Annual Meeting of the Japanese Society of Toxicology

This investigation was carried out to determine the effects of aflatoxin B₁ (AFB₁) on fetuses of mice and rats, and maternal tissues of rats. The pregnant ICR mice were injected intraperitoneal (ip.) at Day-13 of gestation with AFB₁ at 20 and 10 mg/kg BW (body weight). The fetuses from mice were examined at 12, 24 and 48 hours after inoculation (HAI). The pregnant Wistar rats were also injected ip. at Day-13 of gestation with AFB₁ at 20 mg/kg BW (sacrificed at 12 HAI), 5mg/kg BW (sacrificed at 24 HAI) and 2.5mg/kg BW (sacrificed at 48 HAI). Fetuses, placenta, and maternal liver, thymus and spleen were collected from rats for histopathology, the in situ detection of apoptotic cells (TUNEL method) and immunohistochemistry. Light microscopic study on fetuses of mice, and placenta and fetuses of rats detected no difference of cell death in AFB₁-treated group and in control group examined at different time points of study. On the contrary, liver, spleen and thymus of rats treated with AFB₁ at different doses of different time points evoked cell death where the lesions of spleen and liver were striking at 24 and 48 HAI, respectively. The lesions of liver were characterized by severe congestion, fatty change and multifocal/diffuse cell death; and at early time these hepatocytes underwent apoptotic type of cell death . The lesions of liver characterized by necrosis with slight apoptotic cells increased in severity with the advancement of time. The striking pyknotic cell death in thymus was evident at 48 HAI. These pyknotic cells showed positive staining with TUNEL method, the in situ detection of apoptotic cells. Immunohistochemistry detected activated caspase-3 in pyknotic cells of spleen, liver and thymus. However, the relation between AFB₁-induced apoptosis, necrosis and tumorigenesis in rats should be elucidated.

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To assess the effects of test substances on reproductive functions and embryo-fetal development in cynomolgus monkeys (Macaca fascicularis), menstrual cycle and fetal/neonatal evaluation control data are particularly important. Menstrual cycles (146 female monkeys for a total of 907 cycles) were variable within and between females (average of all individual cycles: 30.5 ± 7.7 days, average of mean value of each female: 30.9 ± 4.5 days). Pregnancy ratio of 3-day mating session (for a total of 463 mating sessions) and the mean abortion/embryo-fetal death incidence (62 dams) were approximately 40% and 8%, respectively. In teratology studies, the mean fetal weight on GD100 was 115 g. There were no external fetal abnormalities, a low incidence of visceral (absence of left thyroid, 2.4%) and skeletal abnormalities (hemi-vertebra or absence of the 12th rib, 5.6%), and a 12.6% incidence of skeletal variations (lumbar ribs). Peripheral lymphocyte subsets (flow cytometry) analysis data indicate that the levels of CD45, CD8, CD3, and CD4 were lower in the fetus (GD100). CD8, CD3 and CD4 in neonates are essentially equivalent to the mothers values by 6 months after birth. CD3 and CD4 were increased as compared to mothers levels in the neonate. CD3 was approximately 30% higher while CD4 was 80% higher in the neonate. Both CD3 and CD4 decreased after the neonatal stage and approached the mothers levels at 6 months.

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Recently we reported that simultaneous treatment of NIH 3T3 cells with the combination of phorbol myristate acetate (PMA) and hydrogen peroxide (H₂O₂) resulted in synergistic activation of Raf-1 kinase. In the present study, we have demonstrated that PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a potent and selective inhibitor of the Src-family tyrosine kinase, greatly potentiated the ability of PMA and/or H₂O₂ to activate Raf-1 kinase while it blocked the tyrosine phosphorylation of Raf-1. Unlike PMA/H₂O₂ treatment, which showed transient activation, PP2-mediated Raf-1 activation was sustained and continued to increase through 4 h of treatment. Transient transfection studies with a dominant-negative mutant of Ras (N19Ras) indicated that this PP2-induced activation of Raf-1 was Ras-independent. Moreover, PP2 showed no effect on platelet-derived growth factor (PDGF)-induced Raf-1 activation. Interestingly, mutation of the reported Raf-1 Src family tyrosine kinase phosphorylation site by conversion of tyrosines 340 and 341 to phenylalanine (YY340/341FF Raf) had limited effect on the ability of PP2 to induce significant stimulation of Raf-1 kinase activity. Taken together, our results suggest that a tyrosine phosphorylation event is involved in the negative feedback regulation of Raf-1. Inhibition of a Src family tyrosine kinase by PP2 appears to alleviate this tyrosine kinase-mediated inhibition of Raf-1 and allow activating modification(s) of Raf-1 to proceed. This PP2 effect resulted in significant and sustained Ras-independent activation of Raf-1 by PMA and H₂O₂.

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The use of nanotechnology in consumer and industrial applications will likely have a profound impact on the quality and utility of a number of commercial products from a variety of industrial sectors. Nanomaterials exhibit unique physical/chemical properties and impart enhancements to engineered materials, including better magnetic properties, improved electrical activity, and increased optical properties. In addition, these materials are much more reactive than their bulk material counterparts as a result of their higher surface area. Consequently, the use of nanotechnology has the potential to facilitate substantial improvements in several critical societal functions such as energy generation and distribution, food processing, and building construction. Nanomaterials are already being used in a variety of commercial products, including computer components, textiles, cosmetics, glass technology, and photovoltaic systems. Given the impending widespread use of nanotechnology, a systematic approach needs to be developed for evaluating the risk to human health and the environment from exposure to nanomaterials. Some of the uncertainties associated with exposure to nanomaterials include the extent to which these materials interact with cellular organelles and the biological impact of those interactions, and the long-term health effects from acute and chronic exposure. There is also very little known about the environmental implications of the use of nanomaterials. Accordingly, the ILSI Health and Environmental Sciences Institute (HESI) has formed a subcommittee to improve the science associated with developing toxicological and safety evaluations for engineered nanomaterials and to improve the fundamental understanding of the behavior of these materials in biological systems and the environment. The broad goal of the effort is to develop a systematic approach to conducting a comprehensive toxicological and safety evaluation for nanomaterials. The specific project areas that form the basis of the subcommittee's activities, and that are the focus of this poster presentation, include nanomaterial characterization, toxicity, solubility, and life-cycle analysis.

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