Polystyrene Nano- and Microplastic Particles Induce an Inflammatory Gene Expression Profile in Rat Neural Stem Cell-Derived Astrocytes In Vitro.

Microplastics are considered an emerging environmental pollutant due to their ubiquitous presence in the environment. However, the potential impact of microplastics on human health warrants further research. Recent studies have reported neurobehavioral and neurotoxic effects in marine and rodent models; however, their impact on the underlying cellular physiology in mammals remains unclear. Herein, we exposed neural stem cells and neural stem cell-derived astrocytes, oligodendrocytes, and neurons to various sizes and concentrations of polystyrene nano- and microplastics. We investigated their cellular uptake, impact on cytotoxicity, and alteration of gene expression through transcriptome profiling. The cell type most affected by decreased viability were astrocytes after 7 days of repeated exposure. Transcriptional analysis showed that 1274 genes were differentially expressed in astrocytes exposed to 500 nm microplastics, but only 531 genes were altered in astrocytes exposed to 50 nm nanoplastics. Both canonical pathway and Kyoto Encyclopedia of Genes and Genomes analysis showed that upregulated pathways were involved in neuroinflammation, innate and adaptive immunity, cell migration, proliferation, extracellular matrix remodeling, and cytoskeleton structures. The downregulated pathways were involved in lipid metabolism, specifically fatty acid oxidation and cholesterol metabolism. Our results show that neural stem cell-derived astrocytes repeatedly exposed to nano- and microplastics for 7 days undergo changes that are hallmarks of astrogliosis.

Dietary acrylamide exposure in F344 rats and colon tumor-bearing nude nu/nu mice: Dataset of gene expression of cancer pathway targets and methylation status of tumor suppressor genes in colon mucosae and tumors.

Dietary acrylamide, a thermally induced food contaminant, at a level (2 mg/kg diet) typifying higher occurrence in certain food products - is neither an independent carcinogen nor a tumor promoter in the colon. This is evidenced by our previous studies using the medium-term azoxymethane (AOM)-induced colon tumorigenesis assay in F344 rats and the human colon tumor xenograft model in athymic nude (nu/nu) mice (https://doi.org/10.1371/journal.pone.0073916) [1]. In addition, we found that acrylamide may act as a colon co-carcinogen in association with a known carcinogen (AOM) in F344 rats. Furthermore, exposure to acrylamide at 2 mg/kg in the diet was not associated with any toxicologically relevant changes in clinical biochemistry, hematology, and apical endpoints in healthy rats (exposed only to saline injections) (https://doi.org/10.1016/j.toxrep.2016.08.010) [2]. Here we report data from our previous investigation [1] on gene expression of cancer pathway targets as well as the methylation status of select tumor suppressor genes. Briefly, mRNA and DNA were extracted from (a) colon mucosae and tumors from F344 rats exposed to AOM or saline and (b) athymic nude (nu/nu) mice bearing human colon tumor xenografts, both exposed to dietary acrylamide at concentrations of 0 or 2 mg/kg diet for 20 and 4 weeks, respectively. RT2 Profiler PCR Cancer PathwayFinder Arrays (Qiagen) and EpiTect Methyl II DNA Restriction kits and PCR Assays (Qiagen) were used to detect cancer-relevant gene expression (84 genes representing 9 pathways) and the methylation status of the CpG islands associated with 22 tumor suppressor genes in colon mucosae, tumors and xenografts. Additionally, RT2 Profiler PCR Arrays (Qiagen) for cell cycle regulation, growth factors, inflammatory cytokines and receptors, and inflammatory response and autoimmunity were used to investigate the gene expression (84 genes in each array) of targets involved in these select cellular pathways in the colon mucosae from AOM-treated F344 rats.

Hexabromocyclododecane (HBCD): A case study applying tiered testing for human health risk assessment.

Current global efforts are aiming to increase use of mechanistic information in regulatory testing. In tiered testing paradigms, in vitro, in silico, and in vivo studies are employed progressively to identify and classify health hazards, which are then compared against human equivalent doses. We used data from three companion papers on the brominated flame retardant hexabromocyclododecane (HBCD) to conduct a case study on tiered testing. We included ToxCast™ and in vitro-in vivo extrapolation (Tier 1), rat liver transcriptomic (Tier 2), and conventional rat (Tier 3) data. Bioactivity-exposure ratios (BERs) were derived by comparing human administered dose equivalents of the measured effects to Canadian exposure levels. Biological perturbations were highly aligned between Tiers 1/2, and consistent with apical effects in Tier 3. Tier 1 had the smallest BERs, and Tiers 2/3 were similar. The study demonstrates the promise of using physiologically-based pharmacokinetic modeling and mechanistic analyses in a tiered framework to identify pathways through which chemicals exert toxicological effects; however, they also point to some shortcomings associated with in vitro and in silico approaches. Additional case studies of chemicals from multiple classes are required to define optimal tiered screening procedures to reduce future in vivo requirements in health hazard assessments.

Hepatic transcriptional dose-response analysis of male and female Fischer rats exposed to hexabromocyclododecane.

Hexabromocyclododecane (HBCD) is a brominated flame retardant found in the environment and human tissues. The toxicological effects of HBCD exposure are not clearly understood. We employed whole-genome RNA-sequencing on liver samples from male and female Fischer rats exposed to 0, 250, 1250, and 5000 mg technical mixture of HBCD/kg diet for 28 days to gain further insight into HBCD toxicity. HBCD altered 428 and 250 gene transcripts in males and females, respectively, which were involved in metabolism of xenobiotics, oxidative stress, immune response, metabolism of glucose and lipids, circadian regulation, cell cycle, fibrotic activity, and hormonal balance. Signature analysis supported that HBCD operates through the constitutive androstane and pregnane X receptors. The median transcriptomic benchmark dose (BMD) for the lowest statistically significant pathway was within 1.5-fold of the BMD for increased liver weight, while the BMD for the lowest pathway with at least three modeled genes (minimum 5% of pathway) was similar to the lowest apical endpoint BMD. The results show how transcriptional analyses can inform mechanisms underlying chemical toxicity and the doses at which potentially adverse effects occur. This experiment is part of a larger study exploring the use of toxicogenomics and high-throughput screening for human health risk assessment.

A reproductive and developmental screening study of the fungal toxin ochratoxin A in Fischer rats.

The presence of the mycotoxin ochratoxin A (OTA) in cereal grains is due to the growth of toxigenic Penicillium mold on stored crops. Human exposure to OTA is higher in infants, toddlers, and children than in adolescents and adults, based on exposure assessments of ng OTA consumed/kg body weight/day. Ochratoxin A is nephrotoxic and teratogenic in animals, but its effects on juveniles exposed during the reproduction and development period have not been studied. To address this, Fischer rats were exposed to 0, 0.16, 0.4, 1.0, or 2.5 mg OTA/kg diet throughout breeding, gestation, and lactation and its adverse effects were assessed in adult rats and their offspring on postnatal day (PND) 21. There were no effects on implantation but post-implantation fetotoxicity was observed in the 2.5 mg/kg dose group, corresponding to a calculated dose of 167.0 µg/kg bw/day in dams. Adverse effects on body and kidney weights and on clinical parameters indicative of renal toxicity were significant in adult rats exposed to 1.0 mg OTA/kg diet (55.2 and 73.3 µg/kg bw/day in adult males and females, respectively) and in PND21 rats at the 0.4 mg/kg dose (33.9 µg/kg bw/day in dams), suggesting that weanling rats were more sensitive to OTA than adults. Overall, nephrotoxicity was the primary effect of OTA in weanling rats exposed throughout gestation and lactation at sub-fetotoxic concentrations in diet.

In vitro immunomodulation of splenocytes from DO11.10 mice by the food colouring agent amaranth.

The chemical amaranth (AM) is permitted as a colouring agent in a variety of foods. Safety was established based on chronic rodent studies. AM and its metabolite naphthionic acid (NA) can be absorbed through the intestine, exposing circulating immune cells including splenocytes. An AM feeding study in rats demonstrated an increase in blood lymphocytes. Yet, in contrast, AM inhibited the delayed-type hypersensitivity reaction to antigen. DO11.10 mice express a T Cell Receptor specific for ovalbumin323-339 peptide (OVAp) presented by I-Ad MHCII. DO11.10 splenocytes were cultured to evaluate mechanisms by which AM and NA modulate immune cell function in vitro. Exposure to OVAp alone for 72 h induced cell proliferation, and combination with 2 or 20 µg/mL AM increased IFN-γ. Cytotoxicity was evident at higher concentrations of AM (200 and 2000 µg/mL) and NA (2000 µg/mL) in combination with OVAp, as both cell number and cytokine secretion decreased. At 200 µg/mL AM with OVAp, immunotoxicity gene expression was modified and OVAp-specific KJ1-26+ CD28+ cells became enriched. The equivalent dose of NA did not modify those parameters. Using an antigen-specific model in vitro, lower concentrations of AM potentiated pro-inflammatory cytokine production, and higher concentrations of AM and NA demonstrated cytotoxicity.

Recommended approaches in the application of toxicogenomics to derive points of departure for chemical risk assessment.

There is increasing interest in the use of quantitative transcriptomic data to determine benchmark dose (BMD) and estimate a point of departure (POD) for human health risk assessment. Although studies have shown that transcriptional PODs correlate with those derived from apical endpoint changes, there is no consensus on the process used to derive a transcriptional POD. Specifically, the subsets of informative genes that produce BMDs that best approximate the doses at which adverse apical effects occur have not been defined. To determine the best way to select predictive groups of genes, we used published microarray data from dose-response studies on six chemicals in rats exposed orally for 5, 14, 28, and 90 days. We evaluated eight approaches for selecting genes for POD derivation and three previously proposed approaches (the lowest pathway BMD, and the mean and median BMD of all genes). The relationship between transcriptional BMDs derived using these 11 approaches and PODs derived from apical data that might be used in chemical risk assessment was examined. Transcriptional BMD values for all 11 approaches were remarkably aligned with corresponding apical PODs, with the vast majority of toxicogenomics PODs being within tenfold of those derived from apical endpoints. We identified at least four approaches that produce BMDs that are effective estimates of apical PODs across multiple sampling time points. Our results support that a variety of approaches can be used to derive reproducible transcriptional PODs that are consistent with PODs produced from traditional methods for chemical risk assessment.

Toxicologic effects of 28-day dietary exposure to the flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (TBECH) in F344 rats.

The brominated flame retardant TBECH is used as an additive to delay ignition and inhibit fires in construction materials and consumer goods. Trends in human exposure are not clear, although humans may be exposed to TBECH via indoor dust and air. In birds and fish there is some evidence of disruption in endocrine and reproductive parameters due to TBECH. In vitro studies indicate that TBECH is an androgen receptor agonist. In this study rats were exposed to 0, 10, 50, 250, 1250 or 5000mg/kg technical TBECH for 28days in diet, corresponding to 0, 0.9, 4.2, 21.3, 98.0 or 328.9mg TBECH/kg bw/d in males and 0, 0.8, 3.9, 19.4, 91.7 or 321.4mg TBECH/kg bw/d in females. Dose-dependent increases in α- and ß- TBECH were detected in serum, liver and adipose. Rats in the 5000mg/kg group lost weight rapidly and were euthanized after 15-18days. At study termination rats displayed dose-dependent clinical and histopathological changes consistent with mild hepatic and renal inflammation. In male rats, evidence of gender-specific alpha2u-globulin nephropathy was not considered predictive of renal toxicity in humans. Frank immunosuppression or inappropriate immunostimulation were not apparent, nor was there a primary effect of TBECH on adaptive immunity. Some evidence of hormone disruption was observed, including changes in serum testosterone levels in males and changes in serum T3 and T4 levels in females. Apparent increases in thyroid follicular cell hypertrophy and hyperplasia in male and female rats were not statistically significant. Benchmark dose (BMD) modelling indicated that clinical changes indicative of mild nephrotoxicity and increased blood monocyte numbers indicative of inflammation and tissue damage were the most sensitive outcomes of TBECH exposure that could be modelled. Preliminary evidence of hormone disruption supports the need for rodent studies using more sensitive models of growth, development and reproduction.

Immunomodulation by gastrointestinal carbon black nanoparticle exposure in ovalbumin T cell receptor transgenic mice.

Humans could become exposed to carbon black nanoparticles (CBNPs) in consumer products or an occupational setting. In rodent models, acute respiratory, subcutaneous, and direct immune cell exposure to CBNPs has been shown to enhance allergic sensitization to co-administered ovalbumin (OVA) protein from chicken egg. However, little is known about the effects of ingested CBNPs on immunological responses and oral tolerance to food antigens. We hypothesized that ingestion of CBNPs would enhance the development of food allergy to OVA. Allergy prone DO11.10 mice were orally exposed to CBNPs every second day for 2 weeks (total dose 10.8 (LOW) or 108 µg (HI)), with and without (±) co-administered OVA. Systemic immune parameters were measured at necropsy. Exposure to OVA resulted in significant increases in serum anti-OVA IgG1, anti-OVA IgM, and anti-OVA IgA antibodies relative to vehicle control. Immunophenotyping revealed a reduction in the number of OVA-specific CD4+ T helper cells upon OVA ± CBNPHI treatment in the spleen. Yet, secretion of the allergy-associated Th2 cytokines IL-4, IL-9, and IL-13 was greater in OVA323-339 peptide-pulsed splenocytes from OVA + CBNPHI-treated mice compared with control. Transcriptome analysis at necropsy of splenocytes from OVA + CBNPHI dose mice compared with OVA mice revealed increases in the allergy associated genes Il4 and Stat6 and decreases in Csf3r and Retnlg. Although oral exposure to high-dose CBNPs did not impact OVA-specific antibody production relative to OVA, we did observe increased expression of genes and cytokines associated with allergy in peripheral splenocytes. This work suggests that CBNP gastrointestinal exposure may potentiate allergy pathways.

Hepatic miRNA profiles and thyroid hormone homeostasis in rats exposed to dietary potassium perfluorooctanesulfonate (PFOS).

Perfluorooctanesulfonate (PFOS) has been widely used in a variety of industrial and commercial applications as a surfactant and stain repellent. PFOS causes liver damage (including liver tumors) in experimental animals, primarily via interaction with PPARα and CAR/PXR. We investigated the involvement of microRNAs (miRNAs) in PFOS-induced hepatotoxicity, and mechanisms involved in abnormal thyroid hormone (TH) homeostasis, in the livers of adult male rats exposed in feed to 50mg PFOS/kg diet for 28 days. PFOS-treated rats exhibited expected histopathological and clinical chemistry changes, and global gene expression changes consistent with the involvement of PPARα and CAR/PXR. Thirty-eight miRNAs were significantly altered. Three members of the miR-200 family were the most increased, while miR-122-5p and miR-21-5p were the most decreased, in PFOS-treated rats. Expression of the miR-23b-3p/27b-3p/24-3p cluster also decreased in PFOS-treated animals. Pathway analysis of miRNAs and associated gene expression changes suggests involvement of epithelial to mesenchymal transition (EMT), which is a primary process of tumor cell motility and cancer metastasis. Our analysis also revealed transcripts that may mediate PFOS-induced effects on TH homeostasis including: activation of the CAR/PXR pathway, phase II/III enzymes, and deiodinase. These changes are consistent with low serum TH due to enhanced metabolic clearance of TH. However, most TH hepatic target genes were not altered in a manner consistent with reduced TH signaling, suggesting that PFOS exposure did not induce functional hypothyroidism. Collectively, the study suggests an important role for miRNAs in PFOS-induced hepatotoxicity and provides insight into the effects of PFOS on TH homeostasis.

Toxicogenomic assessment of liver responses following subchronic exposure to furan in Fischer F344 rats.

Furan is a widely used industrial chemical and a contaminant in heated foods. Chronic furan exposure causes cholangiocarcinoma and hepatocellular tumors in rats at doses of 2 mg/kg bw/day or greater, with gender differences in frequency and severity. The hepatic transcriptional alterations induced by low doses of furan (doses below those previously tested for induction of liver tumors) and the potential mechanisms underlying gender differences are largely unexplored. We used DNA microarrays to examine the global hepatic mRNA and microRNA transcriptional profiles of male and female rats exposed to 0, 0.03, 0.12, 0.5 or 2 mg/kg bw/day furan over 90 days. Marked gender differences in gene expression responses to furan were observed, with many more altered genes in exposed males than females, confirming the increased sensitivity of males even at the low doses. Pathway analysis supported that key events in furan-induced liver tumors in males include gene expression changes related to oxidative stress, apoptosis and inflammatory response, while pathway changes in females were consistent with primarily adaptive responses. Pathway benchmark doses (BMDs) were estimated and compared to relevant apical endpoints. Transcriptional pathway BMDs could only be examined in males. These median BMDs ranged from 0.08 to 1.43 mg/kg bw/day and approximated those derived from traditional histopathology. MiR-34a (a P53 target) was the only microRNA significantly increased at the 2 mg/kg bw/day, providing evidence to support the importance of apoptosis and cell proliferation in furan hepatotoxicity. Overall, this study demonstrates the use of transcriptional profiling to discern mode of action and mechanisms involved in gender differences.

Effects of Chronic Ochratoxin A Exposure on p53 Heterozygous and p53 Homozygous Mice.

Exposure to the mycotoxin ochratoxin A (OTA) causes nephropathy in domestic animals and rodents and renal tumors in rodents and poultry. Humans are exposed to OTA by consuming foods made with contaminated cereal grains and other commodities. Management of human health risks due to OTA exposure depends, in part, on establishing a mode of action (MOA) for OTA carcinogenesis. To further investigate OTA's MOA, p53 heterozygous (p53+/-) and p53 homozygous (p53+/+) mice were exposed to OTA in diet for 26 weeks. The former are susceptible to tumorigenesis upon chronic exposure to genotoxic carcinogens. OTA-induced renal damage but no tumors were observed in either strain, indicating that p53 heterozygosity conferred little additional sensitivity to OTA. Renal changes included dose-dependent increases in cellular proliferation, apoptosis, karyomegaly, and tubular degeneration in proximal tubules, which were consistent with ochratoxicosis. The lowest observed effect level for renal changes in p53+/- and p53+/+ mice was 200 µg OTA/kg bw/day. Based on the lack of tumors and the severity of renal and body weight changes at a maximum tolerated dose, the results were interpreted as suggestive of a primarily nongenotoxic (epigenetic) MOA for OTA carcinogenesis in this mouse model.

Effects of furan on male rat reproduction parameters in a 90-day gavage study.

Furan is produced in foods during processing and preservation techniques that involve heat treatment. Previously, we reported that furan-exposed rats exhibited dose-dependent gross and histological changes in liver which correlated with changes in liver serum enzymes ALT, AST and ALP. Here we report the effects of furan on the male reproductive system. There were no histological or weight changes in the reproductive organs. Serum testosterone levels were increased in a dose-dependent manner whereas serum LH was decreased. There were no changes in 17-OHase, 3ß-HSD and 17ß-HSD activities or serum FSH. Furan did not alter mRNA expression levels for the LH receptor or Tspo but in contrast, mRNA levels of StAR were increased in all doses of furan. The mRNA for the cholesterol side-chain cleavage enzyme (Cyp11a1) was increased by furan at the high dose, as was the level of intratesticular testosterone. We conclude that subchronic furan exposure affects testicular steroidogenesis.

Dietary soy isoflavones increased hepatic protein disulfide isomerase content and suppressed its enzymatic activity in rats.

Protein disulfide isomerase (PDI) is a multifunctional protein and plays important roles in protein folding, triglyceride transfer, insulin degradation, and thyroid hormone transportation. This study examined the modulation of PDI expression by soy consumption using rat as a model. Sprague-Dawley male and female rats at 50 days (d) of age were fed diets containing either 20% casein or alcohol-washed soy protein isolate (SPI, containing 50 mg isoflavones (ISFs)/kg diet) or SPI plus ISF (250 mg/kg diet) and mated at age of 120 d. The offspring (F1) were fed the same diets as their parents. Addition of ISF to SPI diet markedly increased PDI protein content in the liver and testis of the adult rats compared with the casein or SPI diet. PDI mRNA abundance in the liver and protein content in the brain, thyroid, heart, and uterus were unchanged by the diets. Two-dimensional Western blot showed that the rats fed diets containing SPI had a diminished hepatic PDI protein with an isoelectric point (pI) of 6.12, a dephosphorylated form, compared with the rats fed diets containing either casein or SPI with supplemental ISF. Soy ISF added into SPI diet remarkably suppressed hepatic PDI activity of the rats compared with the casein diet. Moreover, soy ISF dose-dependently increased PDI and thyroid hormone receptor (TR) ß protein content, whereas reduced TR DNA binding ability in human hepatocytes. Overall, this study shows that soy ISF increased hepatic PDI protein content, but addition of ISF into SPI diet inhibited its enzymatic activities and this effect may be mediated through a post-transcriptional mechanism.

In vitro enhancement of mouse T helper 2 cell sensitization to ovalbumin allergen by carbon black nanoparticles.

Agglomerated carbon black nanoparticles (CBNPs) administered via respiratory or subcutaneous routes have been shown to promote allergic sensitization to coadministered ovalbumin (OVA) protein in rodents. In the present study, we aimed to model and elucidate the mechanism of this adjuvanticity using an in vitro assay based on T cell sensitization to ovalbumin323₋339 peptide (OVA(p)). CBNP base particles of 22 and 39 nm were characterized and termed CBNP22 and CBNP39 powders. Splenic leukocytes derived from transgenic DO11.10 mice were exposed to suspensions of media alone, concanavalin A mitogen, CBNP agglomerates smaller than 220 nm, OVA(p) alone, OVA(p) + anti-CD28 costimulant, OVA(p) + cyclosporin A immunosuppressant, or OVA(p) + CBNPs. Samples were analyzed at 72 h post-exposure. Proliferation rate, a marker of cellular mitosis, was assessed. Polymerase chain reaction arrays were used to assess genes involved in allergic response pathways. The mitogen control, costimulatory control, and immunosuppressive control chemicals modified the T helper cell proliferation rate. CBNP22 mildly reduced proliferation at 12 µg/ml, but CBNP39 did not. Gene expression analysis of cells treated with OVA(p) showed that coincubation with 12 µg/ml CBNP22 enhanced gene expression of interleukin-4 (IL-4), IL-10, and IL-13, all allergy-associated Th2 cytokines. Coincubation of OVA(p) with 12 µg/ml CBNP39 significantly enhanced IL-13 gene expression concurrent with downregulation of the Th1-associated transcription factor Stat4. IL-4 and IL-13 protein secretion reflected the mRNA trends. The changes were consistently higher in cells exposed to CBNP22 than CBNP39, suggesting that smaller particle size, higher surface area, and higher purity were associated with the direct adjuvant effect on Th2 cells in this genetically susceptible model of OVA allergy.

Hexabromocyclododecane concentrations in Canadian human fetal liver and placental tissues.

Detectable concentrations of the flame retardant hexabromocyclododecane (HBCD) have been reported in human tissues worldwide, but investigations to determine fetal exposure to this brominated flame retardant are lacking. This study was undertaken to determine the concentrations of α-, ß- and γ-HBCD in human tissues (fetal liver and placenta) from Canada. Tissue samples were collected over a thirteen year period following elective pregnancy terminations in Montreal, Quebec, Canada. Samples were extracted using homogenisation with solvent, cleaned up using adsorption chromatography and analysis was performed with liquid chromatography-tandem mass spectrometry. Total HBCD concentrations ranged from below the limit of detection (

Bisphenol A in human placental and fetal liver tissues collected from Greater Montreal area (Quebec) during 1998-2008.

In this study, the presence of bisphenol A (BPA) in human placental and fetal liver samples collected from 1998 to 2008 was investigated to provide a more detailed analysis of the transfer of BPA across the placenta and fetal exposure to BPA. The average concentrations in placental samples were 12.6 ng g(-1) for free BPA, 17.2 ng g(-1) for BPA-glu, and 30.2 ng g(-1) for total BPA. The highest concentrations in placental samples were 165 ng g(-1) for free BPA, 178 ng g(-1) for BPA-glu, and 280 ng g(-1) for total BPA. Samples with higher levels of BPA-glu had higher levels of free BPA in general. Fetal age was observed to have a significant effect on BPA-glu levels in placental samples, but not on free or total BPA. The percentages of free BPA relative to total BPA for the placental samples varied considerably from 4.2% to 100%, suggesting that the ability of maternal liver and/or the placenta to conjugate BPA is highly variable during early to mid-gestation. The average concentrations in fetal liver samples were 9.02 ng g(-1) for free BPA, 19.1 ng g(-1) for BPA-glu, and 25.8 ng g(-1) for total BPA. The highest concentrations in fetal liver samples were 37.7 ng g(-1) for free BPA, 93.9 ng g(-1) for BPA-glu, and 123 ng g(-1) for total BPA. The percentages of free BPA level relative to total BPA for all fetal liver samples varied from 12.4% to 99.1%, indicating extensive variability in the ability of the human feto-placental unit to glucuronidate BPA.

Distribution of isoflavones in samples of serum, liver and mammary glands of rats or pigs fed dietary isoflavones.

BACKGROUND/AIMS: There has been great interest in the potential beneficial and adverse health effects of dietary isoflavones. Determination of tissue concentrations of isoflavone metabolites provides an insight into the potential bioactivity of dietary isoflavones. However, data on the distribution of isoflavones in animal models fed dietary isoflavones are limited. In this study, additional data on the distribution of isoflavones in serum and/or tissues of rats and pigs fed dietary isoflavones were generated. METHODS: Rats (male and female) were fed a casein control diet (containing no isoflavones) and an isoflavone-supplemented diet (containing an alcohol-washed soy protein isolate plus NOVASOY, providing a total of 1,047 mg/kg of total isoflavones). Female pigs were fed a control diet (without soy) containing 17.5 mg/kg of isoflavones, a soy diet containing 582.8 mg/kg of isoflavones or a soy diet supplemented with a daily dose of 2.3 g (equivalent to 42.0 and 14.5 mg/kg of body weight at the onset and end of treatment, respectively) of crystalline genistein. The concentrations of isoflavones in serum and tissues (liver and mammary gland) and in tissues (liver and mammary gland) of pigs were determined via a sensitive and rapid method using liquid chromatography/mass spectrometry. RESULTS: Rats fed the control diet containing no isoflavones had nondetectable levels of isoflavone metabolites in serum, liver and mammary gland samples. Rats fed the isoflavone-supplemented diet had the greatest levels of equol, followed by genistein, daidzein and glycitein, respectively, in their serum, livers and mammary glands. The concentrations of total isoflavones (daidzein, equol and genistein plus glycitein) in serum were significantly (p < 0.05) greater in male rats vs. female rats, but the reverse was true in the case of livers. Concentrations of daidzein, equol, genistein and glycitein were lowest (p < 0.05) in the livers of pigs fed the control diet, and in the mammary glands of female pigs there was only an effect of feeding soy plus genistein on the concentrations of daidzein and equol (p <0.05). CONCLUSIONS: The current data therefore show gender as well as species differences in the tissue distribution of isoflavones.

Sex- and age-specific immunomodulatory effects of dietary soya protein isolate and isoflavones in rats.

The present study examined, using rats as a model, the effects of sex and age of exposure to dietary soya components on serum total and soya-specific antibody content. In Expt 1, Sprague-Dawley rats at 50 d of age were fed diets containing 20 % casein or 20 % alcohol-washed soya protein isolate (SPI) with or without supplemental isoflavones (ISF, 250 mg/kg diet) for 70, 190 or 310 d. The offspring were fed the same diets as their parents. In Expt 2, juvenile Sprague-Dawley rats at 30 d of age were fed diets containing 20 % casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10 or 20 %) for 90 d. Exposure of rats to dietary SPI before the age of 28 d increased serum total IgA and IgM, and induced the production of SPI-specific IgA, IgG, IgM and IgE antibodies. Feeding of juvenile or adult rats with SPI elevated serum total IgA in females, while the opposite occurred in males, and markedly stimulated the production of SPI-specific IgM in females and IgG in males. Our data suggest that the effects of soya proteins and ISF on the production of serum total and SPI-specific antibodies appear to be sex dependent and also related to the age of exposure to soya in rats. However, the physiological significance of these immune responses remains to be determined.

GC-MS analysis of bisphenol A in human placental and fetal liver samples.

A method based on extraction with acetonitrile, followed by solid-phase extraction, derivatization with acetic anhydride, and isotope dilution gas chromatography-mass spectrometry (GC-MS) analysis was applied to determine levels of free and conjugated BPA in human tissues. ß-Glucuronidase was used to de-conjugate the glucuronized BPA in the samples. The method was validated using various animal organ meat samples including pork liver and kidney, beef and calf liver, chicken liver and heart; recoveries were from 85% to 112% at two spiking levels. The average method limit of quantification (LOQ) was estimated at 0.77 ng/g for placenta samples and 1.2 ng/g for fetal liver samples based on 10 times the signal to noise ratio. BPA was detected in all animal tissue samples, with concentrations ranging from 1.8 ng/g in beef and calf livers to 17.1 ng/g in pork kidney. The method was used successfully to determine both free and conjugated BPA levels in human placental and fetal liver tissue samples. BPA was detected in 86% of the placental samples; concentrations of free BPA in the positive samples ranged from 0.60 ng/g to as high as 64 ng/g with an average of 9.5 ng/g and a median of 3.0 ng/g, and conjugated BPA was as high as 7.8 ng/g. BPA was also detected in most of the fetal liver samples (57%); concentrations of free BPA in the positive samples ranged from 1.3 to 27 ng/g with an average of 8.5 ng/g and a median of 3.2 ng/g. Conjugated BPA was also detected in most of the liver samples analysed for total BPA, ranging from 0.64 to 20 ng/g with an average of 3.9 ng/g and a median of 1.5 ng/g. This study, while primarily designed as a method validation, has demonstrated that BPA can be detected in human fetal liver samples as early as the third month of fetal life. Further work will be conducted to validate these preliminary findings.