Evaluating the toxicokinetics of some metabolites of a C6 polyfluorinated compound, 6:2 fluorotelomer alcohol in pregnant and nonpregnant rats after oral exposure to the parent compound.

The 6:2 fluorotelomer alcohol (6:2 FTOH) is a common impurity in per- and polyfluoroalkyl substances (PFASs) used in many applications. Our previous toxicokinetic (TK) evaluation of 6:2 FTOH calculated times to steady state (tss) of one of its metabolites, 5:3 fluorotelomer carboxylic acid (5:3A), in the plasma and tissues of up to a year after oral exposure to rats. Our current work further elucidated the TK of 5:3A and other metabolites of 6:2 FTOH in pregnant and nonpregnant rats after repeated oral exposure and examined the role of renal transporters in the biopersistence of 5:3A. The tss values for 5:3A in serum and tissues of adult nonpregnant animals ranged from 150 days to over a year. 4:3 fluorotelomer carboxylic acid (4:3A) was an additional potentially-biopersistent metabolite. 5:3A was the major metabolite of 6:2 FTOH in serum of pregnant dams and fetuses at each time interval. 5:3A was not a substrate for renal transporters in a human kidney cell line in vitro, indicating that renal reuptake of 5:3A is unlikely contribute to its biopersistence. Further research is needed to identify the underlying processes and evaluate the impact of these 6:2 FTOH metabolites on human health.

Isoliquiritigenin Decreases Bone Resorption and Osteoclast Differentiation.

SCOPE: A dose-ranging study is performed using young estrogen-depleted rats to determine whether dietary isoliquiritigenin (ILQ) alters bone metabolism and if the effects are associated with estrogen receptor signaling. METHODS AND RESULTS: Six-week-old rats (ovariectomized at 4 weeks of age) are fed diets containing 0, 100, 250, or 750 ppm ILQ (n = 5/treatment) for 7 days. Gene expression in femur and uterus, blood markers of bone turnover, body composition, and uterine weight and epithelial cell height are determined. Because ILQ lowers bone resorption, the effect of ILQ on in vitro differentiation of osteoclasts from bone marrow of mice is assessed. Treatment resulted in a dose-dependent increases in serum ILQ but no changes in serum osteocalcin, a marker of global bone formation. Contrastingly, ILQ administration results in reduced serum CTX-1, a marker of global bone resorption, and reduces tartrate resistant acid phosphatase expression in osteoclast culture. ILQ treatment and endogenous estrogen production had limited overlap on gene expression in femur and uterus. However, uterine epithelial cell hyperplasia is observed in two of five animals treated with 750 ppm. CONCLUSIONS: In conclusion, dietary ILQ reduces bone resorption in vivo and osteoclast differentiation in vitro, by mechanisms likely differing from actions of ovarian hormones.

Reduction by, ligand exchange among, and covalent binding to glutathione and cellular thiols link metabolism and disposition of dietary arsenic species with toxicity.

Arsenic (As) is a common contaminant in the earth's crust and widely distributed in food and drinking water. As exposures have been associated with human disease, including cancer, diabetes, lung and cardiovascular disorders, and there is accumulating evidence that early life exposures are important in the etiology. Mode-of-action analysis includes a critical role for metabolic activation of As species to reactive trivalent intermediates that disrupt cellular regulatory systems by covalent binding to thiol groups. The central role of glutathione (GSH) in the chemical reactions of metabolism and disposition of arsenic species was investigated here. The chemical kinetics were measured for reactions in which GSH is a ligand for trivalent As complex formation, a reductant for pentavalent As species, and a participant in ligand exchange reactions with other biological As-thiol complexes. The diverse reactions of GSH with As species demonstrate prominent roles in: (1) metabolic activation via reduction; (2) transport from tissues that are the primary sources of reactive trivalent As intermediates following ingestion (intestine and liver) to downstream target organs (e.g., lung, kidney, and bladder); and (3) oxidation to the terminal metabolite, dimethylarsinic acid (DMAV), which is excreted. Studies of As metabolism and disposition emphasize the link between metabolic activation vs. excretion of As (i.e., internal dosimetry of reactive species) and the disruption of critical cellular thiol-based regulatory processes that define the dose-response characteristics of disease in human epidemiological studies and animal models and underpin risk assessment.

Characterizing biopersistence potential of the metabolite 5:3 fluorotelomer carboxylic acid after repeated oral exposure to the 6:2 fluorotelomer alcohol.

Our previous report on pharmacokinetic (PK) evaluation of 6:2 fluorotelomer alcohol (6:2 FTOH) examined the biopersistence potential of its metabolites based on data published from single inhalation and occupational 6:2 FTOH exposure studies. We calculated internal exposure estimates of three key metabolites of 6:2 FTOH, of which 5:3 fluorotelomer carboxylic acid (5:3 acid) had the highest internal exposure and the slowest clearance. No oral repeated 6:2 FTOH exposure data were available at the time to fully characterize the biopersistence potential of the metabolite 5:3 acid. We recently received additional data on 6:2 FTOH and 5:3 acid, which included a 90-day toxicokinetic study report on repeated oral 6:2 FTOH exposure to rats. We reviewed the study and analyzed the reported 5:3 acid concentrations in plasma, liver, and fat using one-compartment PK modeling and calculated elimination rate constants (kel), elimination half-lives (t1/2) and times to steady state (tss) of 5:3 acid at three 6:2 FTOH doses. Our results showed that tss of 5:3 acid in plasma and evaluated tissues were approximately close to 1 year, such that the majority of highest values were observed at the lowest 6:2 FTOH dose, indicating its association with the biopersistence of 6:2 FTOH. The results of our PK analysis are the first to characterize biopersistence potential of the 5:3 acid after repeated oral exposure to the parent compound 6:2 FTOH based on steady state PK parameters, and therefore, may have an impact on future study designs when conducting toxicity assays for such compounds.

(±)-Equol does not interact with genistein on estrogen-dependent breast tumor growth.

Equol (EQ) is a prominent microbial metabolite of the soy isoflavone, daidzein, with estrogen-like properties. The major soy isoflavone, genistein (GEN), stimulated growth of estrogen-dependent breast cancer (EDBC) cells in vitro and tumor growth in vivo but EQ did not. To understand possible interactions of EQ and GEN on EDBC, EQ was used with GEN in combination in vitro and in vivo. Effects of EQ, GEN and EQ + GEN were evaluated using MCF-7 and T47D EDBC. Ovariectomized athymic mice were used as a model for in vivo tumor growth. Dietary EQ had no effect on MCF-7 tumor growth and the absence of effect was confirmed using a T47D EDBC in vivo model. EQ alone or in combination with GEN increased EDBC cell proliferation in vitro. EQ alone neither stimulated EDBC tumor growth in vivo at various doses nor suppressed tumor growth induced by dietary GEN. In summary, EQ has similar estrogenic effect as GEN in vitro but does not interact with GEN on EDBC tumor growth. Based on the evidence presented here, dietary EQ is unlikely to have estrogenic effects in vivo.

Metabolism and disposition of arsenic species from controlled dosing with sodium arsenite in adult and neonatal rhesus monkeys. VI. Toxicokinetic studies following oral administration.

Arsenic is a common toxic contaminant in food and drinking water. Metabolic activation of arsenic species produces reactive trivalent intermediates that can disrupt cellular regulatory systems by covalent binding to thiol groups. Arsenic exposures have been associated with human diseases including cancer, diabetes, lung and cardiovascular disorders and there is accumulating evidence that early life exposures are important in the etiology. Previous toxicokinetic studies of arsenite ingestion in neonatal CD-1 mice showed consistent evidence for metabolic and physiologic immaturity that led to elevated internal exposures to trivalent arsenic species in the youngest mice, relative to adults. The current study in rhesus monkeys showed that metabolism and binding of trivalent intermediates after arsenite ingestion were similar between adult monkeys and CD-1 mice. Unlike neonatal mice, monkeys from the age of 5-70 days showed similar metabolism and binding profiles, which were also similar to those in adults. The absence of evidence for metabolic immaturity in monkeys suggests that toxicological effects observed in mice from early postnatal exposures to arsenic could over-predict those possible in primates, based on significantly higher internal exposures.

A single or short time repeated arsenic oral exposure in mice impacts mRNA expression for signaling and immunity related genes in the gut.

Arsenic is prevalent in contaminated drinking water and affects more than 140 million people in 50 countries. While the wide-ranging effects of arsenic on neurological development and cancer draw the majority of concern, arsenic's effects on the gut mucosa-associated immune system are often overlooked. In this study, we show that 24 h after a single dose [low dose (50 µg/kg bw), medium dose (100 µg/kg bw) or high dose (200 µg/kg bw)] of arsenic by oral gavage, mice show significantly reduced gut mucosa-associated mRNA expression for the key genes involved in the signaling pathways central to immune responses, such as Nuclear factor κB (NFκB), Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), p38 and Myeloid differentiation protein 88-dependent (Myd88) pathways. Additionally, mRNA expression of apoptosis, inflammasomes and inflammatory response genes are significantly downregulated in the animals exposed to arsenic. Comparisons of time-dependent effects (24 h vs 48 h) from low dose arsenic exposed animals showed a significant shift in expression of Myd88 alone, suggesting that the down regulation was sustained for the key genes/signaling pathway. An extended eight-day exposure to arsenic showed a decreased state of immune preparedness, though not as diminished as seen in the single dose exposure.

Metabolism and disposition of arsenic species from controlled dosing with dimethylarsinic acid (DMAV) in adult female CD-1 mice. V. Toxicokinetic studies following oral and intravenous administration.

Arsenic species contaminate food and water, with typical dietary intake below 1 µg/kg bw/d. Exposure to arsenic in heavily contaminated drinking water is associated with human diseases, including cardiovascular and respiratory disorders, diabetes, and cancer. Dietary intake assessments show that rice and seafood are the primary contributors to intake of both inorganic arsenic and dimethylarsinic acid (DMAV) and at similar magnitudes. DMAV plays a central role in the toxicology of arsenic because enzymatic methylation of arsenite produces DMAV as the predominant metabolite, which may promote urinary clearance but also generates reactive intermediates, predominantly DMAIII, that bind extensively to cellular thiols. Both inorganic arsenic and DMAV are carcinogenic in chronically exposed rodents. This study measured pentavalent and trivalent arsenic species in blood and tissues after oral and intravenous administration of DMAV (50 µg As/kg bw). DMAV underwent extensive first-pass metabolism in the intestine and liver, exclusively by reduction to DMAIII, which bound extensively to blood and tissues. The results confirm a role for methylation-independent reductive metabolism in producing fluxes of DMAIII that presumably underlie arsenic toxicity and indicate the need to include all dietary intake of inorganic arsenic and DMAV in risk assessments.

Metabolism and disposition of arsenic species from oral dosing with sodium arsenite in neonatal CD-1 mice. IV. Toxicokinetics following gavage administration and lactational transfer.

Arsenic is a ubiquitous contaminant, with typical human dietary intake below 1 µg/kg bw/d and extreme drinking water exposures up to ∼50 µg/kg bw/d. The formation and binding of trivalent metabolites are central to arsenic toxicity and strong human evidence suggests special concern for early life exposures in the etiology of adult diseases, especially cancer. This study measured the metabolism and disposition of arsenite in neonatal mice to understand the role of maturation in metabolic activation and detoxification of arsenic. Many age-related differences were observed after gavage administration of arsenite, with consistent evidence in blood and tissues for higher exposures to trivalent arsenic species in neonatal mice related to the immaturity of metabolic and/or excretory functions. The evidence for greater tissue binding of arsenic species in young mice is consistent with enhanced susceptibility to toxicity based on metabolic and toxicokinetic differences alone. Lactational transfer from arsenite-dosed dams to suckling mice was minimal, based on no dosing-related changes in the levels of arsenic species in pup blood or milk collected from the dams. Animal models evaluating whole-life exposure to inorganic arsenic must use direct dosing in early neonatal life to predict accurately potential toxicity from early life exposures in children.

A Summary Report of FSCJ Workshop "Future Challenges and Opportunities in Developing Methodologies for Improved Human Risk Assessments".

This is a summary report of FSCJ (Food Safety Commission of Japan) workshop entitled "Future Challenges and Opportunities in Developing Methodologies for Improved Human Risk Assessments, which held in November 2018. Scientific advancements have facilitated the development of new methods for chemical risk assessments with the expansion of toxicological databases. They are promising tools to overcome challenges, such as situations of data insufficiency, estimation of internal exposure and prediction of hazard, and enable us to improve our human health risk assessment in food safety. In this review, current understandings on developments in chemical risk assessments, especially focusing on Threshold of Toxicological Concern (TTC) approach, non-testing and in-silico approaches (e.g. read-across), and physiologically based pharmacokinetics (PBPK) modeling are discussed as possible promising tools. It also discusses future challenges and opportunities regarding social environment buildings in which all stakeholders including scientific experts, risk managers and consumers are able to accept these new risk assessment technologies. International collaboration would increase and enhance the efficiency in forming innovative ideas and in translating them into regulatory practices. It would strengthen technical capacity of experts who contribute to regulatory decisions and also promote acceptance of new methodologies among stakeholders. Cross-sectional collaboration such as making good use of human data of pharmaceutical drugs will facilitate a development of fresh tools for food safety domains. Once a new methodology is recognized in risk assessment agencies as implementable, it needs to be acknowledged and accepted by wider range of different stakeholders. Such stakeholders include scientific experts who conduct risk assessment for the risk assessment agencies, food industries and consumers. Transparency in the risk assessment work performed by regulatory agencies should strengthen their credibility and promote the acceptance of risk assessment including the new methodologies used in it. At the same time, efforts should be continued by regulatory agencies to further communicate with consumers about the concept of risk-based assessment as well as the concept of uncertainty.

Advancing human health risk assessment.

The current/traditional human health risk assessment paradigm is challenged by recent scientific and technical advances, and ethical demands. The current approach is considered too resource intensive, is not always reliable, can raise issues of reproducibility, is mostly animal based and does not necessarily provide an understanding of the underlying mechanisms of toxicity. From an ethical and scientific viewpoint, a paradigm shift is required to deliver testing strategies that enable reliable, animal-free hazard and risk assessments, which are based on a mechanistic understanding of chemical toxicity and make use of exposure science and epidemiological data. This shift will require a new philosophy, new data, multidisciplinary expertise and more flexible regulations. Re-engineering of available data is also deemed necessary as data should be accessible, readable, interpretable and usable. Dedicated training to build the capacity in terms of expertise is necessary, together with practical resources allocated to education. The dialogue between risk assessors, risk managers, academia and stakeholders should be promoted further to understand scientific and societal needs. Genuine interest in taking risk assessment forward should drive the change and should be supported by flexible funding. This publication builds upon presentations made and discussions held during the break-out session 'Advancing risk assessment science - Human health' at EFSA's third Scientific Conference 'Science, Food and Society' (Parma, Italy, 18-21 September 2018).

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women II: Total estrogenicity calculations accounting for competitive protein and receptor binding and potency.

Evaluating the biological significance of human-relevant exposures to environmental estrogens involves assessing the individual and total estrogenicity of endogenous and exogenous estrogens found in serum, for example from biomonitoring studies. We developed a method for this assessment by integrating approaches for (i) measuring total hormone concentrations by mass spectrometry (Fleck et al., 2018), (ii) calculating hormone bioavailable concentrations in serum and, (iii) solving multiple equilibria between estrogenic ligands and receptors, and (iv) quantitatively describing key elements of estrogen potency. The approach was applied to endogenous (E1, E2, E3, E4), environmental (BPA), and dietary Genistein (GEN), Daidzein (DDZ) estrogens measured in the serum of thirty pregnant women. Fractional receptor occupancy (FRO) based estrogenicity was dominated by E1, E2 and E3 (ER-α, 94.4-99.2% (median: 97.3%), ER-ß, 82.7-97.7% (median: 92.8%), as was the total response (TR), which included ligand specific differences in recruitment of co-activator proteins (RCA). The median FRO for BPA was at least five orders of magnitude lower than E1, E2 and E3, and three orders of magnitude lower than the fetal derived E4 and GEN and DDZ. BPA contributed less than 1/1000th of the normal daily variability in total serum estrogenicity in this cohort of pregnant women.

Acrylamide Production in Autoclaved Rodent Feed.

Sterilization of rodent feed by steam autoclaving is a common practice in many research institutions. Often we only considerthe beneficial effects of this process-the reduction of microbial contamination-and forget that the high temperatures andpressures can have negative effects on diet quality. The purpose of our study was to assess both the physical and chemicalchanges to a standard rodent feed autoclaved at multiple sterilization temperatures and the effects of the treated diets on mice. Pelleted NIH31 rodent feed was autoclaved at 4 sterilization temperatures (230, 250, 260, and 270 °F). Feed pellet hardness and the acrylamide concentrations of the diets were tested and compared with irradiated NIH31 feed. Study diets were fed to mice for 28 d, after which tissue samples were collected for analysis of acrylamide, glycidamide (the active metabolite of acrylamide), and genotoxicity. Both feed pellet hardness and acrylamide concentration increased with increasing sterilization temperatures; however, neither affected feed intake or body weight gain. Plasma acrylamide and glycidamide weresignificantly elevated only in mice fed NIH31 diet autoclaved at 270 °F compared with the irradiated feed, whereas urineacrylamide and glycidamide metabolites were significantly elevated in most autoclaved diets. Liver DNA adducts, whichcorrelate with genotoxicity, were significantly elevated in all autoclaved diets compared with the irradiated diet. Institutionsthat autoclave their animal diets should carefully consider the temperatures necessary to achieve feed sterilization and thetype of studies in which these autoclaved diets are used.

Metabolism and disposition of arsenic species from controlled dosing with sodium arsenite in adult female CD-1 mice. III. Toxicokinetic studies following oral and intravenous administration.

Arsenic is a ubiquitous contaminant, with typical dietary intake below 1 µg/kg bw/d and drinking water exposures up to 50 µg/kg bw/d. Arsenic exposures are associated with human diseases and doses of toxicological concern are similar to typical dietary intake. Metabolism of arsenite to dimethylarsinate (DMAV) by arsenite-3-methyltransferase (As3MT) promotes clearance, but also generates reactive trivalent intermediates that bind extensively to cellular thiols. This study measured pentavalent and trivalent arsenic species in blood and tissues after oral and intravenous administration of arsenite (50 µg/kg bw). After oral administration, the intestine and liver contained elevated levels of AsIII and MMAIII, relative to erythrocytes, lung, and kidney, suggesting incomplete conversion to DMA during first-pass metabolism. However, blood concentrations of the predominant species, DMA, were similar for oral and intravenous dosing. While all tissues examined contained DMAIII, muscle, brain, and plasma had undetectable levels of MMAIII. Tissue levels of arsenic species were similar following intravenous vs. oral administration, except lower in the intestine. The results confirm the role of metabolism in producing fluxes of putatively toxic trivalent arsenic intermediates. Tissue dosimetry suggests that the intestine, liver, lung, and kidney could be more susceptible to effects of bound arsenic, relative to muscle and brain.

Exposure to Arsenite in CD-1 Mice during Juvenile and Adult Stages: Effects on Intestinal Microbiota and Gut-Associated Immune Status.

Intestinal microbiota composition and gut-associated immune response can contribute to the toxicity of arsenic. We investigated the potential toxicity of short-term arsenic exposure on gut microbiome composition, intestinal immune status, microbial arsenic resistance gene, and arsenic metabolic profiles in adult and developmental stages of CD-1 mice. The potential toxicity of arsenite [As(III)] was determined for two life stages: (i) adult animals at 24 or 48 h after single gavage (0.05 mg/kg body weight [b.w.] [low dose], 0.1 mg/kg b.w. [medium dose], and 0.2 mg/kg b.w. [high dose]) and repeated exposure at 1 mg/liter for 8 days and (ii) postnatal day 10 (PND10) and PND21 after single gavage (0.05 mg/kg b.w.). Dose- and time-dependent responses in bacterial recovery/microbial composition were observed in adults after a single gavage. Repeated exposure caused a transient decrease in the recovery of intestinal bacteria, a shift in the bacterial population with abundance of arsenic resistance genes, and evidence for host metabolism of arsenite into less-reactive trivalent methylated species. Arsenic exposure in adult animals induced high levels of CC chemokines and of proinflammatory and anti-inflammatory cytokine secretion in intestine. Arsenic exposure at PND21 resulted in the development of distinct bacterial populations. Results of this study highlight significant changes in the intestinal microbiome and gut-associated immune status during a single or repeated exposure to arsenic in juvenile and adult animals. The data warrant investigation of the long-term effects of oral arsenic exposure on the microbiome and of immune system development and responses.IMPORTANCE Transformation of organic arsenic to toxic inorganic arsenic (iAs) is likely carried out by intestinal bacteria, and iAs may alter the viability of certain microbial populations. This study addressed the impact of arsenic exposure on intestinal microbiota diversity and host gut-associated immune mediators during early development or adulthood using scenarios of acute or repeated doses. During acute arsenic exposure, animals developed defense functions characterized by higher abundances of bacteria that are involved in arsenic resistance or detoxification mechanisms. Arsenite had a negative effect on the abundance of bacterial species that are involved in the conversion of protein to butyrate, which is an alternative energy source in the intestine. The intestinal mucosal immune cytokine profile reflected a mechanism of protection from arsenic toxicity.

Licorice root components mimic estrogens in an object location task but not an object recognition task.

This study investigated the efficacy of components of licorice root to alter performance on two different recognition tasks, a hippocampus-sensitive metric change in object location (MCOL) task and a striatum-sensitive double object recognition (DOR) task. Isoliquiritigenin (ISL), licorice root extract (LRE), and whole licorice root powder (LRP) were assessed. Young adult female rats were ovariectomized (OVX) and exposed to ISL, LRE or LRP at 0.075%, 0.5% or 5% respectively in the diet. An estradiol group was included as a positive control based on our prior findings. Rats were allowed to explore two objects for three 5-min study trials (separated by 3-min intervals) before a fourth 5-min test trial where the objects were moved closer together (MCOL task) or replaced with two new objects (DOR task). Rats typically habituate to the objects across the three study trials. An increase in object exploration time in the test trial suggests the rat detected the change. Estradiol improved MCOL performance and impaired DOR performance, similar to previously shown effects of estradiol and other estrogens, which tend to improve learning and memory on hippocampus-sensitive tasks and impair striatum-sensitive cognition. LRP had no effect on recognition while exposure to ISL and LRE improved MCOL performance. Exposure to ISL, LRE and LRP failed to attenuate DOR, contrary to effects of estradiol shown here and to previous reports in young-adult OVX rats. These findings suggest components of licorice root may prove to be effective therapies targeting memory enhancement without unintended deleterious cognitive effects.

Metabolism and disposition of arsenic species after repeated oral dosing with sodium arsenite in drinking water. II. Measurements in pregnant and fetal CD-1 mice.

Arsenic is ubiquitous in the earth's crust, and human diseases are linked with exposures that are similar to dietary intake estimates. Metabolic methylation of inorganic arsenic facilitates excretion of pentavalent metabolites and decreases acute toxicity; however, tissue binding of trivalent arsenic intermediates is evidence for concomitant metabolic activation. Pregnant and fetal CD-1 mice comprise a key animal model for arsenic carcinogenesis since adult-only exposures have minimal effects. This study evaluated inorganic arsenic and its metabolites in pentavalent and trivalent states in blood and tissues from maternal and fetal CD-1 mice after repeated administration of arsenite through drinking water. After 8 days of exposure, DMA species were ubiquitous in dams and fetuses. Despite the presence of MMAIII in dams, none was observed in any fetal sample. This difference may be important in assessing fetal susceptibility to arsenic toxicity because MMA production has been linked with human disease. Binding of DMAIII in fetal tissues provided evidence for metabolic activation, although the role for such binding in arsenic toxicity is unclear. This study provides links between administered dose, metabolism, and internal exposures from a key animal model of arsenic toxicity to better understand risks from human exposure to environmental arsenic.

Comparative estrogenicity of endogenous, environmental and dietary estrogens in pregnant women I: Serum levels, variability and the basis for urinary biomonitoring of serum estrogenicity.

Biomonitoring of human exposure to estrogens most frequently focuses on environmental and dietary estrogens, and infrequently includes measures of exposure to potent endogenous estrogens present in serum. Pregnancy is a developmentally sensitive period during which "added" serum estrogenicity exceeding normal intra-individual daily variability may be of particular relevance. We made repeated measurements of serum concentrations of estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), daidzein (DDZ), genistein (GEN) and bisphenol A (BPA) in thirty pregnant women using ultra-performance liquid chromatography coupled with tandem mass spectrometry detection (UPLC-MS/MS) and electrospray ionization (ESI). Serum E1, E2, and E3 concentrations varied significantly (coefficients of variation 9-10%) with broad ranges across the cohort: 1.61-85.1 nM, 9.09-69.7 nM, and 1.5-36.3 nM respectively. BPA (undetected, estimated from total exposure), DDZ and GEN concentrations were 1-5 orders of magnitude lower. The 24-h urinary elimination profiles of endogenous estrogens were each strongly correlated with their corresponding serum concentrations (Pearson's Correlation Coefficients of 0.83 (E1), 0.84 (E2) and 0.94 (E3)). A multivariate regression analysis produced equations for estimating serum concentrations of E1, E2, E3, E4, GEN and DDZ from urinary elimination rates and gestation period, an important step towards non-invasive biomonitoring for assessment of "added" estrogenicity during pregnancy.

The effects of the botanical estrogen, isoliquiritigenin on delayed spatial alternation.

Age-related declines in cognitive function can impair working memory, reduce speed of processing, and alter attentional resources. In particular, menopausal women may show an acceleration in the rate of cognitive decline as well as an increased vulnerability to brain diseases as estrogens may play a neuroprotective and neurotrophic role in the brain. To treat menopausal symptoms, many women turn to botanical estrogens that are promoted as a safe and natural alternative to traditional hormone replacement therapy. However, the majority of these compounds have not been systematically evaluated for efficacy and safety. The current study investigated the efficacy of the commercially available botanical estrogenic compound isoliquiritigenin (ISL) to alter performance on an operant working memory task, delayed spatial alternation (DSA). ISL is a compound found in licorice root that has been shown to have a wide range of effects on different biological systems, including estrogenic properties. This botanical is currently being used in over the counter dietary supplements. Middle-aged (12-month old) Long-Evans female rats were ovariectomized and orally dosed with either 0 mg, 6 mg, 12 mg or 24 mg of ISL 60 min before testing on the DSA task. The DSA task required the rat to alternate its responses between two retractable levers in order to earn food rewards. Random delays of 0, 3, 6, 9 or 18 s were imposed between opportunities to press. ISL treatment failed to alter DSA performance. Previous work from our research group has found that estrogenic compounds, including 17ß-estradiol and the botanical estrogen genistein impair performance on the DSA task. The goal of our botanical estrogens research is to find compounds that offer some of the beneficial effects of estrogen supplementation, without the harmful effects. This work suggests that ISL may not carry the cognitive risks associated with most other estrogenic compounds tested to date.

Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

Inorganic arsenic is a human carcinogen associated with several types of cancers, including liver cancer. Inorganic arsenic has been postulated to target stem cells, causing their oncogenic transformation. This is proposed to be one of the key events in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human hepatic HepaRG cells, at progenitor and differentiated states, were continuously treated with a noncytotoxic concentration of 1 µM sodium arsenite (NaAsO2). The HepaRG cells demonstrated active intracellular arsenite metabolism that shared important characteristic with primary human hepatocytes. Treatment of proliferating progenitor-like HepaRG cells with NaAsO2 inhibited their differentiation into mature hepatocyte-like cells, up-regulated genes involved in cell growth, proliferation, and survival, and down-regulated genes involved in cell death. In contrast, treatment of differentiated hepatocyte-like HepaRG cells with NaAsO2 resulted in enhanced cell death of mature hepatocyte-like cells, overexpression of cell death-related genes, and down-regulation of genes in the cell proliferation pathway, while biliary-like cells remained largely unaffected. Mechanistically, the cytotoxic effect of arsenic on mature hepatocyte-like HepaRG cells may be attributed to arsenic-induced dysregulation of cellular iron metabolism. The inhibitory effect of NaAsO2 on the differentiation of progenitor cells, the resistance of biliary-like cells to cell death, and the enhanced cell death of functional hepatocyte-like cells resulted in stem-cell activation. These effects favored the proliferation of liver progenitor cells that can serve as a source of initiation and driving force of arsenic-mediated liver carcinogenesis.