The overall goal of the present research project was to investigate the physiology of dietary iron absorption and its interactions with the uptake and metabolism of other divalent metals, especially cadmium, in freshwater fish, using rainbow trout (Oncorhynchus mykiss) as a model species. Using intestinal sac preparations, iron absorption was found to occur along the entire intestinal tract of fish, with anterior intestine being the major site of absorption compared to either mid or posterior intestine. Ferrous iron was more bioavailable than ferric iron, and the uptake of ferrous iron was significantly reduced at alkaline pH (p < 0.05). These findings suggested that a homolog of the mammalian apical ferrous iron transporter, divalent metal transporter-1 (DMT1, a Fe2+/H+ symporter), is involved in the absorption of iron in the fish intestine. Ferric iron appeared to be absorbed through the same pathway as ferrous iron following reduction by an apical ferric reductase. Several divalent metals, both essential (nickel, copper and zinc) and non-essential (cadmium and lead), inhibited intestinal ferrous iron absorption in fish. Importantly, elevated luminal iron reciprocally reduced the accumulation of cadmium in the fish intestine, indicating the significance of the iron transport pathway in dietary cadmium absorption. Two different DMT1 isoforms, Nramp-â and -ã, were found to be expressed along the entire gastrointestinal tract of fish. My study showed that in isolated rainbow trout enterocytes, ferrous iron uptake occurred through a saturable and proton-dependent process, providing further evidence of DMT1-mediated ferrous iron transport. Both cadmium and lead inhibited ferrous iron uptake in the enterocytes in a concentration-dependent manner. Kinetic characterization revealed that the apparent affinity for ferrous iron uptake is significantly decreased (increased Km) in the presence of either cadmium or lead (p < 0.05), whereas the maximum uptake rate (Jmax) remains unchanged. These results indicated that the interaction between ferrous iron and cadmium or lead is competitive in nature, and the uptake of these metals occurs through a common transport pathway (likely DMT1). The uptake characteristics of cadmium were further examined in isolated rainbow trout enterocytes, and my findings indicated that in addition to DMT1, cadmium uptake can be mediated by zinc transport pathway (ZIP8, a Zn2+/HCO3- symporter). My study also showed that cysteine-conjugated cadmium was readily bioavailable to fish enterocytes, possibly via a cysteine-specific transport pathway. The efflux of cadmium from the enterocytes was found to occur via an ATPase-driven pathway. On the other hand, chronic exposure to dietary cadmium at an environmentally-relevant concentration significantly increased cadmium burden in target organs as well as in the whole-body of fish (p < 0.05). Exposure to dietary cadmium increased the mRNA expression level of key stress-inducible proteins such as metallothioneins (MT-A and MT-B) and heat shock proteins-70 (HSP-70a and HSP-70b). Interestingly, each MT and HSP-70 mRNA isoform responded differently in various target organs of fish following dietary cadmium exposure. Fish exposed to dietary cadmium also exhibited an increase in the hepatic transferrin mRNA level as well as the plasma transferrin protein level, indicating the role of transferrin in cadmium handling in fish. Importantly, an iron-supplemented diet reduced cadmium burden in the gut and the whole-body, and ameliorated the expression of MT and HSP-70 genes in fish. These results suggested the protective effects of elevated dietary iron against chronic dietary cadmium toxicity in fish. Overall, findings from the present research project provided novel and important physiological and molecular insights into the uptake, interactions and homeostasis of dietary divalent metals in freshwater fish. This information greatly enhances our current understanding of the toxicological implications for dietary metal exposure in metal contaminated wild fish populations, and may ultimately help the regulators to develop better strategies for ecological risk assessment of metals.

The overall goal of the present research project was to investigate the physiology of dietary iron absorption and its interactions with the uptake and metabolism of other divalent metals, especially cadmium, in freshwater fish, using rainbow trout (Oncorhynchus mykiss) as a model species. Using intestinal sac preparations, iron absorption was found to occur along the entire intestinal tract of fish, with anterior intestine being the major site of absorption compared to either mid or posterior intestine. Ferrous iron was more bioavailable than ferric iron, and the uptake of ferrous iron was significantly reduced at alkaline pH (p < 0.05). These findings suggested that a homolog of the mammalian apical ferrous iron transporter, divalent metal transporter-1 (DMT1, a Fe2+/H+ symporter), is involved in the absorption of iron in the fish intestine. Ferric iron appeared to be absorbed through the same pathway as ferrous iron following reduction by an apical ferric reductase. Several divalent metals, both essential (nickel, copper and zinc) and non-essential (cadmium and lead), inhibited intestinal ferrous iron absorption in fish. Importantly, elevated luminal iron reciprocally reduced the accumulation of cadmium in the fish intestine, indicating the significance of the iron transport pathway in dietary cadmium absorption. Two different DMT1 isoforms, Nramp-â and -ã, were found to be expressed along the entire gastrointestinal tract of fish. My study showed that in isolated rainbow trout enterocytes, ferrous iron uptake occurred through a saturable and proton-dependent process, providing further evidence of DMT1-mediated ferrous iron transport. Both cadmium and lead inhibited ferrous iron uptake in the enterocytes in a concentration-dependent manner. Kinetic characterization revealed that the apparent affinity for ferrous iron uptake is significantly decreased (increased Km) in the presence of either cadmium or lead (p < 0.05), whereas the maximum uptake rate (Jmax) remains unchanged. These results indicated that the interaction between ferrous iron and cadmium or lead is competitive in nature, and the uptake of these metals occurs through a common transport pathway (likely DMT1). The uptake characteristics of cadmium were further examined in isolated rainbow trout enterocytes, and my findings indicated that in addition to DMT1, cadmium uptake can be mediated by zinc transport pathway (ZIP8, a Zn2+/HCO3- symporter). My study also showed that cysteine-conjugated cadmium was readily bioavailable to fish enterocytes, possibly via a cysteine-specific transport pathway. The efflux of cadmium from the enterocytes was found to occur via an ATPase-driven pathway. On the other hand, chronic exposure to dietary cadmium at an environmentally-relevant concentration significantly increased cadmium burden in target organs as well as in the whole-body of fish (p < 0.05). Exposure to dietary cadmium increased the mRNA expression level of key stress-inducible proteins such as metallothioneins (MT-A and MT-B) and heat shock proteins-70 (HSP-70a and HSP-70b). Interestingly, each MT and HSP-70 mRNA isoform responded differently in various target organs of fish following dietary cadmium exposure. Fish exposed to dietary cadmium also exhibited an increase in the hepatic transferrin mRNA level as well as the plasma transferrin protein level, indicating the role of transferrin in cadmium handling in fish. Importantly, an iron-supplemented diet reduced cadmium burden in the gut and the whole-body, and ameliorated the expression of MT and HSP-70 genes in fish. These results suggested the protective effects of elevated dietary iron against chronic dietary cadmium toxicity in fish. Overall, findings from the present research project provided novel and important physiological and molecular insights into the uptake, interactions and homeostasis of dietary divalent metals in freshwater fish. This information greatly enhances our current understanding of the toxicological implications for dietary metal exposure in metal contaminated wild fish populations, and may ultimately help the regulators to develop better strategies for ecological risk assessment of metals.

Runoff and effluent discharge from mining activities has resulted in elevated concentrations of selenium in aquatic ecosystems. Bioavailability is dependent on chemical speciation. Although dissolved inorganic Se species are not directly toxic to organisms, uptake by primary producers and subsequent biotransformation to organo-selenium species substantially increase risk and bioaccumulation potential, potentially impairing reproduction in high-order organisms. The effects of dietary selenium exposure were assessed in two aquatic invertebrates: Ceriodaphnia dubia and Chironomus dilutus. Two generations of these organisms were exposed to seleniferous algae grown in 0-40 μg L-1 selenate. Dissolved selenate was readily absorbed and concentrated by algal species Pseudokirchneriella subcapitata and Chlorella kesslerii. Se had no effect on survival at treated concentrations. Dietary selenium exposures had little effect on reproductive endpoints in either invertebrate species, suggesting that invertebrates have the ability to regulate chronic Se exposures from dietary sources. These results provide valuable information concerning the effects of dietary selenium in aquatic invertebrates.

Runoff and effluent discharge from mining activities has resulted in elevated concentrations of selenium in aquatic ecosystems. Bioavailability is dependent on chemical speciation. Although dissolved inorganic Se species are not directly toxic to organisms, uptake by primary producers and subsequent biotransformation to organo-selenium species substantially increase risk and bioaccumulation potential, potentially impairing reproduction in high-order organisms. The effects of dietary selenium exposure were assessed in two aquatic invertebrates: Ceriodaphnia dubia and Chironomus dilutus. Two generations of these organisms were exposed to seleniferous algae grown in 0-40 μg L-1 selenate. Dissolved selenate was readily absorbed and concentrated by algal species Pseudokirchneriella subcapitata and Chlorella kesslerii. Se had no effect on survival at treated concentrations. Dietary selenium exposures had little effect on reproductive endpoints in either invertebrate species, suggesting that invertebrates have the ability to regulate chronic Se exposures from dietary sources. These results provide valuable information concerning the effects of dietary selenium in aquatic invertebrates.

Arsenic (As) exposure is associated with cancer, lung and cardiovascular disease, yet the mechanisms involved are not clearly understood. Elevated matrix metalloproteinase-9 (MMP-9) levels are also associated with these diseases, as well as with exposure to water As. Our objective was to evaluate the effects of dietary components of inorganic As (iAs) intake on serum MMP-9 concentration at differing levels of tap water As. In a cross-sectional study of 214 adults, dietary iAs intake was estimated from 24-h dietary recall interviews using published iAs residue data; drinking and cooking water As intake from water samples and consumption data. Aggregate iAs intake (food plus water) was associated with elevated serum MMP-9 in mixed model regression, with and without adjustment for covariates. In models stratified by tap water As, aggregate intake was a significant positive predictor of serum MMP-9 in subjects exposed to water As≤10 μg/l. Inorganic As from food alone was associated with serum MMP-9 in subjects exposed to tap water As≤3 μg/l. Exposure to iAs from food and water combined, in areas where tap water As concentration is ≤10 μg/l, may contribute to As-induced changes in a biomarker associated with toxicity.

要旨ファイルを差し替え（2022-01-24） / 京都大学 / 新制・課程博士 / 博士(工学) / 甲第23492号 / 工博第4904号 / 新制||工||1766(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 高野 裕久, 教授 松井 康人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

Polychlorinated biphenyls (PCBs) are a group of 209 persistent organic pollutants, whose documented carcinogenic, neurological and respiratory toxicities are expansive and growing. Existing inhalation estimates demonstrate ubiquitous exposure to World Health Organization (WHO) indicator PCBs and limited other PCB congeners in North America and Europe. However, inhalation exposure estimates of most lower-chlorinated congeners are lacking, and continuing release of PCBs from urban areas demands location-specific assessments of PCB exposure in ambient air and contaminated environments. Using paired indoor and outdoor airborne PCB measurements and activity questionnaires from the AESOP Study, we assess congener-specific exposure rates for adolescent children and their mothers in East Chicago, Indiana and Columbus Junction, Iowa. Our cohorts of 129 (EC) and 135 (CJ) and our detection of 202 individual congeners and coelutions allows unprecedented quantification of congener-specific inhalation exposure, which we compare to dietary exposure using Total Diet Survey PCB concentrations. ∑PCB inhalation is greater for children than for their mothers in both locations, and is greater for East Chicago mothers and children than for Columbus Junction mothers and children, respectively. Schools attended by AESOP Study children have higher indoor PCB concentrations than do homes, and contribute to more than half of children's inhalation PCB exposure. Inhalation of the potentially neurotoxic congeners PCB 11, 40/41/71, and 51 was apparent among individuals at each location. Additional, congener-specific and biological inferences are possible via comparison with sera-based PCB concentrations for these cohorts.

This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

Human exposure to methylmercury (MeHg) through diets (e.g., fish and rice) is a global health concern. Although MeHg exposure through fish consumption has long been considered the major route of mercury health risks, studies concerning the long-term changes in MeHg exposure from fish remain lacking. In sharply contrast to the fish MeHg issue, the presence of MeHg in rice has only been reported recently and its implications on MeHg exposure, albeit probably important, are still in infancy. Focusing on the discrepancies in the studies of MeHg exposure through fish and rice consumption, this study was aimed to assess the MeHg exposure of human infants through consumption of rice cereals and to evaluate the long-term changes in fish MeHg. The presence of MeHg in rice prompted the studies on MeHg concentrations and bioaccessibility in rice cereals and potential infant dietary exposure to MeHg through cereal consumption, which is believed to be the first of its kind. The analysis of a variety of infant cereals sampled from the common markets in the United States and China showed that the concentrations of MeHg in the cereals ranged from 0.07 to 13.9 µg/kg with a mean of 1.61 µg/kg. On the basis of these MeHg concentrations, the daily intake of MeHg through rice cereal consumption for infants was estimated to be 4-122% of the reference dose (RfD). The MeHg bioaccessibility in the cereals, determined using an in vitro digestion method, ranged from 25 to 74% with a mean of 48±16%. A further examination on these results, however, revealed the occurrence of MeHg re-adsorption during extraction steps, which leads to the underestimation of MeHg bioaccessibility and warrants cautions to be exercised when using these procedures to evaluate bioaccessibility in general. The long-term changes in fish MeHg were investigated through conducting a comprehensive data analysis on datasets for the Everglades, a well-studied aquatic ecosystem for Hg contamination. The results showed a clear decline of MeHg in mosquitofish in the Everglades during the past two decades, which was probably related to changes in environmental conditions (e.g., periphyton, dissolve organic matter, and sulfate) instead of mercury deposition.