

p value of Groups I and III p value of Groups II and III

0.002 0.050

0.206 0.239

Data are expressed as mean ± S.D., Student’s t-test, chi-square tests, Mann–Whitney U and significance at p < 0.05.

MT %72.7 (24/33) %69.0 (29/20) %36 (11/30) 0.004 0.013

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Table 3 Characteristics of groups related to placental villus, syncytial knots and vasculo-syncytial membrane

Smokers (Group I) Residents of Denizli (Group II) Rural residents (Group III) p value of Groups I and III p value of Groups II and III

Villus

Syncytial knots

Vasculo-syncytial membrane

9.521 ± 0.928 10.266 ± 1.453 11.940 ± 1.496

9.318 ± 3.544 10.897 ± 3.335 7.720 ± 2.219

30/33 23/29 4/30

0.000 0.000

0.094 0.000

0.000 0.000

Data are expressed as mean ± S.D., Student’s t-test, chi-square tests and significance at p < 0.05.

of Group I (p ≤ 0.05). Mean placental Zn amounts, on the other hand, did not show any statistically significant relationship between groups (p > 0.05) (Table 2). Immunohistochemical staining of MT on sections has shown a staining rate of 72.7% (24/33) in placentas of Group I (Fig. 1A) and 69.0% (20/29) in Group II (Fig. 1B). The staining rate in the placentas of Group III subjects who comprised controls was 36.0% (11/30) (Fig. 1C). A statistically significant positive relationship was found when MT staining of Group I and Group III was compared (p ≤ 0.05). A statistically significant positive correlation was also established between MT staining of Group II and Group III (p ≤ 0.05) (Table 2). Analysis of placenta sections under a light microscope has shown that the mean villus number was highest in Group III. The lowest villus number was found in Group I. When villus values of Group I and Group II were compared with those of Group III, a statistically prominent significance was established in the former two groups (p ≤ 0.05) (Table 3). The lowest mean syncytial knot number belonged to Group III. The highest mean syncytial knot number was observed in the placentas of Group II subjects. When the mean syncytial knot numbers were compared between these two groups, a high statistical significance was observed (p ≤ 0.05). On the other hand, no statistically significant relationship was found between the mean syncytial knot numbers of Group I and Group III (p ≤ 0.05) (Table 3). Vasculo-syncytial membrane thickening was most observed in Group I (30/33). Vasculo-syncytial membrane thickening was seen in 23 of Group II and only 4 of Group III members (p ≤ 0.05) (Table 3). 4. Discussion This study demonstrates that Cd accumulates in the placentas of women who smoke or are exposed to air pollution. Placental Cd accumulation was greatest among smokers, followed by those exposed to air pollution.

Many studies carried out on the placentas of women smokers showed that Cd concentration was higher than in non-smokers (Kantola et al., 2000; Kuhnert et al., 1987a; Lagerkvist et al., 1992; Loiacono et al., 1992). Kantola et al. (2000) similar to the present study, showed that placental Cd was higher in residents of industrial big cities compared to those living in other cities. In the current study, we found that placental Zn increased as Cd increased in all groups. In the smokers group the placental Cd was highest, so was the Zn concentration. Placental Zn in subjects exposed to air pollution was higher than in the control group (women living in rural areas) but lower than of the smokers. Various studies have demonstrated that when the placental Cd increases, Zn level increases as well (Kuhnert et al., 1987a, 1987b; Ronco et al., 2005; Sorell and Graziano, 1990). On the other hand, Piasek et al. (2001) reported that placental Zn concentration was not different in smokers. A decrease in the placental transfer of zinc, iron and copper to the fetus has been reported by Sorell and Graziano (1990) even when exposed to low levels of Cd. Low mean birth weight and placenta weight observed in subjects exposed to air pollution may be due to these factors. This result shows that subjects exposed to air pollution are exposed to other toxic chemicals, in addition to Cd and these toxic chemicals also affect Zn level. It has been shown that binding of Cd by the placenta is generally related to MT biosynthesis (Boadi et al., 1991; Goyer, 1991). Placental MT increases Zn transport and limits Cd (Goyer et al., 1992). A study on MT deficient mice revealed that Cd accumulation in fetuses was higher than in normal mice, and that there was an increase in immunohistochemical expression of placental MT (Lau et al., 1998), and this played a role in reducing the cadmium transfer from the mother to the fetus (Osman et al., 2000). A human study employing Western blots technique (Ronco et al., 2005) showed almost two-fold increase in MT levels secondary to increased Cd in the placentas

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of women who smoked during pregnancy compared to non-smokers. In this study, in order to understand the presence of MT, we observed areas of dense immunohistochemical staining in the placentas of subjects who smoked or exposed to air pollution. In that regard, our finding of increased MT in groups that showed increased Cd concentration was in agreement with the findings of parallel studies (Chan et al., 1993; Goyer and Cherian, 1992; Kuhnert et al., 1987a; Lau et al., 1998; Ronco et al., 2005). Many studies have argued that the concentrations of toxic materials had negative effects on birth weight, birth height and head circumference (Ward et al., 1990; Osman et al., 2000). Though statistically not significant, mean birth weight of babies born to mothers exposed to air pollution being lower than that of babies born to mothers living in rural areas, as shown in our study, is an indicator of this. Many studies reported that Cd of tobacco origin in the placenta has a negative effect on birth weight and placenta weight (Demir et al., 1994; Kuhnert et al., 1987a; Loiacono et al., 1992; Ronco et al., 2005; Roquer et al., 1995). Our finding that mean birth weight of babies and placental weight of women who smoked were lower than those women who lived in rural areas lends support to the findings of these researchers. According to Vander Veen and Fox (1982), necrosis and increased syncytial knots are not important but they are signs of hypoxia or degenerative changes due to ischemic conditions. We found that mean number of syncytial knots was highest in the placentas of women exposed to air pollution (10.920 ± 3.280), followed by those of smokers group (9.247 ± 3.577). Mean number of villi was found lowest in the placentas of smokers (9.525 ± 0.942) and highest in the placentas of women living in rural areas (11.940 ± 1.496). Results of these quantitative comparisons are in agreement with the findings that the number of syncytial knots was higher while the number of villi was lower in smokers (Demir et al., 1994) and that the number of syncytial knots in passive smokers was higher than the control group (Rath et al., 2001). Increase in the number of syncytial knots depending on the number cigarettes smoked a day has been shown by Spira et al. (1977). When we compared the number of syncytial knots, the number was higher in mothers exposed to air pollution and this suggests that air pollution is more effective on syncytial knot formation than smoking. This also suggests that there may be other factors influencing syncytial knot formation besides Cd. Thickening of the villous membrane was most pronounced in the placentas of smokers (30/33), followed

by those of exposed to air pollution (23/29). These findings are not only consistent with the study of Bush et al. (2000) in which they reported thickening of the villous membranes of placentas of smoking mothers, but also show that effects of exposure to air pollution can be harmful. Indeed, studies that reported the negative effects of toxic materials as a result of air pollution (Osman et al., 2000; Ward et al., 1990) lend support to our findings. These results suggest that there is a need for future studies to compare the effects of smoking and exposure to air pollution. A study on pregnant women who smoked showed that Cd decreased the levels of progesterone that is necessary for the progress of pregnancy (Piasek et al., 2001). In vitro studies in the literature (Jolibois et al., 1999a, 1999b) have demonstrated that decrease in low-density lipoprotein (LDL) receptor mRNA in trophoblastic cells was important for the potential role of Cd in decreasing progesterone levels. We found, in our study, that the lowest mean blood progesterone level was in the smokers group. However, blood progesterone level of the subjects exposed to air pollution was also low, nearly as low as the smokers. Progesterone level of women who lived in rural areas and served as a control group was higher than the other two groups. Our findings are in line with those of Piasek et al. (2001) who reported low placental progesterone among smokers. We did not find any reports on the relation between air pollution and placental progesterone level. When we compared mean Hgb and Hct, we found that they were lower in the smokers group. Subjects exposed to air pollution had higher Hgb and Hct than those living in rural areas. This suggests that women exposed to air pollution lived in city centers and had access to better healthcare. Maternal and fetal (umbilical) Hct levels were found significantly higher in smokers compared to non-smokers (Bush et al., 2000). We did not find such a relation between smokers and the control group. Hgb and Hct levels of subjects exposed to air pollution were higher than the control group living in rural areas but the difference was not statistically significant. To the best of our knowledge, this study is the first to compare immunohistochemical MT protein expression and blood progesterone levels in the placentas of women who smoked and of those who were exposed to air pollution. We have shown that smoking increased placental Cd. In line with this increase, there was an increase in placental MT, as demonstrated immunohistochemically. The present study has also established that the consequences of exposure to air pollution were as harmful as the effects of smoking.

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