Laurie Coady

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.

Toxicologic and immunologic effects of perinatal exposure to the brominated diphenyl ether (BDE) mixture DE-71 in the Sprague-Dawley rat

Brominated diphenyl ethers (BDEs) are persistent environmental contaminants found in human blood, tissues, and milk. To assess the impact of the commercial BDE mixture DE‐71 on the developing immune system in relation to hepatic and thyroid changes, adult (F0) rats were exposed to DE‐71 by gavage at doses of 0, 0.5, 5, or 25 mg/kg body weight (bw)/d for 21 weeks. F0 rats were bred and exposure continued through gestation, lactation and postweaning. F1 pups were weaned and exposed to DE‐71 by gavage from postnatal day (PND) 22 to 42. On PND 42, half of the F1 rats were assessed for toxicologic changes. The remaining F1 rats were challenged with the T‐dependent antigen keyhole limpet hemocyanin (KLH) and immune function was assessed on PND 56. Dose‐dependent increases in total BDE concentrations were detected in the liver and adipose of all F0 and F1 rats. In F0 rats, increased liver weight, hepatocellular hypertrophy, and decreased serum thyroxine (T4) were characteristic of DE‐71 exposure. In F1 rats perinatal DE‐71 exposure caused a nondose‐dependent increase in body weight and dose‐dependent increases in liver weight and hepatocellular hypertrophy. Serum T3 and T4 levels were decreased. In spleen from DE‐71 exposed rats the area occupied by B cells declined while the area occupied by T cells increased; however, cellular and humoral immune responses to KLH challenge were not altered. Thus hepatic and thyroid changes in rats exposed perinatally to DE‐71 were associated with altered splenic lymphocyte populations, an effect which has been linked to hypothyroidism.

Effects of long term exposure to the mycotoxin fumonisin B1 in p53 heterozygous and p53 homozygous transgenic mice

The fungal toxin fumonisin B1 (FB1) is a potential human carcinogen based on evidence of renal carcinogenicity in rats and hepatocarcinogenicity in mice. The toxicity and carcinogenicity of FB1 is linked to ceramide synthase inhibition. Based on this mechanism of action and on lack of evidence of genotoxicity, FB1 is considered a non-genotoxic carcinogen. The p53 heterozygous (p53+/-) mouse is a cancer-prone model used for carcinogenesis. The effects of chronic dietary FB1 exposure were characterized in p53+/- mice to confirm non-genotoxicity using a model which is more sensitive to genotoxic than non-genotoxic carcinogens and to clarify the relationship between p53 expression, altered sphingolipid metabolism, and FB1-induced carcinogenesis. Responses to FB1 were similar in p53+/- and p53+/+ mice after 26 weeks exposure to 0, 5, 50 or 150 mg FB1/kg diet, supporting a non-genotoxic mechanism of action. Hepatic adenomas and cholangiomas were observed in mice exposed to 150 mg/kg FB1. For a 10% increase in hepatic megalocytosis, the estimated 95% lower confidence limit of the benchmark dose (BMDL10) ranged from 0.15 and 1.11 mg FB1/kg bw/day. Based on similar responses in p53+/- and p53+/+ mice, p53 and related pathways play a secondary role in responses to FB1 toxicity and carcinogenesis.

Enantioselective and Gender-Dependent Depletion of Chlordane Compounds from Rat Tissues

Isomers and metabolites of the organochlorine pesticide chlordane persist in the environment and bioaccumulate in Arctic marine food webs. Rodent studies indicate that there are gender-related differences in trans-nonachlor and oxychlordane metabolism. Thus, comparative tissue depletion studies were undertaken in male and female rats exposed to trans-nonachlor, oxychlordane, or trans-chlordane at 2.5 mg/kg body weight/d by gavage for 28 d followed by two consecutive 28-d depletion periods. None of the test chemicals were overtly toxic at this dose, although increased liver weights in some groups were consistent with microsomal enzyme induction. The metabolite oxychlordane accumulated in tissues from rats exposed to trans-nonachlor and trans-chlordane. trans-Nonachlor and oxychlordane residue levels were highest in tissues from female rats at each time point; however, trans-chlordane was completely eliminated from males and females by the end of the study. Body burden calculations showed no significant clearance of oxychlordane in females over 56 d postdosing, whereas males lost approximately half their oxychlordane body burden in the same period. For the chiral contaminants oxychlordane and trans-chlordane, tissues from male and female rats were selectively depleted of the (+)-enantiomer; however, there were gender-related differences in enantiomer depletion patterns over time. In general, residue analyses confirmed that gender-related metabolic differences and contaminant structural properties, including chirality, influenced chlordane contaminant elimination from rat tissues. The study points to a need for similar knowledge of gender-related responses in humans in order to provide relevant dietary recommendations for populations exposed to chlordane-related contaminants in foods.

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.

Brominated diphenyl ether (BDE) levels in liver, adipose, and milk from adult and juvenile rats exposed by gavage to the DE‐71 technical mixture

Brominated diphenyl ethers (BDEs) are used as flame retardants in consumer products. Rodent studies indicate that the liver, thyroid, and nervous system of developing animals are targets of BDEs. To explore the relationship between exposure and health in developing animals, BDE accumulation in adult and juvenile rats was examined in conjunction with changes in liver weight and serum thyroxine (T4). Adult (F0) rats received the commercial BDE mixture DE‐71 by gavage at doses of 0.5, 5, and 25 mg kg−1 body weight (bw)/day for 21 weeks. F0 rats were mated and exposure continued throughout breeding, pregnancy, lactation, and postweaning until the pups (F1 generation) reached postnatal day (PND) 42. Milk was collected from lactating dams. Adipose and liver samples were collected from F0 and F1 males and females for BDE congener analysis. Congener prevalence in rat tissues mimicked congener prevalence in wildlife and humans. Tissue concentrations of all congeners except BDE‐153 were lower than would be expected based on dose proportionality, confirming that BDE‐153 has a high capacity for bioaccumulation. BDEs were transferred from maternal tissues to milk during lactation. Milk congener profiles differed from maternal tissue profiles indicating that degree of bromination and maternal sequestration influenced BDE transfer to milk. Female F1 rats accumulated more BDEs than F1 males, indicating that female rats were less able to metabolize and/or excrete BDEs. Significant effects on liver weight and serum T4 levels were observed in adults and juveniles in the middle and high dose groups, corresponding to BDE levels in the μg g−1 range. Although it remains to be determined how human liver and thyroid are affected by exposure to much lower BDE levels, the present study confirmed that gender and reproductive status influence BDE accumulation in tissues and BDE transfer to the neonate via milk.

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 β- 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

Abstract 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.

Testing an emerging animal model for use in the allergenicity assessment of food

Results Mice treated with 2 mg peanut developed peanut-specific IgE levels which were significantly higher than control mice (p<0.001, n=10/group). Mice treated with 2 mg turkey developed a similar IgE response to turkey (p<0.001, n=10/group). In the second study, allergy was only triggered in one of ten mice treated with 2 mg peanut. Two of ten mice exposed to 1 mg potato had a response. There were no IgE responders to spinach. Spleen cells from both the peanutand the spinach-treated mice secreted more allergy-promoting interleukin-4 than controls (p<0.01, n=7-24/group). Levels were not modified in potato-treated mice.