Martine Ferguson

Analysis of food-allergic and anaphylactic events in the National Electronic Injury Surveillance System

BACKGROUND The National Electronic Injury Surveillance System (NEISS) captures a nationally representative probability sample from hospital emergency departments (EDs) in the United States. OBJECTIVE Emergency department data from NEISS were analyzed to assess the magnitude and severity of adverse events attributable to food allergies. METHODS Emergency department events describing food-related allergic symptomatology were identified from 34 participating EDs from August 1 to September 30, 2003. RESULTS Extrapolation of NEISS event data predicts a total of 20,821 hospital ED visits, 2333 visits for anaphylaxis, and 520 hospitalizations caused by food allergy in the United States during the 2-month study period. The median age was 26 years; 24% of visits involved children < or =5 years old. Shellfish was the most frequently implicated food in persons > or =6 years old, whereas children < or =5 years old experienced more events from eggs, fruit, peanuts, and tree nuts. There were no reported deaths. Review of medical records found that only 19% of patients received epinephrine, and, using criteria established by a 2005 anaphylaxis symposium, 57% of likely anaphylactic events did not have an ED diagnosis of anaphylaxis. CONCLUSION Analysis of NEISS data may be a useful tool for assessing the magnitude and severity of food-allergic events. A criteria-based review of medical records suggests underdiagnosis of anaphylactic events in EDs.

A method to rank order water soluble compounds according to their toxicity using Caenorhabditis elegans, a Complex Object Parametric Analyzer and Sorter, and axenic liquid media

Complex Object Parametric Analyzer and Sorter (COPAS) parameters Time of Flight (TOF) and Extinction (EXT) were utilized to assess growth and development in Caenorhabditis elegans exposed to (in order of decreasing toxicity) sodium arsenite, sodium fluoride, caffeine, valproic acid, sodium borate or DMSO in C. elegans Habitation Medium (CeHM) for 72h. Using multivariate statistical modeling and unique sub sampling procedures mean p-value ratios were calculated for each compound. Comparison of mean p-value ratios and/or the percent change in mean-p value ratios to controls were utilized to assess test compound toxicity. Using this assay 5 of the 6 compounds tested (83.3%) were correctly ranked according to their toxicity based on oral rat LD50 data. Test compounds were ranked from most toxic to least toxic as follows: sodium arsenite, sodium fluoride, sodium borate, valproic acid, caffeine and DMSO. Sodium borate was found to be more toxic than caffeine and valproic acid in this bioassay. This study suggests that axenic liquid culture may be used to expose large numbers of nematodes to water soluble toxicants and the COPAS parameters TOF and EXT may be used as functional biomarkers to assess a toxins effect on growth and development in C. elegans.

In vitro toxicity screening of chemical mixtures using HepG2/C3A cells

Traditional toxicological methods that utilize only single pure compounds may not accurately predict risks from substances with multiple chemical constituents. A complementary approach to conventional methodologies includes in vitro systems that assess toxicity of chemical mixtures and identify components that may adversely impact biological processes. Compared to animal models, in vitro assays are inexpensive, rapid, and reduce and refine related animal testing. We utilized HepG2/C3A cells as a hepatotoxicity screening model to evaluate the cytotoxic and metabolic effects of three commercially available oil dispersants, Corexit EC9500A and EC9527A and ZI-400. The surfactant DOSS, a primary active constituent of the Corexit dispersants, was also evaluated. Biologically relevant endpoints were measured including cell viability, oxidative stress, and mitochondrial activity. Significant increases in cytotoxicity were observed with Corexit dispersants (LC(50)∼250 ppm), whereas ZI-400 was moderately cytotoxic (LC(50) >>400 ppm). Each dispersant caused an accumulation of reactive oxygen species and altered mitochondrial activity and other cellular processes. Generally, DOSS made notable contributions to the effects of EC9500A and EC9527A, however, they were observed at concentrations higher than those used in most consumer products. Overall, this system may represent a valuable complementary tool for predicting the toxicity of complex mixtures.

Multiendpoint mechanistic profiling of hepatotoxicants in HepG2/C3A human hepatoma cells and novel statistical approaches for development of a prediction model for acute hepatotoxicity

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.

Reproductive toxicity in rats with crystal nephropathy following high doses of oral melamine or cyanuric acid.

The industrial chemical melamine was used in 2007 and 2008 to raise the apparent protein content in pet feed and watered down milk, respectively. Because humans may be exposed to melamine via several different routes into the human diet as well as deliberate contamination, this study was designed to characterize the effect of high dose melamine or cyanuric acid oral exposure on the pregnant animal and developing fetus, including placental transfer. Clear rectangular crystals formed following a single triazine exposure which is a different morphology from the golden spherulites caused by combined exposure or the calculi formed when melamine combines with endogenous uric acid. Crystal nephropathy, regardless of cause, induces renal failure which in turn has reproductive sequelae. Specifically, melamine alone-treated dams had increased numbers of early and late fetal deaths compared to controls or cyanuric acid-treated dams. As melamine was found in the amniotic fluid, this study confirms transfer of melamine from mammalian mother to fetus and our study provides evidence that cyanuric acid also appears in the amniotic fluid if mothers are exposed to high doses.

Effects of dietary phenolics and botanical extracts on hepatotoxicity-related endpoints in human and rat hepatoma cells and statistical models for prediction of hepatotoxicity

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.

Timing and route of exposure affects crystal formation in melamine and cyanuric exposed male and female rats: gavage vs. feeding.

Effects of the dosing matrix and timing on the onset of renal crystal formation were evaluated in male and non-pregnant female rats (Fisher 344) exposed to both melamine (MEL) and cyanuric acid (CYA) for 28 days. Rats were fed ground feed containing 60 ppm MEL and 60 ppm CYA, (5 mg/kg bw/day equivalent), or exposed via oral gavage to carboxymethylcellulose containing 5 mg/kg bw MEL followed by 5 mg/kg bw CYA either consecutively (<1 min apart) or delayed 45 min after MEL. Staggered gavage exposure to MEL/CYA caused extensive renal crystal formation as compared to when the two compounds were administered consecutively or in feed. Treatment related effects included reduced weight gain, feed consumption, and testicular weight and increased kidney weight, water consumption and urine output. Animals from the staggered MEL/CYA gavage exposure group became ill and were removed after 9 days of exposure. Approximately 1 week after the initiation of exposure microscopic urinalysis revealed MEL/CYA crystals in both groups of gavaged animals but not in the MEL/CYA feed treatment groups. Urinary crystals were smaller (10 μm) in animals consecutively gavaged. In contrast the urinary crystals were larger (20-40 μm) and frequently clumped in the animals in the staggered gavage group.

Inhibition of monoamine oxidase (MAO) by β-carbolines and their interactions in live neuronal (PC12) and liver (HuH-7 and MH1C1) cells.

Interactions among monoamine oxidase (MAO) inhibitors in drugs, botanicals, and dietary supplements may lead to unpredictable neurochemical dysfunction due to excessive inhibition or therapeutic invalidation. Often recombinant MAO or brain tissue homogenates have been used to evaluate MAO inhibitors such as the β-carboline alkaloids (harmane, harmine, harmaline, and harmalol). However, there is a lack of cellular systems for evaluation of MAO activity, which represents a more advanced in vitro model compared to recombinant enzymes or tissue lysates. Using kynuramine assays, intracellular MAO inhibition by β-carbolines was measured in PC12 (rat pheochromocytoma), MH1C1 (rat liver), and HuH-7 (human liver) cell lines, which were compared with human recombinant MAO and cell lysates. β-Carbolines (1 μM, 90 min incubations) inhibited MAO by 40-99% in live PC12 cells where MAO A was the active isoform, and <12% in HuH-7 and MH1C1 cells where MAO B was primarily active. The combination index (CI), which serves as a quantitative indicator of pharmacological interactions, was determined for harmaline/harmine (CI, 1.01-1.25) and methylene blue/harmine (CI, 0.74-1.07) in PC12 cells. Overall, this study illustrates applications of cell-based in vitro assay platforms to gain a comprehensive understanding of intracellular MAO inhibitors and their interactions.

Use of the Combination Index to determine interactions between plant-derived phenolic acids on hepatotoxicity endpoints in human and rat hepatoma cells.

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.

Performance of urinary and gene expression biomarkers in detecting the nephrotoxic effects of melamine and cyanuric acid following diverse scenarios of co-exposure.

Although standard nephrotoxicity assessments primarily detect impaired renal function, KIM-1, clusterin, NGAL, osteopontin and TIMP-1 were recently identified biomarkers proposed to indicate earlier perturbations in renal integrity. The recent adulteration of infant and pet food with melamine (MEL) and structurally-related compounds revealed that co-ingestion of MEL and cyanuric acid (CYA) could form melamine-cyanurate crystals which obstruct renal tubules and induce acute renal failure. This study concurrently evaluated the ability of multiplexed urinary biomarker immunoassays and biomarker gene expression analysis to detect nephrotoxicity in F344 rats co-administered 60ppm each of MEL and CYA in feed or via gavage for 28days. The biomarkers were also evaluated for the ability to differentiate the effects of the compounds when co-administered using diverse dosing schedules (i.e., consecutive vs. staggered gavage) and dosing matrixes (i.e., feed vs. gavage). Our results illustrate the ability of both methods to detect and differentiate the severity of adverse effects in the staggered and consecutive gavage groups at much lower doses than previously observed in animals co-exposed to the compounds in feed. We also demonstrate that these urinary biomarkers outperform traditional diagnostic methods and represent a powerful, non-invasive indicator of chemical-induced nephrotoxicity prior to the onset of renal dysfunction.