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Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on neonatal asthma susceptibility
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Abstracts / Toxicology Letters 221S (2013) S59–S256
P08-24 Effects of different endocrine disruptor (EDC) mixtures on gene expression in neonatal rat brain regions: Focus on developing excitatory synapses Walter Lichtensteiger 1,∗ , Catherine Bassetti-Gaille 1 , Oliver Faass 1 , Julie Boberg 2 , Sofie Christiansen 2 , Ulla Hass 2 , Andreas Kortenkamp 3 , Margret Schlumpf 1 1
GREEN Tox and Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 National Food Institute, Technical University of Denmark, Søborg, Denmark, 3 Institute for the Environment, Brunel University, Uxbridge, UK Sexual brain differentiation is a potential EDC target. It depends on a combination of estrogen receptor- and androgen receptormediated effects in males and on estrogens in females. It is not known how these processes are affected by real-world mixtures of EDCs. We investigated the effect of three EDC mixtures on gene expression in developing brain. Amix (8 antiandrogenic chemicals), Emix (4 estrogenic chemicals) and Tmix (Amix + Emix + paracetamol recently identified as anti-androgenic) were administered by oral gavage to rat dams from gestational day 7 until weaning, at doses corresponding to 450×, 200× and 100× high end human intakes (S. Christiansen et al., 2012. International Journal of Andrology 35, 303). At postnatal day 6, during the last part of sexual brain differentiation, exon microarray analyses were performed in medial preoptic area (MPO) in the highest dose group, and real time RT PCR of selected mRNA species in MPO and ventromedial hypothalamus (VMH) of all dose groups. Microarray analyses revealed mixture- and sex-specific effects on gene expression patterns. The majority of genes affected by an individual mixture was selective for that mixture. Real time RT PCR of individual mRNAs demonstrated treatment- and sex-dependent differences between MPO and VMH. Effects were dose-dependent. Prominent are effects on the expression of genes involved in excitatory glutamatergic synapse formation and function. These data indicate that effects of complex EDC mixtures on developing brain can be characterized by mixture-, sex- and region-specific gene expression patterns. Excitatory synapse development emerged as a potentially relevant target. http://dx.doi.org/10.1016/j.toxlet.2013.05.163
P08-25 Effects of endocrine disruptors bisphenol A and di(2-ethylhexyl) phthalate in a combination with 17-estradiol on apoptosis-related genes in the MCF-7 breast cancer cell line Alzbeta Mlynarcikova ∗ , Tomas Havranek, Maria Fickova Institute of Experimental Endocrinology SAS, Bratislava, Slowakia The role of estrogen signaling in mammary carcinogenesis is well established. Compounds named endocrine disruptors (EDs) can exert estrogen-like activities affecting hormone-controlled homeostasis. Bisphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP) are employed in the manufacture of plastics for consumer products (food packaging, etc.). The correlation between increased EDs exposure and cancer was shown, however, studies with breast cancer cells have yielded inconsistent data. BPA and DEHP can mimic the effects of 17-estradiol (E2) through estrogen receptors or may exert E2-independent actions. Besides mitogenic effects of E2, its antiapoptotic action may contribute to its role in the can-
cer promotion. Since the breast tissue is simultaneously exposed to endogenous and exogenous agents, in the present study, the human breast cancer cells MCF-7 were exposed to E2, BPA/DEHP, or their combinations with E2 (all at concentrations 10−12 , 10−9 , 10−6 M) for 24–120 h. The effects of combinations of E2 with BPA or DEHP on several apoptosis-related genes (Bcl-2, Bax, p53) were analyzed. The results indicate that BPA alone (10−9 , 10−6 M) or in combination with E2 could reduce the expression of proapoptotic p53 and Bax genes. DEHP (10−6 M) was also able to down-regulate Bax gene expression. The presence of E2 increased antiapoptotic Bcl-2 transcripts. These data could partially explain our previous results where the BPA + E2 combinations stimulated de novo DNA synthesis and MCF-7 cell proliferation. Thus, the final proliferative effects induced by EDs in combinations with E2 in MCF-7 cells could partially result from the antiapoptotic action. http://dx.doi.org/10.1016/j.toxlet.2013.05.164 P08-26 Effects of formaldehyde exposure on human epithelial cells all along the respiratory tract Gaëlle Bardet ∗ , Charles Persoz, Thomas Loret, Isabelle Momas, Sophie Achard, Nathalie Seta Université Paris Descartes, Faculté de Pharmacie, EA4064 Impact sanitaire des pollutions, 75006 Paris, France The respiratory tract is directly exposed to inhaled environmental pollutants such as chemicals. Formaldehyde (FA) is a ubiquitous indoor air pollutant known as irritant. Some epidemiological studies revealed an association between FA exposure at low levels and respiratory diseases. Our aim was to assess the cellular inflammatory response to FA exposure at environmental levels of epithelial cells from different parts of the respiratory tract. For this purpose, alveolar A549, bronchial BEAS-2B cell lines and human nasal primary cell cultures (hAECN; Epithelix® ), were cultured on insert and exposed in air/liquid interface (Vitrocell System® ), with air or FA (50 g/m3 ) during 30 min for A549 and BEAS-2B and 60 min for hAECN. 24 h after exposure, apical medium in which IL-8 and MCP-1 levels were measured (ELISA assay), was collected, and cellular viability was assessed by XTT assay. Whatever the conditions and cell type, cell viability was unchanged. No significant variation in inflammatory response was noted after FA exposure compared to air exposure for the A549, BEAS-2B cells while we observed an increase in IL8 for hAECN without any change in MCP-1. Our results suggest that hAECN is more appropriate as a cellular model to mimic the effect of inhaled pollution, since it is a human primary cell culture, and not an immortalized cell line. Mainly nasal epithelium is first in line in contact with inhaled pollutants and the first defensive barrier, which makes this epithelium the most relevant when studying the response to these chemicals. http://dx.doi.org/10.1016/j.toxlet.2013.05.165 P08-27 Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on neonatal asthma susceptibility Kyoung-youl Lee ∗ , Eun-young Choi Kongju National University, Chungnam, Korea Di(2-ethylhexyl) phthalate (DEHP) is widely distributed in the environment and used as a plasticizer. In this study, we investigate
Abstracts / Toxicology Letters 221S (2013) S59–S256
the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using ovalbumin (OVA) induced asthma murine model. Pregnant Balb/c mice received DEHP from Gestation Day 13 to Lactation Day 21. Their offspring was sensitized on Postnatal Day (PND) 9 and 15 by intraperitoneal injection of 0.5 g OVA with 200 g aluminum hydroxide. On PND 22, 23 and 24, live pups were received an airway challenge with OVA for 30 min. Offspring from pregnant mice received DEHP showed the reduction in inflammatory cell count, interleukin (IL)-4, IL-13 and eotaxin in BALF, and total immunoglobulin (Ig) E and OVAspecific IgE in plasma compared with those of offspring from non DEHP-treated pregnant mice. These results were consistent with histological analysis and immunoblot. Maternal exposure to DEHP reduces airway inflammation and mucus production with decrease in inducible nitric oxide synthase (iNOS) in lung tissue of their offspring. This study suggests that maternal exposure to DEHP during pregnancy reduces the risk of asthma in their offspring. These effects were considered to be closely related with suppression of Th2 immune responses and iNOS expression. http://dx.doi.org/10.1016/j.toxlet.2013.05.166
P08-28 Ellagic acid protects against cyclophosphamide induced acute lung injury in Wistar rats by inhibiting oxidative stress, NF-kB activation and inflammatory cell production Sheikh Raisuddin, Falak Beigh Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard University), New Delhi, India Use of cyclophosphamide (CP) as chemotherapeutic drug is associated with side effects including toxicity to respiratory system. Upon administration it enhances lung toxicity which may subsequently progress to the lung fibrosis. Natural compounds have shown mitigating effects against toxicity of anticancer drugs. Ellagic acid (EA), a polyphenolic compound present in many fruits and nuts has shown promising protective effect against toxicity of drugs and chemicals. We studied the ameliorative effect of EA on lung toxicity in rats exposed to CP (50 mg/kg b.w., i.p.) and both were administered for seven days. There was a significant decrease in reduced glutathione level and activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase and co-treatment of EA prevented oxidative stress by restoring the level of antioxidant enzymes. EA also modulated enhanced hydroxyproline level, lipid peroxidation, myeloperoxidase activity, nitric oxide production and protein carbonyl formation in BLM exposed rat lungs. Bronchoalveolar lavage fluid showed increased level of cytotoxic markers and inflammatory cell production in terms of total cell count in drug treated groups and EA attenuated these changes. Further damage was observed by monitoring histopathological as well as immunohistochemical analysis of expression level of molecular markers of inflammation in rat lung tissue. In conclusion, BLM exposure to rat lung tissue showed a toxic effect, especially disruption of lung antioxidant profile and EA emerged as a natural protectant with an ability to protect lungs from onslaught of pulmonary toxicity of anticancer drugs. http://dx.doi.org/10.1016/j.toxlet.2013.05.167
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P08-29 Endocrine disruptor bisphenol A toxicity study in testicular mitochondria of Swiss albino mice Sameya Anjum ∗ , Sheikh Raisuddin Dept of Toxicology, Faculty of Science, Jamia Hamdard, New Delhi, India Introduction: BPA a monomer of polycarbonate plastic used to manufacture plastic baby bottles, water bottles, cans, adhesives, coatings, paints, building materials, thermal paper, etc. Aim and objective: To investigate BPA induced oxidative stress and toxicity in the testicular mitochondria of adult male mice. Methodology: Swiss Albino male mice were exposed to standardized dose of BPA (5, 10, 100 mg/kg body weight), orally for 14 day. Effect of BPA on LPO, GSH, SOD, GPx, GR were observed. Ultrastructural changes in testes on exposure of BPA were observed. Apoptosis marker protein expression levels were observed by IHC. Results: BPA caused lipid peroxidation (LPO) and decrease in reduced glutathione (GSH) content of testicular mitochondria. Significant differences P < 0.01 was observed in the LPO and GSH parameters when compared with control values. Besides, it also affected activities of antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione peroxidase dose dependently. Ultra-structural changes observed by transmission electron microscopy showed BPA caused abnormalities like deformed acrosome and nucleus of spermatids and apoptotic cells were observed in the testes of treated animals. Apoptotic marker proteins like cytochrome c and caspase 3 levels were increased in BPA treated groups as compared to control group of animals. Conclusions: Hence we can conclude that BPA has induced oxidative stress in testicular mitochondria of exposed group and the results were further confirmed by the observations of transmission electron microscopy. BPA caused apoptosis via the intrinsic mitochondrial pathway and this was confirmed by the expression of apoptotic marker proteins. http://dx.doi.org/10.1016/j.toxlet.2013.05.168
P08-30 Heavy metal burdens of public primary school children in Ibadan north-west local government area, Nigeria related to playground soils and classroom dusts O. Ademuyiwa 1 , F. Akinwunmi 2 , Z. Atobatele 1 , O. Adewole 1 , K. Odekunle 1 , L. Arogundade 2 , O.O. Odukoya 2 1
Department of Biochemistry, Nigeria, 2 Department of Chemistry, Federal University of Agriculture, Abeokuta, Nigeria Information about heavy metal intake of children in Nigeria related to playground soils and classroom dusts compared to economically advanced countries is lacking. Playground soil and classroom dust samples were therefore collected from 8 public primary schools – 6 urban and 2 semi-rural – located within Ibadan North-West Local Government Area of Oyo State in Nigeria. Blood and spot urine samples were also collected from the pupils of the selected schools (n = 253). The samples were analysed for Pb, Cu, Zn, Fe and Mn by flame atomic absorption spectrophotometry. Higher Pb levels were detected in blood of the children (mean blood Pb level of 57.40 ± 28.56 g/dl) as well as in dust and soil samples from the 6 schools located in urban areas while lower levels were obtained in the other 2 schools located in semi-rural area (mean blood Pb of 17.43 ± 8.54 g/dl). Mean metal concentrations
Abstracts / Toxicology Letters 221S (2013) S59–S256
P08-24 Effects of different endocrine disruptor (EDC) mixtures on gene expression in neonatal rat brain regions: Focus on developing excitatory synapses Walter Lichtensteiger 1,∗ , Catherine Bassetti-Gaille 1 , Oliver Faass 1 , Julie Boberg 2 , Sofie Christiansen 2 , Ulla Hass 2 , Andreas Kortenkamp 3 , Margret Schlumpf 1 1
GREEN Tox and Institute of Anatomy, University of Zurich, Zurich, Switzerland, 2 National Food Institute, Technical University of Denmark, Søborg, Denmark, 3 Institute for the Environment, Brunel University, Uxbridge, UK Sexual brain differentiation is a potential EDC target. It depends on a combination of estrogen receptor- and androgen receptormediated effects in males and on estrogens in females. It is not known how these processes are affected by real-world mixtures of EDCs. We investigated the effect of three EDC mixtures on gene expression in developing brain. Amix (8 antiandrogenic chemicals), Emix (4 estrogenic chemicals) and Tmix (Amix + Emix + paracetamol recently identified as anti-androgenic) were administered by oral gavage to rat dams from gestational day 7 until weaning, at doses corresponding to 450×, 200× and 100× high end human intakes (S. Christiansen et al., 2012. International Journal of Andrology 35, 303). At postnatal day 6, during the last part of sexual brain differentiation, exon microarray analyses were performed in medial preoptic area (MPO) in the highest dose group, and real time RT PCR of selected mRNA species in MPO and ventromedial hypothalamus (VMH) of all dose groups. Microarray analyses revealed mixture- and sex-specific effects on gene expression patterns. The majority of genes affected by an individual mixture was selective for that mixture. Real time RT PCR of individual mRNAs demonstrated treatment- and sex-dependent differences between MPO and VMH. Effects were dose-dependent. Prominent are effects on the expression of genes involved in excitatory glutamatergic synapse formation and function. These data indicate that effects of complex EDC mixtures on developing brain can be characterized by mixture-, sex- and region-specific gene expression patterns. Excitatory synapse development emerged as a potentially relevant target. http://dx.doi.org/10.1016/j.toxlet.2013.05.163
P08-25 Effects of endocrine disruptors bisphenol A and di(2-ethylhexyl) phthalate in a combination with 17-estradiol on apoptosis-related genes in the MCF-7 breast cancer cell line Alzbeta Mlynarcikova ∗ , Tomas Havranek, Maria Fickova Institute of Experimental Endocrinology SAS, Bratislava, Slowakia The role of estrogen signaling in mammary carcinogenesis is well established. Compounds named endocrine disruptors (EDs) can exert estrogen-like activities affecting hormone-controlled homeostasis. Bisphenol A (BPA) and di(2-ethylhexyl) phthalate (DEHP) are employed in the manufacture of plastics for consumer products (food packaging, etc.). The correlation between increased EDs exposure and cancer was shown, however, studies with breast cancer cells have yielded inconsistent data. BPA and DEHP can mimic the effects of 17-estradiol (E2) through estrogen receptors or may exert E2-independent actions. Besides mitogenic effects of E2, its antiapoptotic action may contribute to its role in the can-
cer promotion. Since the breast tissue is simultaneously exposed to endogenous and exogenous agents, in the present study, the human breast cancer cells MCF-7 were exposed to E2, BPA/DEHP, or their combinations with E2 (all at concentrations 10−12 , 10−9 , 10−6 M) for 24–120 h. The effects of combinations of E2 with BPA or DEHP on several apoptosis-related genes (Bcl-2, Bax, p53) were analyzed. The results indicate that BPA alone (10−9 , 10−6 M) or in combination with E2 could reduce the expression of proapoptotic p53 and Bax genes. DEHP (10−6 M) was also able to down-regulate Bax gene expression. The presence of E2 increased antiapoptotic Bcl-2 transcripts. These data could partially explain our previous results where the BPA + E2 combinations stimulated de novo DNA synthesis and MCF-7 cell proliferation. Thus, the final proliferative effects induced by EDs in combinations with E2 in MCF-7 cells could partially result from the antiapoptotic action. http://dx.doi.org/10.1016/j.toxlet.2013.05.164 P08-26 Effects of formaldehyde exposure on human epithelial cells all along the respiratory tract Gaëlle Bardet ∗ , Charles Persoz, Thomas Loret, Isabelle Momas, Sophie Achard, Nathalie Seta Université Paris Descartes, Faculté de Pharmacie, EA4064 Impact sanitaire des pollutions, 75006 Paris, France The respiratory tract is directly exposed to inhaled environmental pollutants such as chemicals. Formaldehyde (FA) is a ubiquitous indoor air pollutant known as irritant. Some epidemiological studies revealed an association between FA exposure at low levels and respiratory diseases. Our aim was to assess the cellular inflammatory response to FA exposure at environmental levels of epithelial cells from different parts of the respiratory tract. For this purpose, alveolar A549, bronchial BEAS-2B cell lines and human nasal primary cell cultures (hAECN; Epithelix® ), were cultured on insert and exposed in air/liquid interface (Vitrocell System® ), with air or FA (50 g/m3 ) during 30 min for A549 and BEAS-2B and 60 min for hAECN. 24 h after exposure, apical medium in which IL-8 and MCP-1 levels were measured (ELISA assay), was collected, and cellular viability was assessed by XTT assay. Whatever the conditions and cell type, cell viability was unchanged. No significant variation in inflammatory response was noted after FA exposure compared to air exposure for the A549, BEAS-2B cells while we observed an increase in IL8 for hAECN without any change in MCP-1. Our results suggest that hAECN is more appropriate as a cellular model to mimic the effect of inhaled pollution, since it is a human primary cell culture, and not an immortalized cell line. Mainly nasal epithelium is first in line in contact with inhaled pollutants and the first defensive barrier, which makes this epithelium the most relevant when studying the response to these chemicals. http://dx.doi.org/10.1016/j.toxlet.2013.05.165 P08-27 Effects of maternal exposure to di(2-ethylhexyl)phthalate (DEHP) during pregnancy on neonatal asthma susceptibility Kyoung-youl Lee ∗ , Eun-young Choi Kongju National University, Chungnam, Korea Di(2-ethylhexyl) phthalate (DEHP) is widely distributed in the environment and used as a plasticizer. In this study, we investigate
Abstracts / Toxicology Letters 221S (2013) S59–S256
the effects of maternal exposure to DEHP during pregnancy on neonatal asthma susceptibility using ovalbumin (OVA) induced asthma murine model. Pregnant Balb/c mice received DEHP from Gestation Day 13 to Lactation Day 21. Their offspring was sensitized on Postnatal Day (PND) 9 and 15 by intraperitoneal injection of 0.5 g OVA with 200 g aluminum hydroxide. On PND 22, 23 and 24, live pups were received an airway challenge with OVA for 30 min. Offspring from pregnant mice received DEHP showed the reduction in inflammatory cell count, interleukin (IL)-4, IL-13 and eotaxin in BALF, and total immunoglobulin (Ig) E and OVAspecific IgE in plasma compared with those of offspring from non DEHP-treated pregnant mice. These results were consistent with histological analysis and immunoblot. Maternal exposure to DEHP reduces airway inflammation and mucus production with decrease in inducible nitric oxide synthase (iNOS) in lung tissue of their offspring. This study suggests that maternal exposure to DEHP during pregnancy reduces the risk of asthma in their offspring. These effects were considered to be closely related with suppression of Th2 immune responses and iNOS expression. http://dx.doi.org/10.1016/j.toxlet.2013.05.166
P08-28 Ellagic acid protects against cyclophosphamide induced acute lung injury in Wistar rats by inhibiting oxidative stress, NF-kB activation and inflammatory cell production Sheikh Raisuddin, Falak Beigh Department of Medical Elementology & Toxicology, Jamia Hamdard (Hamdard University), New Delhi, India Use of cyclophosphamide (CP) as chemotherapeutic drug is associated with side effects including toxicity to respiratory system. Upon administration it enhances lung toxicity which may subsequently progress to the lung fibrosis. Natural compounds have shown mitigating effects against toxicity of anticancer drugs. Ellagic acid (EA), a polyphenolic compound present in many fruits and nuts has shown promising protective effect against toxicity of drugs and chemicals. We studied the ameliorative effect of EA on lung toxicity in rats exposed to CP (50 mg/kg b.w., i.p.) and both were administered for seven days. There was a significant decrease in reduced glutathione level and activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase and co-treatment of EA prevented oxidative stress by restoring the level of antioxidant enzymes. EA also modulated enhanced hydroxyproline level, lipid peroxidation, myeloperoxidase activity, nitric oxide production and protein carbonyl formation in BLM exposed rat lungs. Bronchoalveolar lavage fluid showed increased level of cytotoxic markers and inflammatory cell production in terms of total cell count in drug treated groups and EA attenuated these changes. Further damage was observed by monitoring histopathological as well as immunohistochemical analysis of expression level of molecular markers of inflammation in rat lung tissue. In conclusion, BLM exposure to rat lung tissue showed a toxic effect, especially disruption of lung antioxidant profile and EA emerged as a natural protectant with an ability to protect lungs from onslaught of pulmonary toxicity of anticancer drugs. http://dx.doi.org/10.1016/j.toxlet.2013.05.167
S107
P08-29 Endocrine disruptor bisphenol A toxicity study in testicular mitochondria of Swiss albino mice Sameya Anjum ∗ , Sheikh Raisuddin Dept of Toxicology, Faculty of Science, Jamia Hamdard, New Delhi, India Introduction: BPA a monomer of polycarbonate plastic used to manufacture plastic baby bottles, water bottles, cans, adhesives, coatings, paints, building materials, thermal paper, etc. Aim and objective: To investigate BPA induced oxidative stress and toxicity in the testicular mitochondria of adult male mice. Methodology: Swiss Albino male mice were exposed to standardized dose of BPA (5, 10, 100 mg/kg body weight), orally for 14 day. Effect of BPA on LPO, GSH, SOD, GPx, GR were observed. Ultrastructural changes in testes on exposure of BPA were observed. Apoptosis marker protein expression levels were observed by IHC. Results: BPA caused lipid peroxidation (LPO) and decrease in reduced glutathione (GSH) content of testicular mitochondria. Significant differences P < 0.01 was observed in the LPO and GSH parameters when compared with control values. Besides, it also affected activities of antioxidant enzymes such as superoxide dismutase, glutathione reductase and glutathione peroxidase dose dependently. Ultra-structural changes observed by transmission electron microscopy showed BPA caused abnormalities like deformed acrosome and nucleus of spermatids and apoptotic cells were observed in the testes of treated animals. Apoptotic marker proteins like cytochrome c and caspase 3 levels were increased in BPA treated groups as compared to control group of animals. Conclusions: Hence we can conclude that BPA has induced oxidative stress in testicular mitochondria of exposed group and the results were further confirmed by the observations of transmission electron microscopy. BPA caused apoptosis via the intrinsic mitochondrial pathway and this was confirmed by the expression of apoptotic marker proteins. http://dx.doi.org/10.1016/j.toxlet.2013.05.168
P08-30 Heavy metal burdens of public primary school children in Ibadan north-west local government area, Nigeria related to playground soils and classroom dusts O. Ademuyiwa 1 , F. Akinwunmi 2 , Z. Atobatele 1 , O. Adewole 1 , K. Odekunle 1 , L. Arogundade 2 , O.O. Odukoya 2 1
Department of Biochemistry, Nigeria, 2 Department of Chemistry, Federal University of Agriculture, Abeokuta, Nigeria Information about heavy metal intake of children in Nigeria related to playground soils and classroom dusts compared to economically advanced countries is lacking. Playground soil and classroom dust samples were therefore collected from 8 public primary schools – 6 urban and 2 semi-rural – located within Ibadan North-West Local Government Area of Oyo State in Nigeria. Blood and spot urine samples were also collected from the pupils of the selected schools (n = 253). The samples were analysed for Pb, Cu, Zn, Fe and Mn by flame atomic absorption spectrophotometry. Higher Pb levels were detected in blood of the children (mean blood Pb level of 57.40 ± 28.56 g/dl) as well as in dust and soil samples from the 6 schools located in urban areas while lower levels were obtained in the other 2 schools located in semi-rural area (mean blood Pb of 17.43 ± 8.54 g/dl). Mean metal concentrations