Scandinavian Journal of Public Health, Jul 13, 2015
Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled d... more Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled drinking water are potential, but yet poorly characterized, sources of human exposure to endocrine disrupting chemicals globally. Here, we investigated the current situation in the most densely populated region in Finland. Influent and effluent bi-monthly samples from a major wastewater treatment plant in Helsinki were obtained over a preceding 2-year period at two time-points (in 2011 and 2014). Equivalent samples from a household water purification plant (located in the same region) were also analyzed, together with various brands of bottled still and mineral water as well as tap water from residential buildings. Samples were obtained in one liter sterile containers, extracted by solid-phase extraction method, and their estrogenic potential determined by a yeast bioluminescent assay. The estrogenic activities of influent samples from the wastewater treatment plant in Helsinki were generally low (from less than limit of detection to 0.7 ng/L estrogen equivalent quantities (EEQ)), except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/L EEQ, respectively) were obtained. Meanwhile, no estrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, influent and effluent samples from the drinking water plant, as well as tap water, bottled still, and mineral waters. These findings indicate that the purification method applied in Helsinki wastewater treatment plant, activated sludge with mechanical, chemical and biological purification steps, is effective in reducing estrogenic activity, and that tap or bottled waters are not a significant source of these compounds to the population in this region.
Food Additives & Contaminants: Part A, Oct 1, 2013
Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly charact... more Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly characterised sources of human exposure to endocrine-disrupting chemicals (EDCs). In this study we investigated the presence of xenoestrogens in commercially processed and packaged Finnish foods, arising from substances deliberately added or inadvertently contaminating the food, substances formed as a result of food processing, or substances leaching from food packaging materials. Samples were obtained in three separate batches of equivalent products from both a supermarket and a local representative of a global chain of hamburger restaurants and extracted by a solid-phase extraction method. Their endocrine-disrupting potential was determined by yeast bioluminescent assay, using two recombinant yeast strains Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc. In this test system, the majority of samples (both foodstuffs and wrappers) analysed proved negative. However, all batches of industrially prepared hamburgers (but not those obtained from a hamburger restaurant) as well as pepper salami significantly induced luciferase activity in the BMAEREluc/ERα yeast strain indicating the presence of xenoestrogens, with estradiol equivalents of these products ranging from 0.2 to 443 pg g(-1). All three products contained soy-based ingredients, which apparently accounted for, or at least contributed to, their high estrogenic activity, since no signal in the assay was observed with extracts of the packaging material, while two different soy sauces tested yielded an intense signal (28 and 54 pg ml(-1) estradiol-equivalent). These findings imply that by and large chemicals arising in the processing or packaging of foodstuffs in Finland constitute an insignificant source of xenoestrogens to consumers. However, soy-derived ingredients in certain food items might render the entire products highly estrogenic. The estrogenic activity of soy is attributed to isoflavones whose health effects - though widely considered beneficial - are controversial. As hamburgers are a popular type of food among children, the findings are noteworthy and possibly of concern.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its mono... more 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its monoglucuronide in moderately TCDD-resistant line B rats, but not in highly TCDD-resistant line A rats. In the mammalian heme degradation process, heme is cleaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXalpha reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is delta-aminolevulinic acid synthetase 1 (ALAS1). The effect of TCDD on HO-1, BVRA and ALAS1 was studied at the levels of mRNA (all three enzymes), protein expression (HO-1), and enzymatic activity (BVRA, liver only) in order to determine whether the accumulation of biliverdin could be due to their altered expression. In both lines A and B, 300 microg/kg TCDD transiently repressed hepatic HO-1 mRNA on day 2 but induced HO-1 protein expression at later time-points; however, the impact emerged earlier (day 14 vs. day 35) in line B rats. In spleen, TCDD repressed HO-1 mRNA and protein expression in lines A and B through days 2-35, but did not affect its mRNA levels in TCDD-sensitive L-E rats (10 days after 100 microg/kg). In all rat strains/lines, there was a strong repression of ALAS1 and a moderate induction of BVRA mRNA in liver, but mostly not in spleen. Hepatic BVRA activity was increased in lines A and B on day 14. At 5 weeks, it was still elevated in line A but reduced to 51% of control in line B. The results suggest that hepatic heme degradation is induced by TCDD in rats; however, this does not alone explain the accumulation of biliverdin in line B rats. Other factors such as the late repression of BVRA found here and possibly oxidative stress may be important contributors to biliverdin accumulation in these rats.
Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chai... more Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.
Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory ani... more Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory animals, including teratogenesis. In mice, the most important teratogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are hydronephrosis and cleft palate. Aryl hydrocarbon receptor (AHR) mediates most of the TCDD-induced effects, but modulation of these effects by other factors such as epidermal growth factor receptor (EGFR) has been propounded. TCDD changes the expression of both EGF and its receptor EGFR, which may be one step in the pathway leading to cleft palate and hydronephrosis. In the present study, the importance of EGFR in TCDD-induced teratogenicity was evaluated. Heterozygous EGFR(+/-)-mice were mated and pregnant females exposed to 1.5-106.0 microg/kg TCDD on gestation day (GD) 10 and killed on GD 18. The fetuses were studied for cleft palate, hydronephrosis, and open eyes. There was no marked difference among the three genotypes in sensitivity to cleft palate or hydronephrosis, but in EGFR(-/-)-mice frequency of the open eye malformation decreased dose-dependently. In conclusion, EGFR signaling is not required for TCDD-induced cleft palate or hydronephrosis but TCDD appears to counteract the effect of EGFR deficiency on eye opening.
We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralizat... more We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.
Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar toot... more Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar tooth development in rats after in utero and lactational exposure, and that the sensitive stage is temporally restricted. To define the stage in which TCDD is able to arrest tooth development and the cellular background of the effect, mouse embryonic molar tooth explants including various early developmental stages from initiation to late cap stage were exposed to TCDD in organ culture. TCDD did not inhibit morphogenesis of the first molar teeth including the early bud-staged E12 first molars, but the teeth were smaller than in control cultures. Accordingly, the second molars underwent morphogenesis in the presence of TCDD when explanted at E15 when they were at the bud stage. TCDD arrested their development when explanted at E14 when they had not yet reached the early bud stage. Immunohistochemical localization of incorporated bromodeoxyuridine in cultured E14 teeth showed that TCDD did not affect cell proliferation. Localization of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) method revealed that TCDD enhanced apoptosis of dental epithelial cells, especially in the dental lamina of both the first and second molars, and in the inner dental epithelium at the cusp tips of the first molars. Thus, TCDD can arrest tooth development in vitro if the exposure starts at the initiation stage, whereas exposure at later stages leads to smaller tooth size and deformation of cuspal morphology. TCDD interferes with tooth development by stimulating apoptosis in those cells of the dental epithelium, which are predetermined to undergo apoptosis during normal development.
We have previously demonstrated a more than 300‐fold difference in acute LD50 values for 2,3,7,8‐... more We have previously demonstrated a more than 300‐fold difference in acute LD50 values for 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) between male Long‐Evans (Turku AB; L‐E) and Han/Wistar (Kuopio; H/W) rats after intraperitoneal exposure. In the present study, we compared the acute lethality of TCDD, 1,2,3,7,8‐pentachlorodibenzo‐p‐dioxin (PCDD) and 1,2,3,4,7,8‐hexachlorodibenzo‐p‐dioxin (HCDD) in these strains by intragastric administration. In agreement with previous data, H/W rats proved to be strikingly resistant to TCDD, since even the highest dose tested, 7200 μg/kg, was below the LD50 level for both genders. The corresponding LD50 values for female and male L‐E rats were 9.8 and 17.7 μg/kg, respectively. A similar strain difference was discovered for PCDD: the LD50 value was > 1620 μg/kg for female H/W rats and between 20 and 60 μg/kg for female L‐E rats. Surprisingly, the acute lethality of HCDD did not follow the same pattern. Female H/W rats turned out to be only about 10 times less susceptible to that congener than female L‐E rats (LD50 values 1871 and between 120 and 360 μg/kg, respectively). These findings do not support the widely accepted concept that sufficiently high doses of all dioxin congeners will produce the same effects. Either the higher chlorinated dioxins have toxic effects distinct from those of TCDD or the relative contribution of toxic impacts varies among these compounds.
Scandinavian Journal of Public Health, Jul 13, 2015
Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled d... more Environmental pollutants appearing in wastewater, bottled mineral water, tap water, and bottled drinking water are potential, but yet poorly characterized, sources of human exposure to endocrine disrupting chemicals globally. Here, we investigated the current situation in the most densely populated region in Finland. Influent and effluent bi-monthly samples from a major wastewater treatment plant in Helsinki were obtained over a preceding 2-year period at two time-points (in 2011 and 2014). Equivalent samples from a household water purification plant (located in the same region) were also analyzed, together with various brands of bottled still and mineral water as well as tap water from residential buildings. Samples were obtained in one liter sterile containers, extracted by solid-phase extraction method, and their estrogenic potential determined by a yeast bioluminescent assay. The estrogenic activities of influent samples from the wastewater treatment plant in Helsinki were generally low (from less than limit of detection to 0.7 ng/L estrogen equivalent quantities (EEQ)), except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/L EEQ, respectively) were obtained. Meanwhile, no estrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, influent and effluent samples from the drinking water plant, as well as tap water, bottled still, and mineral waters. These findings indicate that the purification method applied in Helsinki wastewater treatment plant, activated sludge with mechanical, chemical and biological purification steps, is effective in reducing estrogenic activity, and that tap or bottled waters are not a significant source of these compounds to the population in this region.
Food Additives & Contaminants: Part A, Oct 1, 2013
Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly charact... more Processed and packaged food items as well as ready-to-eat snacks are neglected and poorly characterised sources of human exposure to endocrine-disrupting chemicals (EDCs). In this study we investigated the presence of xenoestrogens in commercially processed and packaged Finnish foods, arising from substances deliberately added or inadvertently contaminating the food, substances formed as a result of food processing, or substances leaching from food packaging materials. Samples were obtained in three separate batches of equivalent products from both a supermarket and a local representative of a global chain of hamburger restaurants and extracted by a solid-phase extraction method. Their endocrine-disrupting potential was determined by yeast bioluminescent assay, using two recombinant yeast strains Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc. In this test system, the majority of samples (both foodstuffs and wrappers) analysed proved negative. However, all batches of industrially prepared hamburgers (but not those obtained from a hamburger restaurant) as well as pepper salami significantly induced luciferase activity in the BMAEREluc/ERα yeast strain indicating the presence of xenoestrogens, with estradiol equivalents of these products ranging from 0.2 to 443 pg g(-1). All three products contained soy-based ingredients, which apparently accounted for, or at least contributed to, their high estrogenic activity, since no signal in the assay was observed with extracts of the packaging material, while two different soy sauces tested yielded an intense signal (28 and 54 pg ml(-1) estradiol-equivalent). These findings imply that by and large chemicals arising in the processing or packaging of foodstuffs in Finland constitute an insignificant source of xenoestrogens to consumers. However, soy-derived ingredients in certain food items might render the entire products highly estrogenic. The estrogenic activity of soy is attributed to isoflavones whose health effects - though widely considered beneficial - are controversial. As hamburgers are a popular type of food among children, the findings are noteworthy and possibly of concern.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its mono... more 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic accumulation of biliverdin and its monoglucuronide in moderately TCDD-resistant line B rats, but not in highly TCDD-resistant line A rats. In the mammalian heme degradation process, heme is cleaved to biliverdin by the rate-limiting enzyme heme oxygenase-1 (HO-1). Subsequently, biliverdin IXalpha reductase (BVRA) catalyzes the reduction of biliverdin to bilirubin. In heme biosynthesis, the rate-limiting enzyme is delta-aminolevulinic acid synthetase 1 (ALAS1). The effect of TCDD on HO-1, BVRA and ALAS1 was studied at the levels of mRNA (all three enzymes), protein expression (HO-1), and enzymatic activity (BVRA, liver only) in order to determine whether the accumulation of biliverdin could be due to their altered expression. In both lines A and B, 300 microg/kg TCDD transiently repressed hepatic HO-1 mRNA on day 2 but induced HO-1 protein expression at later time-points; however, the impact emerged earlier (day 14 vs. day 35) in line B rats. In spleen, TCDD repressed HO-1 mRNA and protein expression in lines A and B through days 2-35, but did not affect its mRNA levels in TCDD-sensitive L-E rats (10 days after 100 microg/kg). In all rat strains/lines, there was a strong repression of ALAS1 and a moderate induction of BVRA mRNA in liver, but mostly not in spleen. Hepatic BVRA activity was increased in lines A and B on day 14. At 5 weeks, it was still elevated in line A but reduced to 51% of control in line B. The results suggest that hepatic heme degradation is induced by TCDD in rats; however, this does not alone explain the accumulation of biliverdin in line B rats. Other factors such as the late repression of BVRA found here and possibly oxidative stress may be important contributors to biliverdin accumulation in these rats.
Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chai... more Dioxins are persistent and ubiquitous environmental poisons that become enriched in the food chain. Besides being acutely lethal, the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is developmentally toxic to many animal species. We have previously found that developing teeth of children may be sensitive to environmental dioxins via their mother's milk and that rat and mouse teeth are dioxin-sensitive throughout their development. The aryl hydrocarbon receptor (AHR) together with the AHR nuclear translocator (ARNT) protein is believed to mediate the toxic effects of dioxins. To study the potential involvement of the AHR-ARNT pathway in the dental toxicity of TCDD, we analysed the expression of AHR and ARNT by in situ hybridization and immunohistochemistry in developing mouse teeth. AHR mRNA first appeared in the epithelium of E12 first molar tooth buds and both proteins were weakly expressed in the bud. After cytodifferentiation the expression was up regulated and became intense in secretory odontoblasts and ameloblasts. The coexpression of AHR and ARNT during early tooth development as well as during the information and mineralization of the dental matrices is suggestive of the AHR-ARNT pathway as a mediator of dental toxicity of TCDD.
Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory ani... more Dioxins are persistent environmental contaminants that cause multiple disorders in laboratory animals, including teratogenesis. In mice, the most important teratogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are hydronephrosis and cleft palate. Aryl hydrocarbon receptor (AHR) mediates most of the TCDD-induced effects, but modulation of these effects by other factors such as epidermal growth factor receptor (EGFR) has been propounded. TCDD changes the expression of both EGF and its receptor EGFR, which may be one step in the pathway leading to cleft palate and hydronephrosis. In the present study, the importance of EGFR in TCDD-induced teratogenicity was evaluated. Heterozygous EGFR(+/-)-mice were mated and pregnant females exposed to 1.5-106.0 microg/kg TCDD on gestation day (GD) 10 and killed on GD 18. The fetuses were studied for cleft palate, hydronephrosis, and open eyes. There was no marked difference among the three genotypes in sensitivity to cleft palate or hydronephrosis, but in EGFR(-/-)-mice frequency of the open eye malformation decreased dose-dependently. In conclusion, EGFR signaling is not required for TCDD-induced cleft palate or hydronephrosis but TCDD appears to counteract the effect of EGFR deficiency on eye opening.
We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralizat... more We have previously shown that dioxins at prevailing levels in mothers' milk may cause mineralization defects in the developing teeth of their children. Developmental dental defects have also been reported in rhesus macaques and rats experimentally exposed to dioxin. The most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is a potent modulator of epithelial cell growth and differentiation. To clarify whether epidermal growth factor receptor (EGFR), implicated in the mediation of the developmental toxicity of TCDD, is involved in dental toxicity, we cultured embryonic molar teeth from EGFR-deficient mice with TCDD, epidermal growth factor (EGF), and both agents in combination. In teeth of the normal embryos, TCDD caused depolarization of odontoblasts and ameloblasts. Consequently, the dentin matrix failed to undergo mineralization, the enamel matrix was not deposited, and cuspal morphology was disrupted. In teeth of the null mutant embryos, only the cuspal contour was mildly modified. EGF alone retarded the molar tooth development of normal embryos, but not that of EGFR-deficient embryos. When coadministered with TCDD, EGF for the most part prevented the adverse effects of TCDD on teeth of the normal embryos. These results show that the interference of TCDD with mouse molar tooth development in vitro involves EGFR signaling. Thus, EGFR may also play a role in the developmental defects that dioxins cause in human teeth. Because EGFR is widely expressed in developing organs, EGFR signaling may even be of general relevance in the mediation of the developmental toxicity of TCDD.
Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar toot... more Previous studies have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can arrest molar tooth development in rats after in utero and lactational exposure, and that the sensitive stage is temporally restricted. To define the stage in which TCDD is able to arrest tooth development and the cellular background of the effect, mouse embryonic molar tooth explants including various early developmental stages from initiation to late cap stage were exposed to TCDD in organ culture. TCDD did not inhibit morphogenesis of the first molar teeth including the early bud-staged E12 first molars, but the teeth were smaller than in control cultures. Accordingly, the second molars underwent morphogenesis in the presence of TCDD when explanted at E15 when they were at the bud stage. TCDD arrested their development when explanted at E14 when they had not yet reached the early bud stage. Immunohistochemical localization of incorporated bromodeoxyuridine in cultured E14 teeth showed that TCDD did not affect cell proliferation. Localization of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) method revealed that TCDD enhanced apoptosis of dental epithelial cells, especially in the dental lamina of both the first and second molars, and in the inner dental epithelium at the cusp tips of the first molars. Thus, TCDD can arrest tooth development in vitro if the exposure starts at the initiation stage, whereas exposure at later stages leads to smaller tooth size and deformation of cuspal morphology. TCDD interferes with tooth development by stimulating apoptosis in those cells of the dental epithelium, which are predetermined to undergo apoptosis during normal development.
We have previously demonstrated a more than 300‐fold difference in acute LD50 values for 2,3,7,8‐... more We have previously demonstrated a more than 300‐fold difference in acute LD50 values for 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) between male Long‐Evans (Turku AB; L‐E) and Han/Wistar (Kuopio; H/W) rats after intraperitoneal exposure. In the present study, we compared the acute lethality of TCDD, 1,2,3,7,8‐pentachlorodibenzo‐p‐dioxin (PCDD) and 1,2,3,4,7,8‐hexachlorodibenzo‐p‐dioxin (HCDD) in these strains by intragastric administration. In agreement with previous data, H/W rats proved to be strikingly resistant to TCDD, since even the highest dose tested, 7200 μg/kg, was below the LD50 level for both genders. The corresponding LD50 values for female and male L‐E rats were 9.8 and 17.7 μg/kg, respectively. A similar strain difference was discovered for PCDD: the LD50 value was > 1620 μg/kg for female H/W rats and between 20 and 60 μg/kg for female L‐E rats. Surprisingly, the acute lethality of HCDD did not follow the same pattern. Female H/W rats turned out to be only about 10 times less susceptible to that congener than female L‐E rats (LD50 values 1871 and between 120 and 360 μg/kg, respectively). These findings do not support the widely accepted concept that sufficiently high doses of all dioxin congeners will produce the same effects. Either the higher chlorinated dioxins have toxic effects distinct from those of TCDD or the relative contribution of toxic impacts varies among these compounds.
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