1.0 Summary Individuals may respond to environmental hazards quite differently due to numerous factors, including proximity to the toxicant, the individual's size, health, past exposures, and heredity. Therefore, exposure monitoring must take these variations in individual response into account. While environmental sampling equipment provides an important indication of a toxicant's presence, it does not monitor individual responses. The development of biomarkers of exposure, effect, and susceptibility (1) is a priority at the U.S. Department of Defense Health Affairs in the area of occupational environmental health surveillance (OEHS). Not only do biomarkers offer the opportunity to quantify toxic exposures, but also to identify possible future adverse health effects by regular or contingent testing before, during, and after deployments. In an effort to rapidly advance biomarker discovery the U.S. Army Center for Environmental Health Research (USACEHR) is working to develop new methods combining multiple approaches. This report demonstrates the feasibility of incorporating in vitro cellular models with label free quantitative proteomic screening technologies, functional genomics and an enzyme-linked immunosorbent assay (ELISA) as biomarker discovery tools. This multi-tiered approach advances not only the knowledge about the effects of trinitrotoluene (TNT) and dinitrobenzene (DNB), but may also identify future biomarkers of exposure and/or effect.

Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured bromodichloromethane (BDCM) concentrations of 0.018, 0.143, or 1.424 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic BDCM concentration. BDCM was not hepatocarcinogenic to medaka at the concentrations tested. Chronic toxicity was evidenced at 6 and 9 months by statistically significant (alpha = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.424 mg/L BDCM. Hepatocellular proliferation was assessed after 1, 4, and 20 days of BDCM exposure. Treatment-related increases or decreases in cellular proliferation were not observed at any time point. Hematocrit, leukocrit, cell viability, and cell counts of treated fish after 9 months of BDCM exposure were not significantly different from control fish. Intrahepatic concentrations were evaluated by gas chromatography after 9 months of BDCM exposure. Fish livers from all three BDCM treatments had detectable amounts of BDCM, with median intrahepatic concentrations of 1.02, 2.89, and 21.25 mg BDCM/kg fish liver in the low, middle, and high concentrations, respectively. Medaka chronic toxicity effects of statistically significant gallbladder and bile duct abnormalities occurred at 1.424 mg/L BDCM, well above median drinking water levels.

Japanese medaka fish ( Oryzias latipes) were used to develop an in vivo method to assess hepatocellular proliferation in a nonmammalian model. Proliferative responses were assessed in medaka at 7, 17, 24, and 94 days after a 48-hour exposure to 10 or 100 mg/L diethylnitrosamine (DEN). Subgroups of medaka were exposed to 50 or 75 mg/L of 5-bromo-2'-deoxyuridine (BrdU) in water for 72 hours, sacrificed, and then processed for immunohistochemical staining. Proliferative indices of BrdU-labeled hepatocytes were quantified and compared using both count and area measurements. There was a significant increase ( p < 0.05) in hepatocellular proliferation in the 100 mg/L DEN-treated fish as compared to controls and 10 mg/L DEN-treated fish for the first 3 time points. Hepatocarcinogenicit y was evaluated 26 weeks post-DEN exposure. There was a significant increase ( p < 0.0001) in hepatocellular neoplasms in 100 mg/L DEN-treated fish compared to other fish. Effective BrdU-labeling of S-phase hepatocytes in medaka was achieved by adding BrdU to the aquarium water, and an increase in hepatocellular proliferation using this method was detected 7 days after exposure to a carcinogenic concentration of DEN. Additionally, the new method of area measurement indices of proliferation were as precise as count indices (R 2 ≥0.92).