Search

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

Importance: A centralized repository of clinically applicable facial images with unrestricted use would facilitate facial aesthetic research. Objective: Using a machine learning neural network, we ...

A routine, selective and sensitive ultra-high performance liquid chromatography-electrospray ionisation tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the quantification of 3-monochloropropane-1,2-diol (3-MCPD) in Caco-2 cell transport buffer (FaSSIF-V2, the second version of a fasted state simulated intestinal fluid) and receiving buffer (HBSS, Hank’s balanced salt solution). The method involves measuring deuterated 3-MCPD (3-MCPD-d5) as internal standard (IS) during the entire analytical procedure to obtain precise and accurate results. The separation was performed on a Poroshell 120 HILIC column (2.7 µm, 3.0 × 50 mm) at a flow rate of 0.3 mL/min using water (containing 0.025% acetic acid) and acetonitrile (containing 0.025% acetic acid) as the mobile phases. Mass spectrometric detection was operated in dynamic multiple reaction monitoring (dMRM) in negative ion mode. The method exhibited high sensitivity. The limits of detection (LOD) for 3-MCPD in FaSSIF-V2 and HBSS were 0.012 and 0.014 µmol/L, and the limits of quantification (LOQ) were 0.039 and 0.045 µmol/L, respectively. Satisfactory results were observed for linearity (R2 > 0.999), intra-day precision (RSD% RSD% RSD%

Graphical abstract, Fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD) are chemical contaminants found in a wide range of edible oils that are thermally processed during industrial manufacturing of infant formula and other lipid-containing foods in the United States. Rodent studies have unequivocally demonstrated a plethora of in vivo toxicological effects including reproductive, neurological and renal dysfunction. To determine if similar effects are observed in human organ systems, in vitro studies using human cell lines are conducted to assess concordance of the data collected. One limitation to performing such in vitro research is the extremely high hydrophobicity of 3-MCPD esters; dissolving them into aqueous cell culture media is a tremendous challenge. To address this obstacle, we developed a simple protocol to circumvent the immiscibility of 3-MCPD esters and their corresponding free fatty acids into aqueous cell culture media in order to assess the effect of these esters on epithelial cells of kidney origin in vitro.

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings.

Caco-2 cells are a commonly used model for estimating the intestinal bioavailability of single chemical entity pharmaceuticals. Caco-2 cells, when induced with calcitriol, also express other biological functions such as phase I (CYP) and phase II (glucuronosyltransferases) drug metabolizing enzymes which are relevant to drug-supplement interactions. Intestinal bioavailability is an important factor in the overall safety assessment of products consumed orally. Foods, including herbal dietary supplements, are complex substances with multiple chemical components. Because of potential interactions between components of complex mixtures, more reliable safety assessments can be obtained by studying the commercial products “as consumed” rather than by testing individual chemical components one at a time. The present study evaluated the apparent intestinal permeability (Papp) of a model herbal extract, Rauwolfia serpentina, using both whole plant extracts and the individual purified Rauwolfia alkaloids. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The Papp values for individual Rauwolfia alkaloids were comparable whether measured individually or as components of the complete extract. Both Rauwolfia extract and all individual Rauwolfia alkaloids except yohimbine inhibited CYP3A4 activity (midazolam 1′-hydroxylation). Both Rauwolfia extract and all individual Rauwolfia alkaloids except corynanthine and reserpic acid significantly increased glucuronosyltransferase activity (glucuronidation of 4-methylumbelliferone). The positive control, ketoconazole, significantly inhibited both CYP3A4 and glucuronosyltransferase activities. These findings suggest that the Caco-2 assay is capable of simultaneously identifying both bioavailability and potentially hazardous intestinal drug-supplement interactions in complex mixtures.

Graphical abstract, Highlights • Diglycolic acid (DGA) is an indirect food additive. • DGA was tested for renal cell toxicity in vitro using HK-2 cells. • Evaluation of toxicity included cellular and mitochondrial effects. • In vitro data are highly concordant with in vivo outcomes., Diglycolic acid (DGA) is present in trace amounts in our food supply and is classified as an indirect food additive linked with the primary GRAS food additive carboxymethyl cellulose (CMC). Carboxymethyl starches are used as a filler/binder excipient in dietary supplement tablets and a thickening ingredient in many other processed foods. We sought to utilize the human proximal tubule HK-2 cell line as an in vitro cellular model system to evaluate its acute nephrotoxicity of DGA. We found that DGA was indeed toxic to HK-2 cells in all in vitro assays in our study, including a highly sensitive Luminex assay that measures levels of an in vitro biomarker of kidney-specific toxicity, Kidney Injury Molecule 1 (KIM-1). Interestingly, in vitro KIM-1 levels also correlated with in vivo KIM-1 levels in urine collected from rats treated with DGA by daily oral gavage. The use of in vitro and in vivo models towards understanding the effectiveness of an established in vitro system to predict in vivo outcomes would be particularly useful in rapidly screening compounds that are suspected to be unsafe to consumers. The merit of the HK-2 cell model in predicting human toxicity and accelerating the process of food toxicant screening would be especially important for regulatory purposes. Overall, our study not only revealed the value of HK-2 in vitro cell model for nephrotoxicity evaluation, but also uncovered some of the mechanistic aspects of the human proximal tubule injury that DGA may cause.

Chloropropanols are chemical contaminants that can be formed during industrial processing of foods, such as lipids used in commercially available infant and toddler formula in the USA. Many researchers have studied the most common chloropropanol contaminant, 3-monochloropropane-1,2-diol (3-MCPD), as well as its lipid ester derivatives. A plethora of toxicological outcomes have been described in vivo, including effects on the heart, nervous system, reproductive organs, and kidneys. To better understand the concordance of some of these effects to in vitro outcomes, we focused our research on using an in vitro cellular model to investigate whether the proximal tubule cells of the kidney would be vulnerable to the effects of free 3-MCPD and nine of its common esters in commercial formula. Using the established human kidney proximal tubule cell line, HK-2, we performed 24-h treatments using 3-MCPD and nine mono- or di-esters derived from palmitate, oleate, and linoleate. By directly exposing HK-2 cells at treatment doses ranging from 0 to 100 μM, we could evaluate their effects on cell viability, mitochondrial health, reactive oxygen species (ROS) production, and other endpoints of toxicity. Since chloropropanols reportedly inhibit cellular metabolism through interference with glycolysis, we also tested the extent of this mechanism. Overall, we found mild but statistically significant evidence of cytotoxicity at the highest tested treatment concentrations, which were also associated with mitochondrial dysfunction and transient perturbations in cellular metabolism. Based on these findings, further studies will be required to better understand the effects of these compounds under conditions that are more physiologically relevant to human infant and toddler proximal tubules in order to mimic their exposure to chloropropanol-containing foods.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

Chemical looping combustion (CLC) is an energy conversion technology that can produce concentrated CO2 stream without the need for a gas separation step, and thus, has the potential to drastically reduce the energy consumption and cost associated with CO2 capture in power generation. The coal-direct chemical looping (CDCL) process is a CLC technology that uses a moving bed reducer configuration that can directly consume coal as a feedstock without requiring an upstream gasification step. An integrated 250 kWth CDCL pilot unit using iron-based oxygen carriers was constructed and demonstrated for over 1000 h of testing. The principles for the CDCL pilot unit design and operation are summarized in this article. During the 288-hour continuous operation testing, the CDCL pilot unit achieved >96% coal conversion with a CO2 purity of >97%. Low carbon carryover into the combustor, i.e.

Ethylene is an important industrial feedstock for the generation of commodity chemicals and is increasingly abundant as a by-product of natural gas production. In some cases, the reaction of ethylene can be enhanced by using it to create a gas-expanded liquid with the co-reactants dissolved in an organic solvent. Here, we present a molecular simulation study of the phase behavior, structure, and transport properties of mixtures of ethylene and methanol, in which the ethylene mole fraction is controlled by changing the pressure. For this ethylene-expanded methanol system, we report phase equilibria, volume expansion, liquid structure, translational diffusion constants, and rotational correlation times. The simulation results show excellent agreement with experimental values, where available, and the ethylene and methanol models show promise for use in further studies on related systems.

Adhatoda zeylanica is a dietary supplement ingredient present in several types of dietary supplements, including weight loss, respiratory relief, and immune regulating products. Due to its reported wide range of uses in folk medicine, it was hypothesized that it may have the potential to target multiple organs and lead to a range of toxicity features. As a preliminary evaluation of the safety of this herbal ingredient, an investigation into its effects on the kidney was sought. An in vitro study of its potential nephrotoxicity using the HK-2 human proximal tubule cell line in a variety of functional indicators was performed to capture both general forms of cellular toxicity as well as ones that are specific to proximal tubules. A. zeylanica was only capable of inducing detrimental short-term toxicity to HK-2 cells at relatively high treatment concentrations when exposed directly to the cells. The lack of acute and potent toxicity of A. zeylanica under our experimental conditions calls for further studies to better define its toxicant threshold and establish safe dosage levels. Keywords: Kidney proximal tubule, Adhatoda zeylanica, Nephrotoxicity, HK-2

3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD esters are contaminants present in a variety of processed foods, including infant formulas. Toxicological data are unavailable in humans, but rodent studies have demonstrated renal and testicular toxicity from 3-MCPD and 3-MCPD esters. There is evidence that 3-MCPD esters are hydrolyzed in the digestive system, releasing 3-MCPD that would be absorbed and induce damage. We assessed absorption and metabolism of 3-MCPD and three 3-MCPD monoesters, 1-oleoyl (1-Ol), 1-linoleoyl (1-Li) and 1-palmitoyl (1-Pa) commonly found in U.S. infant formula using differentiated Caco-2 cells. After 1-hour incubation, all three monoesters released free 3-MCPD and free fatty acids (FFA) into Caco-2 cell supernatants. Free 3-MCPD had a high apparent permeability (Papp = 30.36 ± 1.31 cm/s × 10−6) suggesting that it is freely diffusible and highly absorbed by intestinal epithelium. 1-Li released 3–4-fold more 3-MCPD than 1-Ol and 1-Pa over 1 h, suggesting that this variable release rates might contribute to the overall in vivo exposure to 3-MCPD. None of the monoesters or FFA were detected in basolateral supernatants, suggesting that these compounds do not cross the intestinal wall without further transformation. In summary, this study provides relevant data to advance knowledge of in vivo intestinal absorption and metabolism of 3-MCPD monoesters.

Highlights • Psoralea corylifolia, psoralen and isopsoralen are in vitro CYP3A4 inhibitors. • CYP3A4 inhibition could happen at concentrations relevant to in vivo exposures. • Multiple in vitro systems are needed when evaluating CYP3A4 inhibition., Psoralea corylifolia (P. corylifolia) is a medicinal plant used primarily in herbal dietary supplements to treat skin diseases, such as vitiligo and psoriasis. Case reports of liver toxicity have recently emerged from its use, which often includes co-administration with other herbal products. In this study, CYP3A4 inhibition and hepatotoxicity of P. corylifolia and its major components were evaluated in human recombinant CYP3A4 enzyme, differentiated human hepatoma HuH-7 and HepaRG cells. LC/MS-TOF was used to identify the major components of P. corylifolia fruit methanol–water extract. P. corylifolia and its major bioactive components psoralen and isopsoralen were then incubated with human recombinant CYP3A4 (10 min) or differentiated HuH-7 and HepaRG cells (24 h) prior to CYP3A4 activity and cytotoxicity assays. P. corylifolia extract, psoralen, and isopsoralen concentration dependently inhibited CYP3A4 activity with different potency in the three in vitro systems. No cytotoxicity was observed at any concentration tested. In vitro CYP3A4 inhibition by P. corylifolia and its major components suggests potential drug–dietary supplement interactions that warrant further investigations in vivo.

Pharmacometabolomics is a relatively new field that measures an individual’s metabolome in biofluids to detect prognostic and diagnostic biomarkers of drug response and to provide an effective means to predict variation in a subject’s response to drug treatment. Pharmacometabolomics has the potential to help clinicians determine the effectiveness and safety of a drug on an individual basis. To provide information from the current literature in pharmocometabolomics relevant to drug safety including factors besides genetics that can play a role in how a subject responds to a drug treatment. Pharmacometabolomics studies on drug-induced liver toxicity, the use of pharmacometabolomics to detect and predict drug interactions, and future applications of pharmacometabolomics in drug safety are discussed. Pharmacometabolomics can play a role in identifying and/or characterizing toxicity at all stages of drug development. These stages include: pharmacokinetics and ADME; initial toxicity; protective mechanisms; adverse events; late injury; and, injury progression or recovery. Pharmacometabolomics also has the ability to detect endogenous metabolites and markers of other exposure factors including alcohol consumption, impact of the gut microbiome, nutrition, other medications (polypharmacy), dietary supplements, and current individual health-to-disease status, all of which could play a role in patient response to a drug. Pharmacometabolomics alone or in combination with pharmacogenomics can be used to develop customized treatment plans for patients (i.e., personalized medicine) that could significantly reduce adverse events that are sometimes associated with the use of pharmaceuticals.

A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,”Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening ofC. zacatechichifor safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects ofC. zacatechichirelative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement.

The range of non-alcoholic fatty liver disease (NAFLD) includes simple hepatic steatosis, the inflammatory non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. The accumulation of specific lipids in hepatocytes has been associated with oxidative stress and progression of the disease. Elevated serum free fatty acids and hepatocyte lipotoxicity can be studied in an in vitro cellular model. For this purpose, we cultured the human liver cell line, HepG2/C3A, in medium supplemented with increasing amounts of oleic acid (C18:1) and evaluated oxidative stress by measuring the content of the cellular antioxidant, glutathione (GSH). We observed a dose-dependent steatosis, as determined by Nile Red staining, with concurrent increases of GSH; similar findings were also observed in cultured human hepatocytes. Cells cultured with palmitic acid (C16:0) or the combination oleic/palmitic acids (2:1 ratio) also exhibited a dose-dependent increase of GSH; however palmitic-supplemented cultures did not sustain the GSH increase after 24h. We also detected an increase in the formation of lipid peroxides (LPO) indicating that the increase of GSH was a cellular mechanism that may be related to the high exposure of fatty acids. The results of this in vitro study suggest an antioxidant response against fat overloading and indicate potential differences in response to specific fatty acid-induced hepatic steatosis and associated lipotoxicity.

Toxicity assessment of botanical materials is difficult because they are typically complex mixtures of phytochemicals. In the present study, 16 phenolics were tested in both human (HepG2/C3A) and rat (MH1C1) hepatoma cells using a battery of eight toxicity endpoints. Cluster analysis was used to group the phenolics into four clusters for each cell type. Comparison of overall and individual liver activity of phenolics on both human and rat hepatoma cell lines showed significant differences for some endpoints. However, the cluster membership was similar across both cell types with the majority of phenolics clustering with the solvent control group (cluster 1). Each cell type produced a cluster of compounds with reported in vivo liver toxicity (cluster 2). Five herbal extracts were prepared and then tested as above. Using the cluster model developed with the phenolics, in the HepG2/C3A cells green tea was assigned to cluster 2 and the remaining four extracts to cluster 1. In the MH1C1 cells, green tea and thyme were assigned to cluster 2, cinnamon to cluster 4, and juniper berry and peppermint to cluster 1. The data suggest that this in vitro model may be useful for identifying hepatotoxic phenolics and botanical preparations rich in phenolics.

Women are more susceptible than men to acute liver injury from drugs and other xenobiotics. The biological mechanisms for this sex difference are unknown, but known sex differences in steroid hormone levels and immune response could play a role. A human hepatocyte cell line, HepG2, was cultured for 8 days in either a male hormone, female hormone, or sex hormone-free medium. The cells were then exposed to a mixture of pro-inflammatory cytokines (interleukin (IL)-1beta, IL-6, TNFalpha) for 72h to simulate acute inflammation. Cell viability (total DNA) and various metabolic functions (reactive oxygen species (ROS), neutral and polar lipid (PL) accumulation, mitochondrial membrane potential, cytochrome P450 (CYP) activities) were measured fluorometrically. Acute phase proteins (albumin, IL-1ra) were measured in the culture medium by ELISA. This model gave both significant hormone only effects (ROS, PL accumulation) and cytokine only effects (total DNA, CYP1A, neutral and PL accumulation, albumin, IL-1ra) consistent with known biological responses. Significant hormone-cytokine interactions were observed for several endpoints (total DNA, ROS, neutral and PL accumulation, albumin). These findings suggest that sex hormones and pro-inflammatory cytokines can interact to alter liver metabolism in ways that may contribute to the marked sex difference in susceptibility to chemical-induced acute liver injury.

HepG2/C3A human hepatoma cells were exposed to serial concentrations of seven known hepatotoxicants for 48h. Six endpoint assays were selected to model different mechanisms of acute hepatotoxicity. Each compound produced a unique concentration-response pattern across all endpoints. The endpoints did not correlate strongly, suggesting that each endpoint monitored an independent cellular process. Prediction models were developed using five statistical methods. The models used only known hepatotoxicants for the training set. The zero concentration (control) and all concentrations not significantly different from control were programmed as non-toxic levels and concentrations significantly different from control as toxic levels. So, rather than a binary classification of each compound (i.e., toxic or non-toxic), the models gave a prediction of the concentration, if any, at which a compound showed behavior similar to liver toxicants at their toxic concentrations. The discriminant analysis model gave the best overall performance with positive and negative predictive values of 1.00 and 0.83, respectively. Ten additional compounds were tested using this prediction model. The model predicted liver active concentrations for each compound that were consistent with their known biologically active concentrations. This model system may be useful for predicting concentration levels at which unknown compounds would display undesirable liver activity.

A meeting was convened so that users of three models for in vitro developmental toxicity (embryonic stem cells, whole embryo culture, and zebrafish) could share their experiences with each model, and explore the areas for improvement. We present a summary of this meeting and the recommendations of the group.

Deoxynivalenol (DON) is a mycotoxin food contaminant found in several cereal grains. The literature on the liver toxicity of DON in vivo is conflicting and does not clearly characterize its hepatotoxic effects. Cultured rat liver clone-9 cells were used as a model to assess the hepatotoxic potential of DON. The cell cultures, seeded onto 96-well plates, were treated at confluence with varying concentrations of DON (0-100 microg ml(-1)) for 48 h at 37 degrees C in 5% CO2. After the treatment period, the cells were assayed for a number of hepatotoxic endpoints that included cytotoxicity, double-stranded DNA (ds-DNA) content, oxidative stress and mitochondrial function. The concentration-dependent toxicity of DON, as measured by cytotoxicity and ds-DNA content, was observed over the entire concentration range studied beginning at 0.5 microg ml(-1). DON also induced a significant concentration-dependent increase in oxidative stress at DON concentrations starting at 10 microg ml(-1). The mitochondrial function of the treated cells decreased with the increasing concentration of DON exposure, but it was not statistically different from that of the control value. Liver histopathology observed at 3, 24 and 72 h following a single intraperitoneal administration dose of DON (10 mg kg(-1) BW) to adult male rats is consistent with early mild hepatotoxicity. The overall results of this study suggest that acute DON exposure has early mild cytotoxic effects on hepatocytes in vivo that are expressed as severe effects in rat liver clone-9 cells in vitro.

Androstenedione, a naturally occurring steroid hormone, has been used to enhance athletic performance. Little is known, however, about its hepatotoxicity. Clone-9 cells, a non-transformed epithelial cell line that was originally isolated from normal liver of a 4-week old Sprague-Dawley rat, were used as an in vitro model to assess the hepatotoxic potential of androstenedione. The cultures were treated with androstenedione for 24 h at 37 degrees C in 5% CO(2) at concentrations of 0-100 microg ml(-1). After the treatment period, the cells and the culture supernatants were assayed for markers of cytotoxicity which included: release of liver enzymes, cell viability, cellular double-stranded DNA content, oxidative stress, steatosis, cellular ATP content, caspase-3 activity, the mitochondrial permeability transition and induction of cytochrome P450 activity. Significant concentration-dependent differences from control were observed in some endpoints at medium concentrations of 10 microg ml(-1) and above. These in vitro findings were compared with comparable endpoints obtained from an in vivo study of androstenedione toxicity in female Sprague-Dawley rats. Of the eight endpoints that could be compared between the two studies, only three (lipid accumulation, ATP depletion and P450 activity) appeared to be concordant. This suggests that, under the experimental conditions used, the clone-9 cells were not a good model for androstenedione hepatotoxicity.

A human hepatoma cell line (HuH-7) was evaluated as a metabolically competent cell model to investigate cytochrome P450 3A4 (CYP3A4) inhibition, induction, and hepatotoxicity. First, CYP3A4 gene expression and activity were determined in HuH-7 cells under three culture conditions: 1-week culture, 3-week culture, or 1% dimethyl sulfoxide (DMSO) treatment. HuH-7 cells treated with DMSO for 2 weeks after confluence expressed the highest CYP3A4 gene expression and activity compared to the other two culture conditions. Furthermore, CYP3A4 activity in DMSO-treated HuH-7 cells was compared to that in a human hepatoma cell line (HepG2/C3A) and human bipotent progenitor cell line (HepaRG), which yielded the following ranking: HepaRG > DMSO-treated HuH-7 >> HepG2/C3A cells. The effects of three known CYP3A4 inhibitors were evaluated using DMSO-treated HuH-7 cells. CYP3A4 enzyme inhibition in HuH-7 cells was further compared to human recombinant CYP3A4, indicating similar potency for reversible inhibitors (IC50 within 2.5 fold), but different potency for the irreversible inhibitor. Next, induction of CYP3A4 activity was compared between DMSO-treated HuH-7 and HepaRG cells using two known inducers. DMSO-treated HuH-7 cells yielded minimal CYP3A4 induction compared to that in the HepaRG cells after 48-h treatments. Finally, the cytotoxicity of five known hepatotoxicants was evaluated in DMSO-treated HuH-7 cells, HepG2/C3A, and HepaRG cells, and significant differences in cytotoxic sensitivity were observed. Overall, DMSO-treated HuH-7 cells are a valuable model for medium- or high-throughput screening of chemicals for CYP3A4 inhibition and hepatotoxicity.

Androstenedione, a steroidal dietary supplement taken to enhance athletic performance, could affect serum and liver lipid metabolism, induce liver toxicity or alter inflammatory response depending on dose and duration of exposure. Pregnancy could further exaggerate these effects. To examine this, mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Serum was collected and livers were removed from dams on gestation day 20 and from non-pregnant rats after 5 weeks of treatment. Androstenedione had no effect on serum total cholesterol, triglycerides or HDL-cholesterol, but significantly decreased C-reactive protein in pregnant rats and prostaglandin E(2) in serum of both pregnant and non-pregnant rats. There were treatment related decreases in liver ATP and, to a lesser degree, caspase-3 and no change in alkaline phosphatase of pregnant female rats. Androstenedione decreased docosahexaenoic acid in both serum and liver phospholipids of pregnant female rats. In conclusion, oral androstenedione did not result in overt hepatotoxicity in pregnant female rats, but produced modest changes in lipid metabolism and may impair regeneration of injured hepatic cells or tissue.

It is unknown whether androstenedione, a steroidal dietary supplement taken to enhance athletic performance, can affect physiological hormone levels by altering liver enzyme activities that metabolize steroid hormones. Altered hormone levels could be especially devastating during pregnancy. Mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Livers were removed from dams on gestation day 20 and from non-pregnant rats after five weeks' treatment. Liver microsomes were incubated with 200 microM testosterone, and the reaction products were isolated and analyzed by HPLC. In pregnant rats, formation of 6alpha-, 15beta-, 7alpha-, 16beta-, and 2beta-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6beta-hydroxytestosterone increased significantly at both the 30 and 60 mg/kg/day dose levels. In non-pregnant rats, 60 mg/kg/day androstenedione significantly increased formation of 15beta-, 6beta-, 16beta-, and 2beta-hydroxytestosterone. The data suggest that high oral doses of androstenedione can induce some female rat liver cytochromes P450 that metabolize steroid hormones and that the response to androstenedione does not differ between pregnant and non-pregnant female rats.

Androstenedione, a naturally occurring steroid hormone, is a dietary supplement used to enhance athletic performance. Little is known, however, about the safety of its use by young adults including women of child bearing age. To test the possible hepatotoxic effects of androstenedione use, this study was undertaken using a rat model. Pregnant rats (six rats/dose) were exposed to androstenedione in corn oil by gastric intubation at 0, 5, 30 or 60 mg/kg body weight/day beginning 2 weeks before mating and continuing through gestation day 19. On gestation day 20, blood and livers were collected from the pregnant rats for analysis of hepatotoxicity endpoints: serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), glutathione (GSH) and glutathione S-transferase (GST), total microsomal P450, nuclear DNA damage and lipid peroxidation. Under these experimental conditions, no significant differences were observed in any of these biomarkers over the concentration range examined.

The effects of dietary flaxseed (FS), and defatted flaxseed meal (FLM) on serum and tissue fatty acid profiles were investigated. Pregnant Sprague–Dawley rats were fed AIN-93 based diets balanced in calories, fat, nitrogen, and fiber. Diets contained 0, 20%, 40% FS or 13% or 26% FLM by weight. The control, FS and FLM diets differed in linoleic acid to α-linolenic acid (ALA) fatty acid ratio. These diets were fed continuously during gestation, suckling period and 8 weeks post-weaning (F 1 ). FS fatty acids were bioavailable and metabolized by pregnant and F 1 rats. ALA and eicosapentaenoic acid increased; linoleic and arachidonic acid decreased; and docosahexaeonic acid was unchanged in serum, ‘gastric milk’ and liver of FS and FLM-fed pregnant and F 1 rats. FS more than FLM, changed fatty acids profiles, but FLM and 40% FS significantly reduced serum cholesterol. Dietary 40% FS may have increased oxidative stress as evidenced by a reduction in liver vitamin E.

Gestation day 9.5 rat embryos were cultured for 45 h in serum obtained from pregnant rats that had been fed throughout gestation with either a control diet (based on the AIN-93 formulation), a diet supplemented with flaxseed (20% or 40%, w/w), or a diet supplemented with de-fatted flaxseed (“flaxseed meal”, 13 or 26%, w/w). The embryos were fixed in neutral formalin at the end of culture. Overall growth and development was assessed, and the presence of abnormalities was noted. A significant inhibition of growth (as determined by crown-rump length) relative to control was observed in embryos cultured in serum from rats fed the 20% flaxseed diet. The incidence of spontaneous heart inversions was increased significantly in the embryos cultured in serum from the 20% flaxseed and 26% flaxseed meal fed rats. The incidence of flexion defects was increased significantly in embryos cultured in serum from 20% flaxseed-fed rats. The lack of an apparent dose response in any of the statistically significant effects suggests that the observed anomalies were chance occurrences unrelated to the treatment group from which serum was obtained. It is therefore concluded that diets high in flaxseed or flaxseed meal do not result in serum factors that are directly embryotoxic to organogenesis-staged rat embryos. This finding is consistent with the findings of a parallel in vivo rat teratology study where no significant embryotoxicity attributable to flaxseed exposure was observed.

Genistein is a widely consumed phytoestrogen in dietary supplements and has been reported to play roles in both cancer prevention and promotion. These conflicting effects may be complicated by sex differences. Cytochrome P450 1A (CYP1A) participates in carcinogen activation and detoxification, and the enzyme may interact with genistein. Therefore, modulation of CYP1A by a combination of genistein and sex hormones could be responsible for sex differences related to cancer prevention and promotion. In the current study, a human liver cell line, HepG2/C3A, cultured in sex hormone-supplemented media was used to investigate the modulatory effect of genistein on CYP1A gene expression and activity. Genistein exerted both long-term (72 h) induction and short-term (immediate) inhibition of CYP1A activity in HepG2/C3A cells. In the long-term study, CYP1A gene expression and enzyme activity were induced to a greater extent in male hormone-supplemented cells than female ones. In the short-term study, CYP1A activity was inhibited more strongly by genistein in the male hormone-supplemented cells than in the female hormone-supplemented cells. These significant differences suggest that male hormones can modulate the effects of genistein on CYP1A gene expression and activity.

Aminopentol (AP1) is the total hydrolysis product of fumonisin B1 (FB1), the major and best characterized of the fumonisins, which are mycotoxins that are common contaminants of corn and corn meal. Some human populations expected to have significant exposure to AP1 have a high incidence of babies born with neural tube defects (NTD). The embryotoxicity of AP1 was evaluated in cultured rat embryos. Gestation day 9.5 embryos were exposed to 0, 3, 10, 30, 100 or 300 microM AP1 throughout the entire 45-hr culture period. At 100 microM AP1, growth and overall development were reduced significantly. There was also a significant increase in the incidence of abnormal embryos. 29% of the embryos had NTD, and 36% of the embryos had other abnormalities. At 300 microM AP1, the incidence of NTD was 15%, and 85% of the embryos had other abnormalities. These findings suggest that AP1, at concentrations of 100 microM and above, can induce NTD in organogenesis-stage cultured rat embryos. However, these NTD are in conjunction with significant overall retardation of growth and development as well as significant increases in the incidence of other defects. These studies also showed, when compared with previous findings, that AP1 is over 100-fold less toxic than FB1 to cultured rat embryos.

Summary A special session at the Toxicology and Risk Assessment Conference in Cincinnati, OH, USA in May, 2012 presented approaches to expand current uses of in vitro toxicity data for risk assessment. Evaluation of xenobiotics through use of in vitro study methods is increasing exponentially and these methodologies offer a relatively fast and considerably cheaper way to determine toxicities in comparison to traditional animal-based approaches. One of the challenges with in vitro data is to effectively use this information for risk assessment purposes. Currently, in vitro studies are used as supportive information for hazard characterization and to identify mechanisms associated with toxicity. Being able to effectively correlate in vitro effects with in vivo observations represents a major challenge for risk assessors. The presentations in this special session provided innovative approaches toward effectively using in vitro data for the human health risk assessment process.

Driven by economic pressures and government emission regulations, the electric power industry is moving toward tighter control of boilers to improve plant efficiency and reduce emissions. Tighter control depends on better boiler diagnostic tools, especially for discriminating dynamic patterns and correlating those patterns with overall performance. Our research indicated that improved discrimination of dynamic pattern in boilers can be achieved by combining traditional data analysis techniques and chaotic time series analysis. Suggested analysis tools and data acquisition procedures are described, along with example results for measurements from a pressurized fluidized bed and a lowx pulverized coal boiler.

The beneficial or adverse effects of isolated phytochemicals are not always concordant with effects of the botanical dietary supplements from which they were derived. This disparity could be due to interactions between the various phytochemicals present in the whole plant. The phenolic acids, rosmarinic acid (RA), caffeic acid (CA) and ferulic acid (FA) are widely present in foods and dietary supplements, and they are assumed to exert various beneficial biological effects. However, there is little data on the potential biological interactions of these three phenolic acids which commonly occur together and are linked metabolically. In the present study, liver toxicity of the three phenolic acids was assessed on the three compounds singly and in various binary and one ternary combinations. A series of in vitro endpoints relevant to liver toxicity were evaluated in both a human (HepG2/C3A) and rat (MH1C1) hepatocyte cell line. The Combination Index (CI) was calculated for each endpoint from both the concentration responses of the single compounds and the responses of the various binary and ternary mixtures. Both synergistic and antagonistic interactions were observed for some endpoints and some combinations of test agents. Interactions were most prevalent in measures of oxidative stress and cytochrome P450 activities in both cell types. There was only a 53% concordance between the rat and human cells which may be suggestive of species differences. The data suggest an approach for better characterizing the beneficial or adverse effects of complex botanical products through evaluation of interactions between individual phytochemical components.

The cause of habitual early pregnancy loss is not known for most affected couples. It has been proposed that a deficiency of amino acids or other nutrients may contribute to early embryo loss, and an assay based on culture of rat embryos in human serum has been proposed to evaluate women with poor reproductive histories. We tested this assay in women with unexplained infertility (n = 27), habitual abortion (n = 15), and normal midtrimester pregnancies (n = 10) by examining the ability of subject's serum to support the normal development of rat embryos in culture with and without supplemental vitamins and amino acids. Nonpregnant women with nutrient deficiencies identified in this manner were given oral supplements or placebo and were retested. A similar proportion of women in each group had serum that was unable to support the normal development of rat embryos without supplemental vitamins and amino acids. When oral supplements were used, most sera were able to support normal embryo growth. There were no seroconversions on placebo. In spite of the apparent success in producing seroconversions on oral supplementation, only two women conceived, one on the placebo treatment and one on nutritional supplements. Because serum nutrient deficiencies identified by rat embryo culture could not distinguish normal pregnant women from women with unexplained infertility or habitual abortion, and because of the low pregnancy rates, we could not confirm the utility of this assay for the general population of women with habitual abortion.(ABSTRACT TRUNCATED AT 250 WORDS)

Approximately 50% of untreated presomite rat embryos in culture have demonstrated inversions of cardiac looping (laeval instead of dextral) or tail flexure (left-sided instead of right-sided), or both. This spontaneous situs inversus (SI) was not accompanied by growth inhibition or any other observable defects. The incidence of SI was directly related to the stage at dissection, and all heart defects and most flexure defects were eliminated by delaying explantation to the early somite stage. The incidence of SI was not lowered significantly either by removal of endogenous catecholamines from the culture serum by dialysis or by inclusion of alpha- or beta-adrenergic antagonists in the medium. However, the alpha-adrenergic agonist L-phenylephrine (50 micrograms/ml) increased the incidence of SI to 73%. These findings appear to rule out adrenergic mechanisms as a cause of spontaneous SI in cultured, neural plate-staged rat embryos but suggest a mechanism, yet unknown, that is additive with SI induced by alpha-adrenergic agonists. The low incidence of non-SI-related defects suggests that the high incidence of SI is not an artifact of suboptimal culture conditions. The virtual absence of SI in embryos cultured in bovine serum, a medium in which overall embryonic growth and development were retarded, provides further evidence against nonspecific artifacts.

Androstenedione, an anabolic steroid used to enhance athletic performance, was administered in corn oil by gastric intubation once daily in the morning to nonpregnant female rats at a dose of 5 or 60 mg/kg/day, beginning two weeks before mating and continuing through gestation day (GD) 19. On GD 20, the distribution of androstenedione and other steroid metabolites was investigated in the maternal plasma and target organs, including brain and liver. The concentration of estradiol in plasma approached a statistically significant increase after treatment as compared with the controls, whereas the levels of androstenedione, testosterone and progesterone were not significantly different from the controls. In the liver, the concentrations of androstenedione and estradiol only were increased in a dose-related manner. None of these steroids was detectable in the brain. Androstenedione treatment also produced changes in the level of selected free fatty acids (FFAs) in the maternal blood, brain, liver and fetal brain. The concentrations of palmitic acid (16:0) and stearic acid (18:0) in the plasma were not significantly different between the controls and treated rats. However, oleic acid (18:1), linoleic acid (18:2) and docosahexaenoic acid (DHA, 22:6) were 17.94 +/- 2.06 microg/ml, 24.23 +/- 2.42 microg/ml and 4.08 +/- 0.53 microg/ml, respectively, in the controls, and none of these fatty acids was detectable in the treated plasma. On the other hand, palmitic, stearic, oleic, linoleic and DHA were present in both control and treated livers. Among the FFAs in liver, linoleic and DHA were increased 87% and 169%, respectively, over controls. Palmitic, stearic and oleic acids were not significantly affected by the 60 mg/kg treatment. These were present in both control maternal and fetal brains, whereas linoleic acid was found only in fetal brain control. DHA was present only in the control maternal brain (0.02 +/- 0.02 microg/mg protein) and fetal brain (0.24 +/- 0.15 microg/mg protein). The results indicated that androstenedione exhibits significantly different effects on the FFA composition among target organs during pregnancy.

In this paper we review the concept of network centricity, and relate it to the evolution that is anticipated in weather and environmental operational remote sensing in the decade ahead. We provide a practical roadmap for implementing concepts that recognize the value of legacy systems, accounting for a spectrum of different interoperability potentials of legacy and future systems, from the perspective of three communities and five levels of integration.

Flaxseed, a rich source of reportedly beneficial n-3 fatty acid and phytoestrogens, has not been thoroughly tested for reproductive effects. High levels of flaxseed (FS, 20 or 40%) or defatted flaxseed meal (FLM, 13 or 26%) added to AIN-93 diet were evaluated in a two-phase study: dosed during gestation only or during gestation and maturation in a lifetime study. At cesarean section on gestation day 20, neither FS nor FLM affected fertility, body weight gain, litter size, or fetal development. FLM, but not FS, decreased gestation length. The offspring of dams allowed to litter were observed to postnatal day (PND) 21 or 90. Neither FS nor FLM affected PND 21 survival indices of F1 pups. FS (20 and 40%), but not FLM, increased the anogenital index (AGI) of F1 females at PND 21. The AGI of F1 males was not affected by either FS or FLM. FLM (13 and 26%), but not FS, delayed puberty in F1 males. Age and weight at the onset of puberty in females were not affected by FS or FLM. FS and FLM caused dose-related increases in the number of F1 females with irregular estrous cycles. During PND 21-90, F1 females fed 20% FS, 13% FLM, or 26% FLM gained more weight than the controls. FS and FLM decreased thymus/body weight and thymus/brain weight ratios in weanling F1 males and females. FS and FLM decreased liver/body weight and liver/brain weight ratios in weanling F1 females, and 26% FLM decreased the same two ratios in F1 males. In conclusion, FS did not affect fetal development but did affect indices of postnatal development such as the estrous cycle.

Since the initial observations over 25 years ago thatalcohol (ethanol) is a human teratogen (Jones andSmith, ’73; Jones et al., ’73), concerns regarding theprenatal effects of alcohol as a significant public healthproblem have been raised throughout the world. Mucheffort has gone into the delineation of a physical andneurobehavioral phenotype, numerous studies have fo-cused on the birth prevalence of those phenotypes invarious populations, and countless experiments havedocumented the prenatal effects of alcohol in a varietyof animal species. Investigations into the risks of drink-ing small and moderate amounts of alcohol duringpregnancy have been undertaken, education and pre-vention campaigns have been implemented, and pro-grams to benefit affected children have been estab-lished.Although there are numerous gaps in knowledgewhen considering the full spectrum of effects of pre-natal exposure to alcohol, several unresolved prob-lems relating to the diagnosis and prevention of themost severe end of the spectrum, fetal alcohol syn-drome (FAS), are of particular importance. The fol-lowing five points describe these issues and the Pub-lic Affairs Committee’s recommendations in responseto each.

We describe techniques for diagnosing the state of coal-fired utility burners using dynamic characteristics of the output of optical flame scanner signals. The analysis techniques are optimized for targeting dynamical features associated with nonlinear instabilities that develop as burner operating parameters are changed. Various specific instability indicators are used, including shifts in probability distributions, temporal asymmetry and coarsed-grained descriptions of unstable periodicities. We show that careful application of such methods can accurately characterize a range of different flame states. Specifically, transitions through bifurcation points between attached and lifted flames are targeted, giving insight into causes of instability such as stoichiometry or feed and flow variations. We demonstrate results from the application of these methods to utility-scale staged pulverized-coal burners in a real-time software package. The Flame Doctor™ burner-monitoring software is presently undergoing plant-implementation trials in a program sponsored by the Electric Power Research Institute and participating utilities. In a practical application, we show how real-time monitoring and intervention can significantly mitigate adverse combustion conditions.