Project description:To provide insights into the mode of action for Ni3S2 lung carcinogenicity by examining gene expression changes in target cells after inhalation exposure. Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer F344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m3 (0.03, 0.06, 0.11, and 0.44 mg Ni/m3) for one and four weeks (6 hours per day, 5 days per week). Results: Broncho-alveolar lavage fluid evaluation and lung histopathology provided evidence of inflammation only at the two highest concentrations, which were similar to those tested in the 2-year bioassay. The number of statistically significant up- and down-regulated genes decreased markedly from one to four weeks of exposure, suggesting adaptation. Cell signal pathway enrichment at both time-points primarily reflected responses to toxicity, including inflammatory and proliferative signaling. While proliferative signaling was up-regulated at both time points, some inflammatory signaling reversed from down-regulation at 1 week to up-regulation at 4 weeks. Conclusions: These results support a mode of action for Ni3S2 carcinogenicity driven by chronic toxicity, inflammation and proliferation, leading to mis-replication, rather than by direct genotoxicity. Benchmark dose (BMD) analysis identified the lowest pathway transcriptional BMD exposure concentration as 0.026 mg Ni/m3, for apoptosis/survival signaling. When conducted on the basis of lung Ni concentration the lowest pathway BMD was 0.64 M-BM-5g Ni/g lung, for immune/inflammatory signaling. Implications: These highly conservative BMDs could be used to derive a point of departure in a nonlinear risk assessment for Ni3S2 toxicity and carcinogenicity. Gene expression changes were determined in micro-dissected lung broncho-alveolar cells from Fischer F344 rats following inhalation of Ni3S2 at 0.0, 0.04, 0.08, 0.15, and 0.60 mg/m3 (0.03, 0.06, 0.11, and 0.44 mg Ni/m3) for one and four weeks (6 hours per day, 5 days per week).

Project description:Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q < 0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p < 0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p < 0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat. Rats were exposed to sub-chronic low doses of the Sertoli cell toxicant 2,5-hexanedione (HD) or water (control for HD) for 3 months. Some rats in each group underwent 3 months of post-exposure recovery.

Project description:Current human reproductive risk assessment methods rely on semen and serum hormone analyses, which are not easily comparable to the histopathological endpoints and mating studies used in animal testing. Because of these limitations, there is a need to develop universal evaluations that reliably reflect male reproductive function. We hypothesized that toxicant-induced testicular injury can be detected in sperm using mRNA transcripts as indicators of insult. To test this, we exposed adult male Fischer 344 rats to low doses of model testicular toxicants and classically characterized the testicular injury while simultaneously evaluating sperm mRNA transcripts from the same animals. Overall, this study aimed to: 1) identify sperm transcripts altered after exposure to the model testicular toxicant, 2,5-hexanedione (HD) using microarrays; 2) expand on the HD-induced transcript changes in a comprehensive time course experiment using qRT-PCR arrays; and 3) test these injury indicators after exposure to another model testicular toxicant, carbendazim (CBZ). Microarray analysis of HD-treated adult Fischer 344 rats identified 128 altered sperm mRNA transcripts when compared to control using linear models of microarray analysis (q < 0.05). All transcript alterations disappeared after 3 months of post-exposure recovery. In the time course experiment, time-dependent alterations were observed for 12 candidate transcripts selected from the microarray data based upon fold change and biological relevance, and 8 of these transcripts remained significantly altered after the 3-month recovery period (p < 0.05). In the last experiment, 8 candidate transcripts changed after exposure to CBZ (p < 0.05). The two testicular toxicants produced distinct molecular signatures with only 4 overlapping transcripts between them, each occurring in opposite directions. Overall, these results suggest that sperm mRNA transcripts are indicators of low dose toxicant-induced testicular injury in the rat.

Project description:The rostral ventrolateral medulla (RVLM), a part of the medullary reticular formation, plays a major role in several physiological responses, including cardiovascular and sympathetic nervous system functions. Although aging causes disturbances in the responses of these physiological systems, RVLM involvement in these age-related changes is not clear. Previous work using high-throughput gene expression analysis of the RVLM in aged animals suggested that chemical neurotransmission-related genes might be downregulated with advancing age. Since the RVLM function involves a balance of inhibitory and excitatory inputs, which is largely mediated by gamma-aminobutyric acid (GABA) and excitatory amino acid (EAA) neurotransmission, we hypothesized that aging is associated with altered excitatory and/or inhibitory neurotransmission-related gene expression in the RVLM. To test this hypothesis, we micropunched an RVLM-containing area from young (3–5 months), middle-aged (12–14 months), and aged (22–26 months) Fischer 344 male rats. RNA purified from these micropunches was analyzed using GABA and Glutamate RT2 Profiler PCR arrays (n= 8–10). In addition, the expression of selected genes was validated at the RNA level using TaqMan® based- qPCR and at the protein level using western blotting. All the genes that displayed significant differential expression (1.5-fold, p < .05, FDR < .05) were identified to be downregulated in the RVLM of aged and middle-aged rats compared to young rats. Among the downregulated genes, the percentage of glutamate neurotransmission-related genes was higher than GABA neurotransmission-related genes. Solute carrier family 1 member 6 (Slc1a6) gene showed the highest fold downregulation at the RNA level in the RVLM of aged compared to young rats, and its protein product, Excitatory amino acid transporter 4 (EAAT4), showed a downregulatory trend in the RVLM of aged and middle-aged rats. These results suggest that molecular constituents of both GABA and glutamate neurotransmission might be altered in the RVLM of aged and middle-aged rats, and the changes in glutamate neurotransmission might be more prominent. Investigating age-associated anatomical and functional changes in RVLM GABA and glutamate neurotransmission might provide a foundation for understanding the effects of aging on physiological function.

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Gene expression profiling of hepatocarcinogenesis initiation in Fischer-344 rats.

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