Lack of adverse health effects following 30-weeks of dietary exposure to acrylamide at low doses in male F344 rats.

Understanding the health hazards following exposure to food-borne acrylamide, especially at low levels typified by human diets, is an ongoing food safety issue. We recently published results from a study that aimed to understand the effects of acrylamide short-term exposure at doses known to cause tumors in rodents, demonstrating that a number of key toxicological end points were altered by acrylamide exposure. Additionally, we reported that at much lower doses for 30 weeks of exposure, dietary acrylamide was 'not a complete carcinogen' to the colon in an organ-specific rodent carcinogenesis study but acted as a co-carcinogen along with azoxymethane (AOM, a colon-specific carcinogen). Here, we present toxicological data from a sub-set of this long-term exposure study from animals that received saline (instead of AOM). Briefly, male F344 rats were randomized to receive acrylamide at 0.5, 1.0 and 2.0 mg/kg diet (∼0.02, 0.04, and 0.09 mg/kg BW/day, respectively) or no acrylamide (control), for 30 weeks; all rats were then euthanized and their tissues harvested and processed for toxicological evaluation. We report that at the doses tested, acrylamide did not cause any changes in general well-being, body weight or food intake. Similarly, acrylamide did not cause any biologically relevant change in parameters associated with immunophenotyping, serum biochemistry or hematology. Histopathology assessment of tissues showed no changes except in the testis, where non-specific mild lesions were observed in all the groups, inclusive of the controls. No neuropathological effects of acrylamide were observed in the brain and nerve tissues. Together, these results suggest that acrylamide administered to rats through the diet at low doses for 30 weeks did not cause any toxicologically relevant changes. Given that the doses of acrylamide in the current study are low and are comparable to human dietary exposure, this null-effect study provides data that contribute to the body of scientific evidence relevant to understanding the health effects of acrylamide.

Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles.

Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20-2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein-AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R(2)=0.9821) and fluorescence intensity (R(2)=0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles.

Toxicological effects of short-term dietary acrylamide exposure in male F344 rats.

We recently reported that acrylamide, a known rodent and probable human carcinogen, does not increase the risk of azoxymethane (AOM)-induced rat colon precancerous lesions when administered through the diet. Here, we present toxicological data from non-AOM-injected rats. Briefly, male F344 rats were randomized into four dietary groups and received experimental diets based on AIN-93G formulation and containing acrylamide at 0 (control), 5, 10 or 50mg/kg diet (wt/wt) ad libitum for 10 weeks, after which they were killed and their blood collected for hematological and biochemical markers. Acrylamide at the higher doses (10 and 50mg/kg diet) significantly lowered (p<0.05) serum total high density lipoprotein and total testosterone and increased serum lipase in comparison to the control. Blood hematocrit values and lymphocyte counts were significantly lower (p<0.05) in the high dose acrylamide (50mg/kg diet) group compared to control, with a concomitant decrease in hemoglobin level, mean corpuscular volume and mean corpuscular hemoglobin. These results provide additional hazard characterization data and strengthen the notion that at high doses, acrylamide may involve systemic toxicity potentiating tumorigenesis in experimental animals. Further studies are required to understand the health effects of food-borne acrylamide, especially at the lower exposures typified by human diets.

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.

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.