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.

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.

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.

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.

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.

Soy isoflavones modulate azoxymethane-induced rat colon carcinogenesis exposed pre- and postnatally and inhibit growth of DLD-1 human colon adenocarcinoma cells by increasing the expression of estrogen receptor-beta.

We studied the effects of lifetime exposure to dietary soy isoflavones in an azoxymethane (AOM)-induced rat colon cancer model. Male pups born to Sprague-Dawley rats exposed (including during pregnancy and lactation) to soy isoflavones at either no (0 mg = control), low (40 mg), or high (1000 mg) doses/kg diet were weaned and continued receiving their respective parental diets until the end of the study. Weaned rats received 2 subcutaneous injections (15 mg/kg body weight) of AOM 1 wk apart. After 26 wk, rats were killed and the coordinates of colon aberrant crypt foci (ACF) and tumors were determined. Expression of estrogen receptor (ER)-beta was assessed in rat colon tumors and in DLD-1 human colon adenocarcinoma cells exposed to soy isoflavones. Compared with the control, soy isoflavones did not affect incidences or multiplicities of colon ACF or tumors. Low-dose soy isoflavones decreased tumor burden and size compared with the control (P < 0.05). Expression of ERbeta increased in colon tumors of soy isoflavone-treated groups compared with the control. Soy isoflavones dose-dependently arrested the growth of DLD-1 cells and at subcytotoxic levels increased the expression of ERbeta. Our results suggest that pre- and postnatal exposure to dietary soy isoflavones suppresses the growth of colon tumors in male rats. The overexpression of ERbeta in both rat colon tumors and DLD-1 cells caused by soy isoflavones suggests that ERbeta is a critical mediator in mitigating its cancer-preventive effects.

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.

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.

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.

Effects of dietary fats and proteins on rat testicular steroidogenic enzymes and serum testosterone levels.

It is known that certain dietary fats can modulate rat testosterone metabolism. In the current study we have investigated testicular steroidogenic enzyme activities and serum testosterone levels in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). The diets examined reflect different marine oils and proteins which are significant components of Northern Canadian diets. Male rats (42-45 days old, 6 per group), were assigned to specific diets for 42 days. On the 43rd day of the study, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17,20-lyase, 17beta-HSD) were measured radiometrically. There were no differences in enzyme activities between the three dietary protein sources. In contrast, compared with the standard casein diet, all lipid sources caused reductions in C-17,20-lyase activity (>50%); seal oil and fish oil reduced 17-OHase activity (approximately 30%) and soybean oil, DHA fish oil and lard reduced 17beta-HSD activity (approximately 30%). No effect on 3beta-HSD activity was evident. Serum testosterone levels were determined using ELISA kits and were not affected by any diet with the exception of the soybean oil diet which was significantly elevated compared with the casein protein diet. Body and testis weights were not affected by diet. In conclusion, these data demonstrate that some dietary lipid sources caused reductions in testicular 17-OHase and C-17,20-lyase activities but not to the extent that serum T levels were affected, while soybean oil caused elevated serum testosterone in the absence of elevated steroidogenic enzyme activities.

An investigation of the effects of methylmercury in rats fed different dietary fats and proteins: testicular steroidogenic enzymes and serum testosterone levels.

Methylmercury (MeHg) is a testicular toxicant causing reduced steroidogenic enzyme activity, reduced serum testosterone (T) and abnormal spermatogenesis in mammals and fowl. It is also known that certain diets can alter androgen metabolism in rats. Previously we have shown that diets used in the current study impact circulating androgen levels and testicular steroidogenic enzyme activities in Sprague Dawley rats in the absence of MeHg. In the present study, we have investigated the impact of imposing an environmental contaminant (MeHg) commonly found in marine mammals and fish onto the rats' dietary intake of different proteins and lipids in order to determine if the different diets could modify MeHg toxicity in rats. Therefore, we examined the effects of MeHg on testicular steroidogenic enzymes and serum testosterone in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). Male rats 42-45 days of age (18 per group) were assigned to different experimental diets for 28 days after which 6 rats in each group were gavaged daily with 0, 1 or 3 mg/kg body weight (BW)/day MeHg chloride in 5 mM Na(2)CO(3) solution for 14 days while being maintained on their diets. On the 43rd day of dosing, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17, 20-lyase, 17beta-HSD) were measured radiometrically. Serum testosterone was determined using ELISA kits. Testis weights were not affected by MeHg. MeHg at 3 mg/kg BW/day caused a reduction (>50%) in the activity of C-17, 20-lyase in all three protein diets and similar reductions in 17-OHase activity were seen in the casein and whey protein fed rats. At 3 mg/kg BW/day, MeHg reduced 17-OHase activity in the DHA diet but had no effect on 3beta-HSD activity and no inhibitory effects on 17beta-HSD activity. MeHg (3 mg/kg BW/day) caused significant reductions in serum T in the whey, soybean oil and fish oil groups. Interestingly, fishmeal protein but not fish oil offered some protection with respect to maintaining steroidogenic enzyme activities and serum T levels in rats dosed with MeHg. In conclusion, these studies show that different lipid diets can alter the toxic effects of MeHg on male rat steroidogenesis in terms of serum testosterone and steroidogenic enzyme activities.

Altered fatty acid homeostasis and related toxicologic sequelae in rats exposed to dietary potassium perfluorooctanesulfonate (PFOS).

Perfluorooctanesulfonate (PFOS) is one of a class of industrial chemicals known as perfluoroalkyl acids, which have a wide variety of uses as surfactants and stain repellants. The presence of fluorochemical residues in human blood, plasma, or serum from sample populations worldwide is indicative of widespread human exposure. Previous studies demonstrated that PFOS alters fatty acid metabolism in the liver of rodents and that this leads to peroxisome proliferation. This study was undertaken to (1) confirm the effects of PFOS on rat liver, (2) identify additional target organs and systems, and (3) further explore the biochemical and molecular changes associated with PFOS exposure. The results confirmed that liver was a primary target for PFOS. Hepatomegaly, decreased serum triglycerides and cholesterol, and increased expression of the genes for acyl-coenzymeA oxidase 1 (ACOX1) and cytochrome P-450 4A22 (CYP4A22) were indicative of exposure to a peroxisome proliferator. Changes in liver fatty acid profiles included increased total monounsaturated fatty acid levels and decreased total polyunsaturated fatty acids, as well as an increase in linoleic acid levels and a decrease in longer chain fatty acids. These changes were similar to those induced by relatively weak peroxisome proliferators. Disruptions in hepatic fatty acid metabolism may contribute to changes in red blood cell membranes, resulting in increased lysis and cell fragility. Serum thyroid hormone levels were decreased in PFOS-treated rats, while the kidney and cardiovascular systems were not significant targets. Residue analyses indicated that PFOS accumulation in tissues was dose dependent, appearing preferentially in the liver at lower doses but increasing in serum and other organs relative to liver at higher doses.

Immunomodulatory effects of dietary potassium perfluorooctane sulfonate (PFOS) exposure in adult Sprague-Dawley rats.

Perfluorooctanesulfonate (PFOS) is a stable and environmentally persistent metabolic or degradation product of perfluorooctanyl compounds that were manufactured for a variety of industrial and consumer applications. PFOS itself was sold for use as a surfactant. The structurally related contaminants perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and N-ethyl perfluorooctane sulfonamide (N-EtPFOSA) were shown to suppress immune responses in laboratory rodents. Relatively low doses of PFOS were found to be immunosuppressive in mice. To assess effects of PFOS on the rat immune system at doses known to alter hepatic function, changes in the morphology and function of immune tissues and cells were measured in adult rats exposed to PFOS in their diet for 28 d at levels ranging from 2 to 100 mg PFOS/kg diet (corresponding to approximately 0.14 to 7.58 mg/kg body weight [bw]/d) and compared to those receiving control diet. Body weight reductions were significant in male and female rats exposed to 50 and 100 mg PFOS/kg diet. Liver/body weight was significantly increased in females exposed to 2 mg PFOS/kg diet and in males exposed to 20 mg PFOS/kg diet. Female rats exposed to 100 mg PFOS/kg diet exhibited a significant increase in spleen weight relative to body weight; these changes lacked a histologic correlate and were not observed in males. While thymus weights relative to body weights were not affected, numbers of apoptotic lymphocytes rose in thymus with increasing dietary PFOS. There was a significant dose-related increase in total peripheral blood lymphocyte numbers in female but not male rats. In both genders the percentages of cells within lymphocyte subclasses were altered. There was a significant trend toward increasing T and T-helper (Th) cells and decreasing B cells with higher PFOS dose. Serum total immunoglobulin (Ig) G1 levels were significantly reduced in males exposed to 2 and 20 mg PFOS/kg diet. The ability of male and female rats to mount delayed-type hypersensitivity (DTH) responses to the T-cell-dependent antigen keyhole limpet hemocyanin (KLH) was not altered by PFOS. There was a significant trend toward elevated KLH-specific IgG in serum from male rats exposed to increasing levels of PFOS in diet. Splenic T- and B-cell proliferation in response to ex vivo mitogen exposure was unaffected by exposure to dietary PFOS. In conclusion, changes in immune parameters in rat did not manifest as functional alterations in response to immune challenge with KLH and may be secondary to hepatic-mediated effects of PFOS in this model.

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.

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.

Effects of ciprofibrate on testicular and adrenal steroidogenic enzymes in the rat.

Testicular and adrenal steroidogenic enzymes were measured radiometrically following oral dosing of rats with ciprofibrate (2-[4-(2,2-dichlorocyclopropyl) phenoxyl]-2-methylpropinoic acid), a peroxisome proliferator. Six-week-old male Fisher 344 rats were fed a diet containing ciprofibrate (0.025%, w/w) for 3, 7, 14, 28, 56, 84, 112 or 140 days leading to a daily ciprofibrate intake of approximately 15 mg/kg body weight/day. Ciprofibrate caused a marked inhibition of testicular 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) activity that was significant after 3 days and subsequently decreased to 40% of control level. Ciprofibrate treatment also reduced 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity to a lesser extent but had no effect on 17-hydroxylase (17-OHase) activity. Immunoblot analyses indicated that ciprofibrate treatment did not alter enzyme protein levels and semi-quantitative RT-PCR analysis also revealed no significant changes in testicular 3beta-HSD mRNA levels. Furthermore, in addition to the enzyme-specific effect of ciprofibrate on 3beta-HSD in the testes, a tissue-specific effect was also evident, since no significant effects of ciprofibrate were seen on the activities of 3beta-HSD or 21-OHase in the adrenal glands from the same animals.

Mammary gland tumor promotion in F1 generation offspring from male and female rats exposed to soy isoflavones for a lifetime.

The effect of dietary isoflavones in the form of NOVASOY (NS) was investigated on methylnitrosourea-initiated mammary gland cancer in F1 generation female Sprague Dawley rats from parents who had undergone lifetime exposure to variable levels of dietary NS. In comparison to NS-free dietary groups, lifetime exposure of F1 rats to 40 and 1000 mg/kg diets of NS reduced tumor latency, but did not significantly affect tumor incidence, tumor size, or tumor multiplicity. A significantly lower tumor multiplicity was, however, observed in rats fed the soy-based, NS-free diet compared to the casein-based, NS-free diet. An evaluation of a dose-response relationship pointed towards a biphasic effect, with a trend showing lower tumor incidence, lower tumor multiplicity, and lower tumor size in rats fed 1000 mg/kg diet NS compared to 40 mg/kg diet NS; however, the data failed to achieve statistical significance. Histologically, tumor type significantly differed according to the administered basal diet variety and NS dose. Our data and that of others provide conflicting evidence for chemopreventive effects of soy isoflavones on mammary gland tumor induction. We suggest standardization of interlaboratory experimental approaches for establishing low dose-response relationships for soy and its isoflavones to aid in risk assessment.

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.