Nanosilver suppresses growth and induces oxidative damage to DNA in Caenorhabditis elegans.

Studies on the effects of nanomaterial exposure in mammals are limited, and new methods for rapid risk assessment of nanomaterials are urgently required. The utility of Caenorhabditis elegans cultured in axenic liquid media was evaluated as an alternative in vivo model for the purpose of screening nanomaterials for toxic effects. Spherical silver nanoparticles of 10 nm diameter (10nmAg) were used as a test material, and ionic silver from silver acetate as a positive control. Silver uptake and localization, larval growth, morphology and DNA damage were utilized as endpoints for toxicity evaluation. Confocal reflection analysis indicated that 10nmAg localized to the lumen and tissues of the digestive tract of C. elegans. 10nmAg at 10 µg ml(-1) reduced the growth of C. elegans larvae, and induced oxidative damage to DNA as measured by 8-OH guanine levels. Consistent with previously published studies using mammalian models, ionic silver suppressed growth in C. elegans larvae to a greater extent than 10nmAg. Our data suggest that medium-throughput growth screening and DNA damage analysis along with morphology assessments in C. elegans could together provide powerful tools for rapid toxicity screening of nanomaterials.

Utilization of a model hepatotoxic compound, diglycolic acid, to evaluate liver Organ-Chip performance and in vitro to in vivo concordance.

Microphysiological systems (MPS) are emerging as potentially predictive models for drug safety and toxicity assessment. To assess the utility of these systems, the Food and Drug Administration partnered with Emulate to evaluate the Human Liver Organ-Chip in a regulatory setting. Diglycolic acid (DGA), a known hepatotoxin, was evaluated in the Liver-Chip and compared to a multi-well plate format to assess the Liver-Chip's capabilities, limitations, overall performance, and concordance with other in vivo and in vitro studies. Cryopreserved primary human hepatocytes were exposed to DGA from 1 to 20 mM in Liver-Chips or traditional multi-well plates. We found that 10 mM or 20 mM of DGA was severely cytotoxic in both platforms, while 5 mM was mildly cytotoxic in Liver-Chips. Additionally, some hepatocyte functions were reduced with 5 mM DGA in Liver-Chips and 1 mM in well plates. Individual well effects were greater or occurred sooner than in the Liver-Chips. Examination of the performance of the Liver-Chip showed that variability was low for biochemical endpoints, but higher for imaging endpoints. Sensitivity and specificity were high. Only 3-4 Liver-Chips were necessary to detect an effect depending on the endpoint and effect size. The specifics of the experiment are found herein.

C. elegans Development and Activity Test detects mammalian developmental neurotoxins.

Due to the high cost and long duration of traditional testing methods for developmental neurotoxicity (DNT), only a small fraction of chemicals that humans are exposed to have been assessed for DNT activity. In order to ensure public safety, human-predictive methods for DNT detection that are faster and less resource intensive are urgently required. Using Caenorhabditis elegans, a novel worm Development and Activity test (wDAT) has been designed that uses a relatively inexpensive small-animal activity tracker and takes less than 4 days to complete. The wDAT was able to detect both developmental delay and hyperactivity for arsenic, lead, and mercury, heavy metals that are known human developmental neurotoxins and have been associated with hyperactivity in children. Lithium was also tested as a control developmental toxin that is not considered a mammalian neurotoxin. With the wDAT, lithium induced developmental delay but not hyperactivity. This initial assessment of a new assay for DNT detection indicates that the wDAT has potential for detecting at least some types of mammalian developmental neurotoxins. A planned 20-compound validation study will clarify the utility of the wDAT for predicitive toxicology.

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.

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.

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.

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.

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.

Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats.

Deoxynivalenol (DON, vomitoxin), is one of the most common contaminants of cereal grains world-wide. The effects of DON on fetal development were assessed in Charles River Sprague-Dawley rats. Pregnant female rats were gavaged once daily with DON at doses of 0, 0.5, 1, 2.5, or 5 mg/kg body weight on gestation days (GD) 6-19. At cesarean section on GD 20, reproductive and developmental parameters were measured. All females survived to cesarean section. DON caused a dose-related increase in excessive salivation by the pregnant females, a reaction probably linked to the lack of emetic reflex in rats. At 5 mg/kg, feed consumption and mean body weight gain were significantly decreased throughout gestation, mean weight gain (carcass weight), and gravid uterine weight were significantly reduced, 52% of litters (12/23) were totally resorbed, the average number of early and late deaths per litter was significantly increased, average fetal body weight and crown-rump length were significantly decreased, the incidence of runts was significantly increased, and the ossification of fetal sternebrae, centra, dorsal arches, vertebrae, metatarsals, and metacarpals was significantly decreased. At 2.5 mg/kg, DON significantly decreased average fetal body weight, crown-rump length, and vertebral ossification. These effects may be secondary to maternal toxicity and the reduced size of the fetuses. The incidence of misaligned and fused sternebrae was significantly increased at 5.0 mg/kg. No adverse developmental effects were observed at 0.5 and 1.0 mg/kg. Dose-related increases in maternal liver weight-to-body weight ratios were observed in all treated groups (significant at 1, 2.5, and 5 mg/kg). The weight changes were correlated with dose-related cytoplasmic alterations of hepatocytes. The NOEL for maternal toxicity for this study is 0.5 mg/kg based on the dose-related increase in liver-body weight ratio at 1 mg/kg. The NOEL for fetal toxicity is 1 mg/kg based on the general reduction in fetal development at 2.5 and 5 mg/kg. DON is considered a teratogen at 5 mg/kg day in Sprague-Dawley rats based on the anomalous development of the sternebrae.

Effects of zearalenone on in utero development in rats.

Zearalenone (ZE), an estrogenic mycotoxin produced by Fusarium graminearum or F. roseum, is one of the most common contaminants of cereal grains world-wide. The objective of this study was to determine the effects of ZE on in utero development of rats. Pregnant female Charles River Sprague-Dawley rats were gavaged once daily with ZE (in corn oil) at doses of 0, 1, 2, 4, or 8 mg/kg body weight on gestation days (GD) 6-19. All females survived to cesarean section on GD 20. At cesarean section, reproductive and developmental parameters were measured and blood was taken for hormone analysis. Dose-related decreases were seen in maternal feed consumption and body weight gain in all treated groups. Delayed fetal development was linked to maternal toxicity. Fetal body weight was significantly decreased in both sexes in all treated groups. ZE retarded skeletal ossification at 4 and 8 mg/kg. Fetal anogenital index (anogenital distance normalized for body weight) was increased in all treated groups, indicating an androgenic effect of ZE during fetal development. Fetal viability was significantly decreased at 8 mg/kg; significant decreases were observed in number of viable fetuses, and number of litters totally resorbed. At 4 and 8 mg/kg, maternal liver-body weight ratios were significantly increased and organ-brain weight ratios for weights of liver, heart, spleen, kidneys, and ovaries were significantly decreased. Gonadotropins (LH, FSH, and prolactin) and sex steroids (progesterone and estradiol) were analyzed from the blood serum obtained at cesarean section. LH in the 0, 1, 2, and 4 mg/kg groups showed minimal variation, and slightly increased at 8 mg/kg. FSH was decreased in the 1, 2, and 4 mg/kg groups, but the level at 8 mg/kg was slightly higher than the control level. Prolactin level was not affected at 1 mg/kg, slightly increased at 2 and 4 mg/kg, and significantly increased at 8 mg/kg. Progesterone was decreased at 2, 4, and 8 mg/kg and the decreases were significant at 2 and 4 mg/kg. Estradiol level was not affected at 1mg/kg, but dose-related decreases were observed at 2, 4, and 8 mg/kg. Only the 8 mg/kg level of estradiol was significantly decreased. In summary, ZE was maternally toxic and fetotoxic but not teratogenic. The increased anogenital distance observed in male and female fetuses was considered a hormonal change rather than a teratologic response. The increased anogenital distance indicated an androgenic effect. Based on the dose-related maternal and fetal toxicity in all treated groups, the NOEL for reproductive and teratogenic effects was less than 1 mg/kg.

Effects of aminopentol on in utero development in rats.

UNLABELLED: Aminopentol (AP1), the backbone and main hydrolysis product of the mycotoxin fumonisin B1 (FB1), is present in corn-based foods which are consumed daily as a substantial part of the diet in some areas of the world. The toxicity of FB1 has been attributed to altered sphingolipid metabolism, but the toxicity of AP1 is less certain. Epidemiological correlations and in vitro studies have suggested that AP1 can increase neural tube defects (NTDs), but no in vivo developmental study of AP1 was done prior to this study. AP1 was given once daily to rats by gavage on gestation days (GD) 3-16 at doses of 0, 15, 30, 60, or 120 mg/kg. Reproductive and developmental parameters were measured at GD 17, one day after the last dose, and on GD 20. In addition, on GD 17, maternal and fetal tissues were analyzed for sphingolipid content. CONCLUSIONS: AP1 reduced dam body weight gain, but was less toxic than FB1. AP1 was not teratogenic, did not affect tissue sphingolipid ratios, did not alter reproduction or development of fetuses, and produced no dose-related histopathological effects in dams.

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.

Maternal exposure to androstenedione does not induce developmental toxicity in the rat.

Thirty-day old female rats received corn oil or androstenedione (in corn oil) at one of four concentrations (5.0, 10.0, 30.0 or 60.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and until gestation day (GD) 19. Caesarean sections were performed on GD 20. No dose related changes were observed in serum androstenedione, estradiol, LH, FSH, testosterone or progesterone. A statistically significant decrease in estrous cycle length was observed in the 60.0 mg/kg dose group only. Feed and fluid consumption, mean body weight gain, organ weight and fetal parameters were not affected by androstenedione treatment. At the doses given, androstenedione had no specific effect on the development of individual bones or soft tissues.

Characterization of the effect of deoxynivalenol on selected male reproductive endpoints.

The effect of deoxynivalenol (DON) exposure on male reproductive function was assessed in the rat. Male rats were divided into a control group (n=15 rats) and four treatment groups (0.5 mg/kg, n=15; 1.0 mg/kg, n=15; 2.5 mg/kg, n=15; and 5.0 mg/kg DON, n=16) and exposed to DON daily for 28 days via gastric intubation. Both body weight gain and the final body weight of animals in the 5.0 mg/kg dose group and feed consumption in animals in the 2.5 mg/kg and 5.0 mg/kg dose groups were significantly reduced compared to controls. Fluid consumption was not affected in any of the treated groups. Epididymal and seminal vesicle weights expressed per gram of body weight and brain weight were significantly reduced, compared to control weights, in animals from the 2.5 and 5.0 mg/kg dose groups while prostate weight expressed per gram of brain weight and body weight was significantly lower than controls only in the 5.0 mg/kg dose group. A statistically significant, dose-related decrease in homogenization resistant testicular spermatid counts, spermatid numbers, absolute cauda epididymal sperm numbers and cauda epididymal sperm numbers per gram of cauda epididymis was observed in the 5.0 mg/kg DON treatment group. Sperm tail abnormalities (broken tails) in the 5.0 mg/kg dose group were significantly higher than in the control group. Sperm swimming speed (VSL and VCL) was significantly increased only in the 2.5 mg/kg dose group. Serum FSH and LH concentrations were increased in a dose dependent manner across all treated groups while serum testosterone concentrations were decreased in a dose-related manner across all dose groups. An increase in germ cell degeneration, sperm retention and abnormal nuclear morphology was observed in the 2.5 mg/kg and 5.0 mg/kg dose groups. Treatment related effects included lesions in the non-glandular stomach, thymic lymphoid depletion and splenic hematopoiesis in the 5.0 mg/kg treatment group.

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.

Substrate dependent inhibition profiles of fourteen drugs on CYP3A4 activity measured by a high throughput LCMS/MS method with four probe drugs, midazolam, testosterone, nifedipine and terfenadine.

The CYP3A4 enzyme is known for its atypical inhibition kinetics; ligand inhibition can differ depending upon the probe drug used. A high throughput-LCMS/MS CYP3A4 inhibition assay with four substrate drugs was developed to minimize the potential oversight of CYP3A4 inhibition. The assay uses a 96-well format, human liver microsomes, and four CYP3A4 substrate drugs, midazolam, testosterone, nifedipine and terfenadine. After incubation of the individual substrate with human liver microsomes, the reaction is stopped by solid phase extraction and the four probe metabolites produced are pooled and measured by LCMS/MS with multiple-ion-monitoring mode. Using this assay, the IC(50) values of fourteen compounds recognized as substrates/inhibitors of CYP3A4, were measured for the CYP3A4 catalyzed-metabolism of probe drugs. IC(50) values were also obtained for the common set of compounds by the microtiter plate fluorescent assays with cDNA-expressed CYP3A4. Comparison of the results from the two methods suggests that decision making should be cautiously executed to predict drug interaction potential caused by inhibition of CYP3A4 considering the gap between the two assays and various other factors.

Effects of androstenedione on in utero development in rats.

This study was conducted to characterize the effect of androstenedione on estrous cyclicity, mating behavior and fetal development. Thirty-day old rats received corn oil alone or androstenedione (in corn oil) at one of four concentrations (0, 1.0, 5.0, 10.0 or 30.0 mg/kg body weight) by gavage for two weeks prior to mating, during the mating period and throughout gestation. Dose related increases in serum androstenedione, estradiol and estrone were observed in all androstenedione treated animals at gestation day 20. A statistically significant increase in serum testosterone concentration was observed in the 30 mg/kg dose group. Feed and fluid consumption were not affected by androstenedione treatment during the pre-mating or gestational periods, however a statistically significant decrease in the number of females with regular estrous cycles was observed in the 10.0 and 30.0 mg/kg dose groups. Exposure to androstenedione did not affect mean body weight gain during pre-mating or gestation. Slight not statistically significant reductions in the number of implants, number of viable fetuses and number of viable male fetuses were observed in the 30.0 mg/kg androstenedione group. Reductions were not observed in the number of corpora lutea. Fetal growth in terms of fetal weight, crown-rump length, anogenital distance and the number of external abnormalities was not affected by androstenedione exposure. At the doses given, androstenedione had no specific effect on the development of individual bones, including sternebrae. Dose related effects of androstenedione were not observed on the development of soft tissues. A statistically significant increase in moderately enlarged ureter at the kidney was observed in both the 1.0 and 5.0 mg/kg dose groups. Organ weights (expressed per gram of body weight or per gram of brain weight) were not affected by androstenedione treatment.

Effects of flaxseed and defatted flaxseed meal on reproduction and development in rats.

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.

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.

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.