We investigated the effects of aflatoxin B1 (AFB1) on isolated splenic lymphocytes and the histo-morphologic changes in the spleens and liver of Fisher-344 male rats. Weaned animals were fed chow diets that contained 0, 0.01, 0.04, 0.4, or 1.6 ppm AFB1, using an intermittent dosing regimen (4 weeks on and 4 weeks off AFB1), for 40 weeks. An additional group of animals was fed the 1.6 ppm AFB1 diet continuously. The intermittent dosing regimen was designed to evaluate effects of cumulative dose and exposure for risk assessment comparisons. The percentages of T and B cells were affected as shown by flow cytometric analysis after the dosing cycles. The observed changes appeared to reverse or compensate to some extent after the off cycles. Lymphocytes were stimulated in culture for analysis of the production of IL-2, IL-1, and IL-6. Significantly increased production of IL-1 and IL-6 was seen in the second dosing cycle (12 weeks) and the second "off" cycle (16 weeks) at the higher doses. Inflammatory infiltrates were seen in the liver after eight weeks of continuous and intermittent dosing and were increased in size and number at 12 weeks in both 1.6 ppm dose groups correlating with the peak production of Il-1 and IL-6. We concluded that AFB1 effects on the immune system can be either stimulatory or suppressive dependent on a critical exposure window of dose and time. Immune cells in spleen such as T-lymphocytes and macrophages, both important mediators of inflammatory responses to tissue damage, were affected differently in the continuous and intermittent exposures to AFB1.

In January 2001 a Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Consultation Committee on Allergenicity of Foods Derived from Biotechnology published a report outlining in detail an approach for assessing the allergenic potential of novel proteins. One component of this decision tree is a determination of whether the protein of interest is resistant to proteolytic digestion. Although these (Italic)in vitro(/Italic) methodologies have been useful, the correlation between resistance to proteolysis and allergenic activity is not absolute. Two views and highlights of supporting research regarding the relationship of resistance to digestion and allergenicity are presented in this article.

The rapid advances in the field of immunology and an understanding of the potential adverse effects of xenobiotics on the immune system have resulted in the development of a discipline in toxicology now referred to as immunotoxicology. This discipline has evolved steadily over the last 2 decades as a result of research in the national and international communities. Various US, European, and Japanese regulatory agencies have recognized a need to promulgate testing guidelines for immunotoxicity in support of the approval process involving toxicological testing. The US Food and Drug Administration "Redbook II" guidelines and some of the research conducted in support of the concepts and testing strategies are presented here. Concerns raised with regard to these guidelines are included, as are on-going initiatives in development of experimental approaches for assessing allergic potential and/or hypersensitivity responses to new foods and food constituents.

We studied the effects of intermittent exposure to aflatoxin B1 (AFB1) on hepatic DNA and RNA adduct formation. Fisher-344 male rats were fed 0.01, 0.04, 0.4, or 1.6 ppm of AFB1 intermittently for 8, 12, 16, and 20 weeks, alternating with 4 weeks of dosing and 4 weeks of rest. Other groups of rats were fed 1.6 ppm of AFB1 continuously for 4, 8, 12, and 16 weeks. Control rats received AFB1-free NIH-31 meal diet. AFB1-DNA and -RNA adducts were measured by HPLC with fluorescence detection. The data are presented as total DNA or RNA adducts. The DNA and RNA adduct levels increased or decreased depending on the cycles of dosing and rest. Rats removed from treatment 1 month after 1 or 2 dosing cycles (8 and 16 weeks of intermittent exposure) showed approximately a twofold decrease in DNA adduct levels and a two- to elevenfold decrease in RNA adduct levels compared with rats euthanized immediately after the last dosing cycle (12 and 20 weeks of intermittent exposure). Our data indicate that DNA and RNA adducts increased linearly, from 0.01 ppm to 1.6 ppm of AFB1 after 12 and 20 weeks of intermittent treatment. A linear dose response was also apparent for DNA but not for RNA adducts after 8 and 16 weeks of treatment. As biomarkers of exposure, AFB1-RNA adducts were three to nine times more sensitive than AFB1-DNA adducts but showed greater variability. These results suggest that binding of AFB1 to hepatic DNA is a linear function of the dose, regardless of the way this is administered. The dose-response relationship for RNA adducts depends on the length of the no-dosing cycles and on the turnover rate of RNA.

Triphenyl phosphate (TPP), a potential food contaminant, was fed to weanling Spartan Sprague-Dawley rats at dose levels of 0, 0.25, 0.5, 0.75, and 1.0% for 120 days. The immunotoxicity evaluation, planned as a minimum testing model in a subchronic study design as well as to provide information on TPP, was performed along with the routine testing of a separate group of animals. Traditional measures were made of growth and food consumption, total protein analysis, electrophoretic analyses of serum proteins, lymphoid organ weights in relation to growth, and histopathology, with expanded immunohistochemical evaluation of B- and T- lymphocyte regions in spleen, thymus, and lymph nodes, using immunoperoxidase staining. Assessment was made of the humoral response to a T-lymphocyte-dependent antigen, sheep red blood cells, and was begun at midterm of the feeding period for the primary response followed by secondary and tertiary booster immunizations at 3-week intervals. The kinetics of the responses were measured by hemolysin assay of relative antibody titers at days 3, 4, 5, and 6 postinjection. No significant effects on the responses were noted for either sex at any of the dose levels tested. The only effects noted were a decreased rate of growth at high levels of TPP and increases in the levels of alpha- and beta-globulins suggestive of increased hepatic activity.