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Anthraquinones exhibit various pharmacological activities (e.g., antioxidant and laxative) and are commonly found in consumer products including foods, dietary supplements, drugs, and traditional medicines. Despite their widespread use, there are limited data available on their toxicokinetic properties. Cytochrome P450 enzymes (CYPs) in the liver play major roles in metabolizing exogenous chemicals (e.g., pharmaceuticals, food ingredients, and environmental pollutants) and endogenous biomolecules (e.g., steroid hormones and cholesterol). Inhibition of CYP activities may lead to serious interactions among these compounds. Here, in silico (quantitative structure-activity relationship modeling) and in vitro (human recombinant enzymes and liver microsomes) methods were used to identify inhibitors of five major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) among 22 anthraquinones. First, in silico prediction and in vitro human recombinant enzyme assays were conducted for all compounds, and results showed that most of the anthraquinones were potent CYP1A2 inhibitors. Second, five selected anthraquinones (emodin, aloe-emodin, rhein, purpurin, and rubiadin) were further evaluated in human liver microsomes. Finally, plasma concentrations of the five anthraquinones in animal and humans were identified in the literature and compared to their in vitro inhibition potency (IC50 values) towards CYP activities. Emodin, rhein, and aloe-emodin inhibited activities of multiple CYPs in human liver microsomes and potential in vivo inhibition may occur due to their high plasma concentrations. These in silico and in vitro results enabled rapid identification of potential CYP inhibitors and prioritized future in-depth studies.

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.

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.

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.

Anthraquinones are found in a variety of consumer products such as dietary supplements, traditional Chinese medicines, and drugs. Along with their widespread use, potential safety concerns have emerged, especially liver toxicity. Therefore, there is a need to conduct rapid and inexpensive safety assessment for anthraquinones due to a lack of animal and human toxicological data. Here, a combined in vitro cytotoxicity and in silico reverse dosimetry approach was adopted to consider the potential human liver toxicity of 16 anthraquinones and derivatives. First, cytotoxicity (EC50) in two human liver cell lines (HepG2/C3A and HuH-7) was measured under two conditions (single and repeated dosing, 72 h). Second, toxic doses (Dtox) required to yield plasma steady-state concentrations (Css) equal to in vitro EC50 values were predicted by reverse dosimetry simulation using a PBPK model. Finally, Dtox was compared to literature-derived estimated daily intake (EDI) of anthraquinones to assess safety. Among the 16 anthraquinones, rhein was identified as a potential hepatotoxicant due to a combination of cytotoxicity, plasma concentration, and daily intake level. These in vitro and in silico findings provide preliminary data and guidance for further animal and clinical studies to confirm liver toxicity of anthraquinones.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12951-017-0265-6) contains supplementary material, which is available to authorized users.

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.

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 60 ppm each of MEL and CYA in feed or via gavage for 28 days. 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.

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 (

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.

Acute toxicity of a single oral dose of sodium arsenite (As), administered in half and half cream (HH), was assessed in male and non-pregnant female rats (0.41, 4.1, 41.0 and 410.0 mg/kg body weight) and pregnant rats (0.41, 4.1 and 41.0 mg/kg body weight). Control rats received deionized water alone, HH alone or 41.0 mg/kg As in deionized water (41 mg/kg As–water). Male and non-pregnant rats were monitored for 14 consecutive days post-dosing. Pregnant rats, dosed on gestation day 10 (GD-10), were monitored until fetuses were collected on GD 20. High mortality (100%) was observed in male and non-pregnant female rats exposed to 410.0 mg/kg As-HH. Low mortality (25%) was observed in non-pregnant female rats exposed to 41 mg/kg As–water. No mortality was observed in other control or treated groups. Reduced female fetal numbers were observed in the 41 mg/kg As–water group but not in the other control groups. Developmental effects were not observed in the controls or the As-HH treatment groups. In conclusion, As toxicity was not reduced when a high dose (410 mg/kg) was administered in HH however, at lower doses (41 mg/kg), HH reduced acute As oral toxicity in the female and developing fetus.

Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment.

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.

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.

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.

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.

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.

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.

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.

Although traditional assessments of renal damage detect loss of kidney function, urinary renal biomarkers are proposed to indicate early changes in renal integrity. The recent adulteration of infant formula and other milk-based foods with melamine revealed a link between melamine ingestion and nephropathy. Thus, the effects of melamine and related analogs (e.g., cyanuric acid) should be assessed in other potentially sensitive groups. We evaluated whether urinary Kim-1, clusterin, and osteopontin could detect the effects of high doses of melamine or cyanuric acid in pregnant and non-pregnant female rats gavaged with 1000 mg/kg bw/day for 10 days. We demonstrate that these biomarkers can differentiate the severity of effects induced by melamine or cyanuric acid. All melamine-treated animals experienced adverse effects; however, pregnant rats were most sensitive as indicated by increased SCr, BUN, and kidney weights, decreased body weight, and presence of renal crystals. These effects coincided with elevated urinary biomarker levels as early as day 2 of exposure. One cyanuric acid-treated rat displayed effects similar to melamine, including increased urinary biomarker levels. This work illustrates that these biomarkers can detect early effects of melamine or cyanuric acid crystal-induced nephropathy and further supports the use of urinary protein immunoassays as a powerful, non-invasive method to assess nephrotoxicity.

Sodium fluoride (NaF) has been used to fluoridate drinking water in the United States since the mid 1940s. Because of the lack of reliable studies on the multigeneration effects of the compound, NaF (0, 25, 100, 175 or 250 ppm in drinking water) was given to rats continuously during three generations. Parental (F0) generation rats were treated for 10 weeks and mated within groups. At gestation day 20, caesarean sections were performed and eight F0 females per group and their litters (F1) were observed for implant status, fetal weight and length, sex and morphological development. The remaining F0 females (29-32 per group) were allowed to litter. F1 offspring (36 of each sex per group) were mated within groups, and caesarean sections were performed at gestation day 20. The F1 females and their litters (F2) were observed for implant status, fetal weight and length, sex and morphological development. In addition, F2 fetuses were evaluated for internal (soft-tissue) and skeletal development. Decreased fluid consumption for F0 and F1 dams at 175 and 250 ppm was attributed to decreased palatability of the solution. No dose-related effects in feed consumption or mean body weight gain were observed in either F0 or F1 females. Numbers of corpora lutea, implants, viable fetuses and fetal morphological development were similar in all groups. No dose-related anomalies in internal organs were observed in F2 fetuses. Ossification of the hyoid bone of F2 fetuses was significantly decreased at 250 ppm. Because of the decreased ossification of the hyoid bone, 250 ppm is considered the effect level.

Fumonisin B1 (FB1), the major mycotoxin from Fusarium moniliforme, has been implicated as a causative agent in several animal and human diseases. Despite animal toxicity studies and human epidemiological studies of FB1, knowledge of its reproductive effects is scarce. In this study, one of a series of proposed studies that will allow extrapolation to humans, pregnant rats were given oral doses of 0, 1.875, 3.75, 7.5 or 15 mg FB1/kg on gestation days 3–16. Caesarean sections were performed on day 17 or 20, and maternal condition, implantation efficiency, foetal viability and foetal development were measured. Dose-related decreases in overall feed consumption and body weight gain were seen, but only the feed consumption decrease at 15 mg/kg, and the decreased body weight gain at 15 mg/kg on days 0–17 were statistically significant. Foetal body weights at day 17 were similar in control and treated groups; but in day-20 foetuses, female weight and crown–rump length were significantly decreased at 15 mg/kg. FB1 was not teratogenic at the doses tested, and no dose-related effects were seen in either skeletal or soft-tissue development. In day-17 animals, maternal and foetal brain, liver and kidney tissues, and maternal serum were preserved to study the levels of sphinganine (Sa), sphingosine (So), and the Sa/So ratios. Dose-related increases were seen in Sa/So ratios in maternal livers, kidneys and serum. Sa/So ratios of maternal brains were not affected, nor were those of foetal kidneys, livers or brains.

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.

The potential of sodium fluoride to affect spermatogenesis in the rat was assessed by intratesticular injection. Experimental rats' left testis was injected with sodium fluoride (50, 175 and 250 ppm) in vehicle (0.9% physiological saline); control testes were injected with vehicle. The right testis served as a non-injected control. Testicular tissues collected ‘at’ and ‘distal to’ the injection site and from the non-injected control testes were evaluated microscopically 24 hr and 1, 2 and 3 wk post-injection. Testicular tissues obtained at and distal to the injection site in all fluoride-injected groups resembled tissues collected from corresponding areas in the controls. Seminiferous tubule damage observed in both the vehicle-injected control testes and the fluoride-injected testes but not in the non-injected testes was attributed to injection trauma. Polymorphonuclear leucocyte infiltration was observed 24 hr post injection only at the injection site in the vehicle- and fluoride-injected groups. Leydig cells were unaffected. Leucocyte infiltration with seminiferous tubule damage was not considered to be a fluoride treatment-related effect because it was observed in both vehicle- and fluoride-injected testes. The results demonstrate that spermatogenesis in the rat is not adversely affected by direct exposure to fluoride at levels 200 times greater than those under normal conditions.

The pyrazolone dye Orange B was given by gavage to pregnant Osborne- Mendel rats throughout gestation. Dose levels of 0, 15, 30, 100, 200, 400, or 700 mglkg body weight were given daily. On gestation day 20, the females were killed and cesarean sections were performed. Feed consumption and maternal weight gain were not affected. No dose-related changes were seen in maternal clinical findings, implantations, fetal viability, or fetal size (weight and length). No compound-related effects were seen in sternebral development. No dose- related effect was seen in the incidence of skeletal variations in fetuses or in the number of litters containing fetuses with skeletal variations. Skeletal development, as measured by the average number of ossified vertebrae, was similar in all groups. No compound-related effects were seen in soft-tissue development.

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.

Despite the chronic exposure of the US population to fluoridated drinking water since the 1940s, existing studies have been judged inadequate to determine any potential reproductive or developmental hazard. This study was conducted to determine the effects of sodium fluoride (NaF) on foetal development. Sperm-positive female rats were given 0, 10, 25, 100, 175 or 250 ppm NaF daily throughout gestation. They were dosed by drinking water to mimic human exposure to fluoridated water. No dose-related behavioural changes or maternal clinical signs were noted. Fluid consumption by females in the 175- and 250-ppm groups was significantly less than that of the control females. Because of this decreased fluid consumption, the daily amount of NaF ingested (0, 1.4, 3.9, 15.6, 24.7 and 25.1 mg/kg body weight) was less than expected at the two high levels. Feed consumption decreased significantly at 250 ppm, and body weights of pregnant females reflected feed consumption trends. The mean number of viable foetuses per female in all treated groups was similar to that of the control group. The significant decrease in the mean number of implants per litter in the 250-ppm group is probably linked to the lower mean number of corpora lutea in this group. The occurrence of in utero deaths was similar in the control and treated groups. Foetal growth (in terms of foetal body weight and crown-rump length) was not affected by NaF, despite the fact that the dams in the 250-ppm group ate significantly less feed and drank significantly less fluid. There was no dose-related increase in the number of external anomalies in foetuses due to NaF ingestion. At the doses given, NaF had no effect on the development of specific bones, including sternebrae. A significant increase was seen in the average number of foetuses with three or more skeletal variations in the 250-ppm group; the number of litters with foetuses with three or more skeletal variations was increased in the 250-ppm group also, but the increase was not significant. There was no dose-related effect of NaF on the incidence of soft tissue variations.

The utility of any model system for toxicity screening depends on the level of correlation between test responses and toxic reactions in humans. Assays in Caenorhabditis elegans can be fast and inexpensive, however few studies have been done comparing toxic responses in this easily cultured nematode with data on mammalian toxicity. Here we report that a screening assay for acute toxicity, using adult C. elegans grown in axenic liquid culture, replicated LD50 toxicity ranking in rat for five metals. This assay utilized the COPAS Biosort and propidium iodide (PI) as a fluorescent indicator of morbidity and mortality after 30-h exposures. We found that chronic toxicity assays of 2-week treatment duration, followed by analysis of PI induced red fluorescence levels, produced less consistent results than the acute assays. However, other chronic toxicity endpoints were compound and concentration specific, including changes in vulval and gonadal morphology, intestinal thickness and integrity, and the presence of retained internal eggs in post-reproductive animals. Some of these endpoints reflect similar findings in mammals, indicating that measurements of morbidity and mortality in conjunction with morphology analyses in C. elegans may have the potential to predict mammalian toxic responses.

Endocrine disrupting chemicals that activate the estrogen receptor are routinely detected in the environment and are a concern for the health of both exposed humans and indigenous wildlife. We exposed the western clawed frog (Xenopus tropicalis) to the weak estrogen octylphenol from Nieuwkoop-Faber (NF) stage 46 tadpoles through adulthood in order to document the effects of a weak estrogen on the life history of an amphibian species. Frogs were exposed to 1, 3.3, 11 and 36 μg/L octylphenol in a continuous flow-through water system. Just prior to completion of metamorphosis (NF 65), a random subsample of froglets was collected and assessed, while the remaining frogs received continued exposure through 31 weeks of exposure when the remaining animals were sampled. Significant induction of the female egg yolk protein precursor vitellogenin was observed in the high treatment at the larval subsampling for both males and females, but not at the final sampling for either sex. No significant deviation from the control sex ratio was observed for either sampling period, suggesting minimal to no effect of octylphenol exposure on gonad differentiation. No effects in the adult frogs were observed for mortality, body mass and size, liver somatic index, estradiol and testosterone serum levels, sperm counts, or oocyte counts. The development and growth of oviducts, a female-specific secondary sex characteristic, was observed in males exposed to octylphenol. These results indicate that octylphenol exposure can induce vitellogenin in immature froglets and the development of oviducts in male adult frogs. The lack of effect observed on the developing gonads suggests that in amphibians, secondary sex characteristics are more susceptible to impact from estrogenic compounds than the developing gonads.

Free fatty acids (FFAs) are known to be markers of cellular membrane degradation through lipid peroxidation and are substrates for the production of reactive oxygen species (ROS). Oxidative stress, due to overproduction of ROS, may facilitate cellular insult by various toxicants. The ability of the rat conceptus to respond to toxic stress may be critical for normal development. In this study, the effects of the environmental toxicant sodium arsenite (NaAsO2) on FFAs were investigated after administering a single oral dose, in water and in a lipid medium, to pregnant rats on gestational day (GD) 10, a time point at mid-organogenesis. NaAsO 2 was administered in deionized water (AsH2O) or in half and half dairy cream (AsHH) at a dose of 41 mg sodium arsenite (NaAsO 2)/kg body weight. Control animals were treated with either dairy cream (HH) or deionized water (H2O). The animals were sacrificed on GD 20. The fetal brain and the maternal liver, brain, plasma and kidneys were harvested. The FFAs were extracted and analyzed by gas chromatography. In the liver, there was an increase of myristic acid (1200%), myristoleic acid (174%), palmitic acid (47%), elaidic acid (456%), oleic acid (165%) and docosahexaenoic acid (224%) in the AsH2O group as compared to the AsHH group. Oleic acid and arachidonic acid were increased by 192% and 900%, respectively, in the AsH2O group as compared to the H 2O group, and myristic acid was decreased by 90% in the AsHH group as compared to the HH group. In the maternal brain, myristoleic acid was decreased by 91% in the AsH2O group as compared to the H2O group, and DHA increased by 148% in the AsHH group as compared to the HH group. In the fetal brain, myristic and stearic acids were decreased by 87% and 89%, respectively, in the AsH2O group as compared to the AsHH group. Myristic, stearic and arachidonic acids were increased by 411%, 265%, and 144%, respectively, in the AsHH group as compared to the HH group. There was no effect on the fatty acids concentrations in the kidney or plasma as compared to controls. This study shows that NaAsO2 produced a differential effect on the fatty acid profiles in rats. Further investigation is needed to elucidate the role of fatty acids in differential signaling and regulation by either the palmitoylation or myristoylation process of cellular functions in these target organs.

BackgroundCholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.Methodology/principal findingsTo determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.Conclusions/significanceOur data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

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 toxin's effect on growth and development in C. elegans.

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 a previous study it was demonstrated that spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult intact rats that lack LH. The studies described herein were designed to examine the extent to which spermatogenesis can be maintained quantitatively with exogenously administered testosterone in adult rats that lack all pituitary hormones. Adult male rats were hypophysectomized and testosterone was administered at the time of hypophysectomy via sustained release polydimethylsiloxane (PDS) capsules of increasing lengths. We used the PDS capsules to clamp testosterone at defined concentrations within the seminiferous tubule fluid over a 2- to 3-month treatment period. Mean testis weights and advanced spermatid numbers per testis stabilized by 8 weeks of testosterone treatment regardless of testosterone dose. Both testis weight and advanced spermatid number responded to testosterone dose, reaching plateaus of 1.2 g and 170 x 10(6) per testis, respectively. These values were 60% of, and significantly less than, the respective control values. This result was in striking contrast to the results of our previous study of LH-suppressed intact rats, in which exogenously administered testosterone resulted in testis weights and advanced spermatid numbers that plateaued at values not significantly different from those in controls. These different effects of testosterone in intact and hypophysectomized rats occurred despite the fact that the seminiferous tubule fluid testosterone concentrations achieved in the hypophysectomized rats (up to 25 ng/ml) were greater than the minimal testosterone concentration found previously to be required to maintain spermatogenesis quantitatively in LH-suppressed intact rats (13 ng/ml). Taken together, these results demonstrate clearly that intratesticular testosterone doses that are as high as or higher than those that maintain spermatogenesis quantitatively in intact rats lacking LH fail to maintain spermatogenesis quantitatively in rats lacking all pituitary hormones.

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.

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.

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.

The Tox21 program calls for transforming toxicology testing from traditional in vivo tests to less expensive and higher throughput in vitro methods. In developmental toxicology, a spectrum of alternative methods including cell line based tests has been developed. In particular, embryonic stem cells (ESCs) have received widespread attention as a promising alternative model for developmental toxicity assessment. Here, we characterized gene expression changes during mouse ESC differentiation and their modulation by developmental toxicants. C57BL/6 ESCs were allowed to differentiate spontaneously and RNA of vehicle controls was collected at 0, 24, 48, 72, 96, 120 and 168 h after embryoid body (EB) formation; RNA of compound-exposed EBs were collected at 24 h. Samples were hybridized to Affymetrix Mouse Gene 2.0 ST Array; using stringent cut-off criteria of Bonferroni-adjusted p2.0, a total of 1996 genes were found differentially expressed among the vehicle controls at different time points. Gene ontology (GO) analysis showed these regulated genes were mostly involved in differentiation-related processes such as development, morphogenesis, metabolism, cell differentiation, cell organization and biogenesis, embryonic development, and reproduction. Biomarkers of all three germ layers or of their derivative early cell types were identified in the gene list. Principal component analysis (PCA) based on these genes showed that the unexposed vehicle controls appeared in chronological order in the PCA plot, and formed a differentiation track when connected. Cultures exposed to thalidomide, monobutyl phthalate, or valproic acid deviated significantly from the differentiation track, manifesting the capacity of the differentiation track to identify the modulating effects of diverse developmental toxicants. The differentiation track defined in this study may be further exploited as a baseline for developmental toxicity testing, with compounds causing significant deviation from the differentiation track being predicted as potential developmental toxicants.

Since the mid 1940s, fluoride has been added to tap water in American communities in an effort to reduce the incidence of dental caries in the population. When the levels of fluoride in drinking water were tested and set, water was the only measurable source of fluoride for most communities. Now, adults and children ingest fluoride with foods and beverages prepared with fluoridated water, and they are exposed to fluoride-containing dental products. As a result, exposure to fluoride is greater than had been anticipated. In the early 1990s, the existing reproductive studies were reviewed in several reports and were considered to be inadequate to determine potential reproductive or developmental hazards. The effects of sodium fluoride ingestion at 0, 25, 100, 175 or 250 ppm in drinking water measured in rats throughout three generations are reported here. Feed and fluid consumption, body weights and clinical signs were recorded at regular intervals. Decreased fluid consumption observed at 175 and 250 ppm was attributed to decreased palatability and did not affect reproduction. No cumulative effects were observed in the three generations. Mating, fertility and survival indices were not affected. Organ-to-body-weight ratios and organ-to-brain weight ratios were not affected. Sodium fluoride up to 250 ppm did not affect reproduction in rats.

Quantitative information was collected on male reproductive effects of maternal and postnatal dietary exposure to flaxseed (20 or 40%), flaxmeal (13 or 26%) or standard NIH AIN-93 feed (0% flaxseed control). Measurements were made on the testes of F1 generation males rats (1) whose mothers were exposed to the diets designated above, and (2) who, after weaning, were placed on the same diet as their mothers for an additional 70 days. The seminiferous tubules comprised 86%, 84%, 84%, 84% and 85% of the total testis volume while the interstitial space comprised 12%, 14%, 14%, 14%, 13% of the total testis volume for the 0% flaxseed/flaxmeal, 20% flaxseed, 13% flaxmeal, 40% flaxseed and 26% flaxmeal groups, respectively. Statistically significant decreases in the absolute volume of the seminiferous tubules were observed in the 20% and 40% flaxseed-treated groups when these groups were compared to controls. Borderline statistically significant differences were also observed when Sertoli cell nucleolar number per tubular cross-section were compared in the 13% flaxmeal and 20% flaxseed treatment groups. These effects were not considered biologically significant because other parameters of male reproductive function appeared normal. Overall, the quantitative information obtained suggests that exposure to flaxseed/flaxmeal at the doses used in the present study does not adversely affect testis structure or spermatogenesis in the rat.

Pregnant Sprague–Dawley rats were exposed to a flaxseed (20 or 40%), flaxmeal (13 or 26%) or standard NIH AIN-93 (0% flaxseed control) diet throughout gestation and until their offspring were weaned. After weaning, F1 generation males were placed in the same diet treatment groups as their mothers for 70 days. Statistically significant differences were not observed between either low-dose or high-dose flaxseed and flaxmeal-treated animals and the 0% flaxseed control animals for testis weights, homogenization resistant spermatid counts, daily sperm production rates, epididymal weights, seminal vesicle weights, seminiferous tubule fluid testosterone concentrations and the percentage of sperm abnormalities. The following statistically significant differences were observed when treated groups and the 0% flaxseed control groups were compared: (1) increases in serum LH in the 20% and 40% flaxseed treatment groups and in serum LH and testosterone in the 26% flaxmeal treatment group; (2) increases in the cauda epididymal weight from the 20% and 40% flaxseed groups; (3) increases in cauda epididymal sperm numbers/g epididymis from the 20% and 40% flaxseed and the 13% and 26% flaxmeal treatment groups; (4) a decrease in prostatic weight from the 20% flaxseed and 13% and 26% flaxmeal treatment groups. Prostate weight in the 40% flaxseed treatment group was lower but not statistically significantly different than the 0% flaxseed control group. Histological effects on spermatogenesis were not observed in either the control group, flaxmeal or the flaxseed treated groups.

The potential of vomitoxin (VT) to affect testicular morphology and testicular and epididymal sperm counts was assessed in three strains of mice: IL-6KO [B6129-IL6 〈tmlKopf〉 (IL-6 gene deficient)], WT [B6129F2 (wild type to B6129-IL6 with an intact IL-6 gene)] and B6C3F 1 mice in a 90-day feeding study. The treated mice received VT at a concentration of 10 ppm in their diet. The body weight of VT-treated animals was significantly reduced compared with control animals. Slight changes, not statistically significant, were observed in relative testis weight and testicular spermatid counts. Histological changes were not apparent in the testes of VT-treated animals. The diameter of the seminiferous tubules, the height of the seminiferous epithelium and the number of Sertoli cell nucleoli per cross-sectioned seminiferous tubule in the VT-treated groups were not significantly different from their respective untreated controls. The IL-6KO and B6C3F 1 VT-treated mice had significantly reduced cauda epididymal weights compared with their respective controls. These changes were not attributed to decreased sperm counts and this finding suggests that VT may exert an adverse affect on the epididymis.

The anatomy of the reproductive tract of the male sand rat, Psammomys obesus , was examined by light microscopy. Histologically, the reproductive tract is similar to other rodent species. Seminiferous tubules in the 1-month-old sand rat do not contain a tubular lumen but Sertoli cells, spermatogonia and spermatocytes are present. A full complement of germ cells is present in the seminiferous tubules by 2.5 months and spermatogenesis is well established. The interstitial space is not well defined until 2.5 months when cell types typical of most rodent species are observed. The epididymis is not noticeably segmented into lobules. An epididymal lumen is not observed until 2.5 months. Cauda epididymal sperm are not observed in the 1 or 2.5-month-old animals and cauda epididymal sperm counts from the 7.5 and 12.5-month-old animals are highly variable. The epididymis, proximal and middle regions of the vas deferens, seminal vesicles and prostate display morphological and histological characteristics similar to other rodent species. The distal end of the vas deferens is not expanded to form an ampulla.

This study provides quantitative information on the effect of sodium fluoride (NaF) on the testes of F 1 generation male rats exposed in utero and during lactation to NaF at one of four concentrations (25, 100, 175, 250 ppm). At weaning, the F 1 generation males were exposed to NaF in their drinking water for 14 weeks, after which time testicular tissues were perfusion-fixed with glutaraldehyde and observed after being embedded in plastic. The seminiferous tubules comprised 89%, 87%, 88%, 88% and 88% of the total testis volume while the interstitial space occupied 9.3%, 11.2%, 10.2%, 9.8% and 9.9% of the total testis volume for the 0, 25, 100,175 and 250 ppm NaF treatment groups, respectively. Statistically significant differences between control and NaF-treated rats were not observed with respect to absolute volume of the seminiferous tubules, interstitial space, Leydig cells, blood vessels boundary layer, lymphatic space, macrophages, tubular lumen or absolute tubular length and absolute tubular surface area, mean Sertoli cell nucleoli number per tubular cross-section, mean seminiferous tubule diameter and the mean height of the seminiferous epithelium. A statistically significant decrease in the absolute volume and volume percent of the lymphatic endothelium was observed in the 175 and 250 ppm NaF-treated groups and in the testicular capsule in the 100 ppm NaF-treated groups. The significance of this finding is unknown at the present time. Overall, the quantitative information obtained suggests that exposure to NaF at the doses used in the present study does not adversely affect testis structure or spermatogenesis in the rat.

The developmental toxicity of purified fumonisin B1 (FB1), a mycotoxin from the common corn fungus Fusarium moniliforme, was examined in Charles River rats. Pregnant rats were dosed orally on gestation days 3-16 at 0, 6.25, 12.5, 25 or 50 mg FB1/kg body weight/day. FB1 was not teratogenic at the doses tested. At 50 mg/kg, maternal toxicity (inappetence, emaciation, lethargy, death, resorption of entire litters) and foetal toxicity (increased number of late deaths, decreased foetal body weight, decreased crown rump length, increased incidence of hydrocephalus, increased incidence of skeletal anomalies) were seen. The foetal toxicity observed at 50 mg/kg may be related to maternal toxicity. Histopathological evaluation of tissues from dams of control and all treated groups revealed dose-related toxic changes in kidney and liver tissues. Acute toxic tubular nephrosis was seen in kidneys from all treated groups. Hepatocellular cytoplasmic alteration and individual cellular necrosis of the liver was seen in the two high-dose groups. Sphinganine (Sa) and sphingosine (So) were measured in day-17 adult and foetal tissues. Dose related increases in Sa/So ratios were seen in maternal liver, kidney, serum and brain, but there was no effect on foetal liver, kidney and brain. These data suggest that FB1 does not cross the placenta and further suggest that the observed foetal toxicity is a secondary response to maternal toxicity.

The potential of sodium fluoride (NaF) to affect spermatogenesis and endocrine function was assessed in P and F1 generation male rats. Male and female experimental rats received sodium fluoride in their drinking water at one of four concentrations (25, 100, 175, 250 ppm). P generation male and female rats were exposed to sodium fluoride in their drinking water for 10 wk and then males were mated to females within the same treatment groups. Reproductive tissues were collected from P generation male rats after approximately 14 wk of treatment. Pregnant females (P) were exposed to sodium fluoride via their drinking water through gestation and lactation. F1 generation weanling male rats remained within the same treatment groups as their parents. F1 generation male rats were exposed to sodium fluoride in their drinking water for 14 wk, at which time reproductive tissues were collected. Dose-related effects were not observed within the P and F1 treatment groups in testis weights, prostate/ seminal vesicle weights, non-reproductive organ weights, testicular spermatid counts, sperm production per gram of testis per day, sperm production per gram of testis, LH, FSH or serum testosterone concentrations. Histological changes were not observed in testicular tissues from either the P or F1 generation. We conclude that prolonged exposure to sodium fluoride in drinking water at the doses administered in this study does not adversely affect spermatogenesis or endocrine function in the P and F1 generation male rats.

The major objective of the studies presented herein was to compare the extent to which exogenously administered testosterone is able to restore spermatogenesis in adult rats made azoospermic by withdrawal of all pituitary hormones (hypophysectomy for 4 weeks) vs. withdrawal of LH alone [testosterone- and estradiol-filled (TE) polydimethylsiloxane implants of 2.5 and 0.1 cm, respectively, for 8 weeks]. In hypophysectornized (Hypox) rats, serum LH and FSH were both undetectable; in the rats that received TE implants, serum LH was undetectable, but FSH was unaffected compared to control values. Seminiferous tubule fluid testosterone concentrations were reduced significantly from their control values of 60-65 to 1.4-1.7 ng/ml in the azoospermic Hypox and TE rats. These rats then received testosterone-filled implants of 4, 12, 18, or 24 cm and were killed 2 months later. In both the Hypox and TE rats, seminiferous tubule fluid testosterone concentrations rose linearly with increasing capsule sizes, and with each of the implant sizes, these concentrations did not differ significantly between the Hypox and TE rats. This made it possible for the first time to examine the effects of comparable intratesticular testosterone concentrations on the numbers of advanced spermatids per testis that could be restored in the azoospermic testes of rats lacking all pituitary factors vs. those lacking only LH. The results that we present demonstrate that the numbers of restored advanced spermatids were consistently and significantly lower in Hypox than in TE rats despite equivalent seminiferous tubule fluid testosterone concentrations. These results provide quantitative conclusive evidence to support the contention that pituitary factors in addition to LH are required for the quantitative restoration of spermatogenesis in the adult rat testis.