Diglycolic acid induces HepG2/C3A liver cell toxicity 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.

Evaluation of the Impact of Varied Carvacrol Concentrations on Salmonella Recovery in Oregano and How Corn Oil Can Minimize the Effect of Carvacrol during Preenrichment.

Phenolic compounds, like carvacrol, in oregano interfere with the detection of foodborne pathogens such as Salmonella enterica. Carvacrol concentration varies based on plant cultivars and growth region. Six oregano cultivars were used to compare the impact of carvacrol concentration on Salmonella and to evaluate the effectiveness of corn oil to help increase Salmonella survival for detection. The results of Agilent 1200 series high-performance liquid chromatography analysis showed that carvacrol concentration in the six oregano cultivars ranged from 64 to 11,200 ppm. Oregano samples were artificially contaminated with S. enterica and were preenriched in Trypticase soy broth with or without 2% (v/v) corn oil. After 18 to 24 h at 37°C, aliquots were transferred to selective enrichment broths. Salmonella was recovered onto xylose lysine Tergitol 4 agar. Six Salmonella serovars were compared, and recovery varied based on carvacrol concentration and serovar. Samples with higher concentrations of carvacrol showed Salmonella recovery only when they were preenriched with corn oil. Based on metagenomic analysis, the microflora associated with the oregano also varied per cultivar. The results show that, as carvacrol levels increased, Salmonella survival decreased. However, the addition of corn oil to the preenrichment broth can minimize the antimicrobial effects of the phenolic compounds, thus allowing for increased detection of Salmonella from oregano cultivars.

In vitro percutaneous penetration of silver nanoparticles in pig and human skin.

In this study, the effects of surface charge, dose, and cosmetic vehicle on the penetration of silver nanoparticles (AgNPs) into pig and human skin were compared. AgNPs (20 nm) with varying surface-charges (polyethylene glycol (PEG; neutral), citrate (CIT; negative), and branched polyethylenimine (bPEI; positive) were dosed onto skin in in vitro diffusion cells using an aqueous solution and an oil-in-water emulsion formulation. Samples were analyzed by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscope (TEM) to assess AgNP skin penetration. The results showed that neutral and positive AgNPs penetrate human skin when applied in a high dose aqueous solution and less with the emulsion vehicle. A mass balance percutaneous penetration study in human skin found the majority of AgNPs were washed from the skin or remained mostly in the stratum corneum (3.4% of the applied dose for AgbPEI and 1.7% for AgPEG). Very little silver was found in the epidermis (1.2% AgbPEI and 0.3% AgPEG) and dermis (0.1% AgbPEI and none detected for AgPEG). These results indicate low dermal penetration of AgNPs that is not greatly affected by surface coating charge. The results will facilitate dermal exposure assessments by better understanding how nanoparticle properties affect skin absorption of nanoparticles found in personal care products.

Comparison of diglycolic acid exposure to human proximal tubule cells in vitro and rat kidneys in vivo.

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.

Toxicity of nano- and ionic silver to embryonic stem cells: a comparative toxicogenomic study.

BACKGROUND: The widespread application of silver nanoparticles (AgNPs) and silver-containing products has raised public safety concerns about their adverse effects on human health and the environment. To date, in vitro toxic effects of AgNPs and ionic silver (Ag+) on many somatic cell types are well established. However, no studies have been conducted hitherto to evaluate their effect on cellular transcriptome in embryonic stem cells (ESCs). RESULTS: The present study characterized transcriptomic changes induced by 5.0 µg/ml AgNPs during spontaneous differentiation of mouse ESCs, and compared them to those induced by Ag+ under identical conditions. After 24 h exposure, 101 differentially expressed genes (DEGs) were identified in AgNP-treated cells, whereas 400 genes responded to Ag+. Despite the large differences in the numbers of DEGs, functional annotation and pathway analysis of the regulated genes revealed overall similarities between AgNPs and Ag+. In both cases, most of the functions and pathways impacted fell into two major categories, embryonic development and metabolism. Nevertheless, a number of canonical pathways related to cancer were found for Ag+ but not for AgNPs. Conversely, it was noted that several members of the heat shock protein and the metallothionein families were upregulated by AgNPs but not Ag+, suggesting specific oxidative stress effect of AgNPs in ESCs. The effects of AgNPs on oxidative stress and downstream apoptosis were subsequently confirmed by flow cytometry analysis. CONCLUSIONS: Taken together, the results presented in the current study demonstrate that both AgNPs and Ag+ caused transcriptomic changes that could potentially exert an adverse effect on development. Although transcriptomic responses to AgNPs and Ag+ were substantially similar, AgNPs exerted specific effects on ESCs due to their nanosized particulate form.

Assessing the effect of oral exposure to Paenibacillus alvei, a potential biocontrol agent, in male, non-pregnant, pregnant animals and the developing rat fetus.

Paenibacillus alvei, a naturally occurring soil microorganism, may be used in the control and/or elimination of human/animal pathogens present on/within produce commodities associated with human consumption. The safety of oral exposure to P. alvei in male, nulliparous females, the pregnant dam and developing fetus was assessed. Adult male and female rats received a single oral dose (gavage) of P. alvei and tissues were collected at post exposure days 0, 3 and 14. To evaluate the effect of the test organism on fetal development, sperm positive female rats received the test organism every 3 days thereafter throughout gestation. As human exposure would be no more than 1 × 103 CFU/ml the following dose levels were evaluated in both study phases: 0 CFU/ml tryptic soy broth (negative control); 1 × 108 CFU/ml; 1 × 104 CFU/ml or 1 × 102 CFU/ml. Neither sex specific dose-related toxic effects (feed or fluid consumption, body weight gain, and histopathology) nor developmental/reproductive effects including the number of implantations, fetal viability, fetal weight, fetal length and effects on ossification centers were observed. The test organism did not cross the placenta and was not found in the amniotic fluid.

28-day repeated dose response study of diglycolic acid: Renal and hepatic effects.

The acute oral toxicity of diglycolic acid (DGA) was evaluated. Groups of female rats (n = 8 rats/group) received 28 consecutive daily single doses of 0.3, 1.0, 3.0, 10.0, 30.0, 100.0 or 300.0 mg DGA/kg body weight by gastric intubation. One group of animals served as vehicle control. Tissues and blood serum were collected at necropsy on day 29. Select organs were weighed and fixed in formalin for histopathological analysis. Animals from the 300 mg/kg bw dose group were removed from the study after 5 consecutive days of treatment as a consequence of adverse treatment related effects. The animals in the remaining treatment groups survived the exposure period. No adverse clinical signs were observed throughout the exposure period in the surviving animals. No significant differences from controls were observed for feed and fluid consumption or body weight gain in the surviving animals. Lesions were observed in the kidneys, liver, stomach, intestine, thymus, spleen and bone marrow in rats from the 300 mg/kg dose group and signs of renal tubular regeneration were observed only in the 100 mg/kg dose group. These results suggest that high levels of pure DGA would need to be consumed before renal and other forms of organ toxicity are observed.

Silver acetate exposure: Effects on reproduction and post natal development.

Effects of oral silver acetate exposure were assessed in P generation and F generation post-natal day 26 rats. Male and female Sprague Dawley rats (n = 20 each) were exposed to silver acetate at 0.4, 4.0 or 40.0 mg/kg bw in their drinking water for 10 weeks prior to and during mating. Females were exposed to silver acetate throughout gestation and lactation. Clinical signs, body weight, feed and fluid consumption were recorded regularly. Decreased mean daily fluid consumption was observed in male and female animals during the 10 week pre mating period and during gestation in the 40 mg/kg bw dose group. Decreased fertility was observed in the 40 mg/kg bw dose group. Decreased feed consumption was observed across all dose groups and decreased mean daily fluid consumption was observed in the 4.0 mg/kg dose group during lactation. Decreased implant numbers, mean numbers of pups born/litter and numbers of live pups born/litter was observed in the 40 mg/kg bw dose group. Pup weight was reduced on lactation days 0, 4 and 7 (males) and 4, 7 and 21 (females) in the 4.0 mg/kg bw dose group and in males at lactation day 21 (40 mg/kg bw dose group). Runting was observed in males (Lactation Day; LD 4) and female (LD 4 and 7) animals in the 4.0 mg/kg bw dose group. Reduced postnatal-day 26 pup weight was observed in male pups in the 40 mg/kg bw dose group and female pups in the 4.0 mg/kg bw dose group.

Effects of maternal silver acetate exposure on immune biomarkers in a rodent model.

Male and female rats (26-day old) were exposed to 0.0, 0.4, 4 or 40 mg/kg body weight silver acetate (AgAc) in drinking water for 10 weeks prior to and during mating. Sperm positive females remained within their dose groups and were exposed to AgAc during gestation and lactation. Splenic and thymic lymphocyte subsets from F1 generation PD (postnatal day) 4 and 26 pups were assessed by flow cytometry for changes in phenotypic markers. Spleens from PD4 pups had lower percentages of CD8+ lymphocytes in 4 and 40 mg/kg AgAc exposed groups and reduced Concanavalin A (Con A) response at all AgAc exposure groups. Splenic maturation increased in PD26 pups compared to PD4 pups. Con A and lipopolysaccharide (LPS) mediated splenic responses were lower in PD26 pups exposed to 40 mg/kg AgAc. Changes in PD 26 pup splenocyte phenotypic markers included lower TCR + cells at 4 and 40 mg/kg AgAc exposure and higher B cell population in the 40 mg/kg AgAc. PD26 pup splenic natural killer cell (NK) activity was higher in the 0.4 AgAc group and unchanged in 4 and 40 mg/kg AgAc groups. In conclusion, maternal exposure to AgAc had a significant impact on rat splenic development during the early lactation period.

Toxicogenomic study in rat thymus of F1 generation offspring following maternal exposure to silver ion.

Male and female rats (26-day-old) were exposed to 0.0, 0.4, 4 or 40 mg/kg body weight silver acetate (AgAc) in drinking water for 10 weeks prior to and during mating. Sperm-positive females remained within their dose groups and were exposed to silver acetate during gestation and lactation. At postnatal day 26, the effect of silver ions on the developing F1 generation rat thymus was evaluated at the transcriptional level using whole-genome microarrays. Gene expression profiling analyses identified a dozen differentially expressed genes (DEGs) in each dose group using a loose criterion of fold change (FC) >1.5 and unadjusted p < 0.05, regardless of whether the analysis was conducted within each gender group or with both gender groups combined. No dose-dependent effect was observed on the number of DEGs. In addition, none of these genes had a false discovery rate (FDR) <0.05 after correction for multiple testing. These results in combination with the observation that thymus-to-body-weight ratios were not affected and no histopathological abnormalities were identified indicate that in utero exposure to silver ions up to 26.0 mg/kg (equivalent to 40.0 mg/kg silver acetate) did not have an adverse effect on the developing thymus.

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.

Bioactivity of nanosilver in Caenorhabditis elegans: Effects of size, coat, and shape.

The in vivo toxicity to eukaryotes of nanosilver (AgNP) spheres and plates in two sizes each was assessed using the simple model organism Caenorhabditis elegans. For each shape, smaller AgNP size correlated with higher toxicity, as indicated by reduced larval growth. Smaller size also correlated with significant increases in silver uptake for silver nanospheres. Citrate coated silver spheres of 20 nm diameter induced an innate immune response that increased or held steady over 24 h, while regulation of genes involved in metal metabolism peaked at 4 h and subsequently decreased. For AgNP spheres, coating altered bioactivity, with a toxicity ranking of polyethylene glycol (PEG) > polyvinylpyrrolidone (PVP) ≅ branched polyethyleneimine (BPEI) > citrate, but silver uptake ranking of PEG > PVP > citrate > BPEI. Our findings in C. elegans correlate well with findings in rodents for AgNP size vs. uptake and toxicity, as well as for induction of immune effectors, while using methods that are faster and far less expensive, supporting the use of C. elegans as an alternative model for early toxicity screening.

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.

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.

Mass spectrometric characterization and activity of zinc-activated proinsulin C-peptide and C-peptide mutants.

Numerous reports have demonstrated an active role for proinsulin C-peptide in ameliorating chronic complications associated with diabetes mellitus. It has been recently reported that some of these activities are dependent upon activation of C-peptide with certain metal ions, such as Fe(II), Cr(III) or Zn(II). In an effort to gain a greater understanding of the structure/function dependence of the peptide-metal interactions responsible for this activity, a series of experiments involving the use of electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) and collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) of C-peptide in the presence or absence of Zn(II) have been carried out. Additionally, various C-peptide mutants with alanine substitution at individual aspartic acid or glutamic acid residues throughout the C-peptide sequence were analyzed. CID-MS/MS of wild type C-peptide in the presence of Zn(II) indicated multiple sites for metal binding, localized at acidic residues within the peptide sequence. Mutations of individual acidic residues did not significantly affect this fragmentation behavior, suggesting that no single acidic residue is critical for binding. However, ESI-MS analysis revealed an approximately 50% decrease in relative Zn(II) binding for each of the mutants compared to the wild type sequence. Furthermore, a significant decrease in activity was observed for each of the Zn(II)-activated mutant peptides compared to the wild type C-peptide, indicated by measurement of ATP released from erythrocytes, with a 75% decrease observed for the Glu27 mutant. Additional studies on the C-terminal pentapeptide of C-peptide EGSLQ, as well as a mutant C-terminal pentapeptide sequence AGSLQ, revealed that substitution of the glutamic acid residue resulted in a complete loss of activity, implicating a central role for Glu27 in Zn(II)-mediated C-peptide activity.

Zinc-activated C-peptide resistance to the type 2 diabetic erythrocyte is associated with hyperglycemia-induced phosphatidylserine externalization and reversed by metformin.

Insulin resistance can broadly be defined as the diminished ability of cells to respond to the action of insulin in transporting glucose from the bloodstream into cells and tissues. Here, we report that erythrocytes (ERYs) obtained from type 2 diabetic rats display an apparent resistance to Zn(2+)-activated C-peptide. Thus, the aims of this study were to demonstrate that Zn(2+)-activated C-peptide exerts potentially beneficial effects on healthy ERYs and that these same effects on type 2 diabetic ERYs are enhanced in the presence of metformin. Incubation of ERYs (obtained from type 2 diabetic BBZDR/Wor-rats) with Zn(2+)-activated C-peptide followed by chemiluminescence measurements of ATP resulted in a 31.2 +/- 4.0% increase in ATP release from these ERYs compared to a 78.4 +/- 4.9% increase from control ERYs. Glucose accumulation in diabetic ERYs, measured by scintillation counting of (14)C-labeled glucose, increased by 35.8 +/- 1.3% in the presence of the Zn(2+)-activated C-peptide, a value significantly lower than results obtained from control ERYs (64.3 +/- 5.1%). When Zn(2+)-activated C-peptide was exogenously added to diabetic ERYs, immunoassays revealed a 32.5 +/- 8.2% increase in C-peptide absorbance compared to a 64.4 +/- 10.3% increase in control ERYs. Phosphatidylserine (PS) externalization and metformin sensitization of Zn(2+)-activated C-peptide were examined spectrofluorometrically by measuring the binding of FITC-labeled annexin to PS. The incubation of diabetic ERYs with metformin prior to the addition of Zn(2+)-activated C-peptide resulted in values that were statistically equivalent to those of controls. Summarily, data obtained here demonstrate an apparent resistance to Zn(2+)-activated C-peptide by the ERY that is corrected by metformin.

Prism-based infrared spectrographs using modern-day detectors.

A comparison of prism-based spectrographs to grating-based spectrographs is made when each of the systems is coupled to a modern-day liquid-nitrogen-cooled photovoltaic array detector. A comparison of the systems is also made using a room-temperature microbolometer array detector. Finally, infrared microspectroscopy of samples whose size is approximately 10 micrometers will be demonstrated using a prism spectrograph outfitted with both types of detectors. The results of the study show that prism-based spectrographs offer an economical alternative to grating-based systems when spectral coverage is more critical than spectral resolution. The results also demonstrate that spectra with good signal-to-noise ratios can be collected on any of the systems with a total integration time of 10 seconds or less.