An in vivo method for using 5-bromo-2'-deoxyuridine (BrdU) as a marker of chemically-induced hepatocellular proliferation in the Japanese medaka (Oryzias latipes).

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. Hepatocarcinogenicity 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 (R2 > or = 0.92).

Disruption of mitochondrial respiration by melatonin in MCF-7 cells.

Clinical and laboratory studies have provided evidence of oncostatic activity by the pineal neurohormone melatonin. However, these studies have not elucidated its mechanism of action. The following series of MCF-7 breast tumor cell studies conducted in the absence of exogenous steroid hormones provide evidence for a novel mechanism of oncostatic activity by this endogenous hormone. We observed a 40--60% loss of MCF-7 cells after 20-h treatment with 100 nM melatonin, which confirmed and extended previous reports of its oncostatic potency. Interestingly, there were no observed changes in tritiated thymidine uptake, suggesting a lack of effect on cell cycle/nascent DNA synthesis. Further evidence of a cytocidal effect came from morphologic observations of acute cell death and autophagocytosis accompanied by degenerative changes in mitochondria. Studies of mitochondrial function via standard polarography revealed a significant increase in oxygen consumption in melatonin-treated MCF-7 cells. Enzyme-substrate studies of electron transport chain (complex IV) activity in detergent permeabilized cells demonstrated a concomitant 53% increase (p < 0.01) in cytochrome c oxidase activity. Additional studies of succinate dehydrogenase activity (complex II) as determined by reduction of (3-4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide demonstrated a significant increase (p < 0.05) in melatonin-treated cells and further confirmed the accelerated ET activity. Finally, there was a 64% decrease (p < 0.05) in cellular ATP levels in melatonin-treated cells. The G-protein-coupled melatonin receptor antagonist luzindole abrogated the cytotoxic and mitochondrial effects. These studies suggest a receptor-modulated pathway of cytotoxicity in melatonin-treated MCF-7 tumor cells with apparent uncoupling of oxidative phosphorylation.

Chronic toxicity of chloroform to Japanese medaka fish.

Japanese medaka (Oryzias latipes) were continually exposed in a flow-through diluter system for 9 months to measured chloroform concentrations of 0.017, 0.151, or 1.463 mg/L. Parameters evaluated were hepatocarcinogenicity, hepatocellular proliferation, hematology, and intrahepatic chloroform concentration. Histopathology was evaluated at 6 and 9 months. Chloroform was not hepatocarcinogenic to the medaka at the concentrations tested. Chronic toxicity was evidenced at these time points by statistically significant ([alpha] = 0.05) levels of gallbladder lesions and bile duct abnormalities in medaka treated with 1.463 mg/L chloroform. We assessed hepatocellular proliferation by exposing test fish to 5-bromo-2'-deoxyuridine in the aquarium water for 72 hr after 4 and 20 days of chloroform exposure; we then quantified area-labeling indices of the livers using computer-assisted image analysis. We observed no treatment-related increases in cellular proliferation. We analyzed cells in circulating blood in medaka after 6 months of chloroform exposure. Hematocrit, leukocrit, cell viability, and cell counts of treated fish were not significantly different from those of control fish. Using gas chromatography (GC), we evaluated intrahepatic concentrations of chloroform in fish after 9 months of exposure. Livers from the 0.151 and 1.463 mg/L chloroform-treated fish had detectable amounts of chloroform, but these levels were always lower than the aquaria concentrations of chloroform. Thus, it appeared that chloroform did not bioaccumulate in the liver. Unidentified presumptive metabolite peaks were found in the GC tracings of these fish livers.

Chronic toxicity of bromodichloromethane to the Japanese medaka (Oryzias latipes).

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.

Histopathology of Japanese medaka (Oryzias latipes) chronically exposed to a complex environmental mixture.

Japanese medaka (Oryzias latipes) were used to evaluate the carcinogenicity of a complex groundwater that contained 5 U.S. Environmental Protection Agency priority pollutant heavy metals and 13 chlorinated aliphatic hydrocarbons. A test protocol that used 10 mg/L diethylnitrosamine (DEN) prior to groundwater exposure was designed to assess both initiation and promotion. The fish were exposed continuously for 9 mo with 0, 1, 5, or 25% groundwater, by volume, with either West Branch of Canal Creek water (Aberdeen Proving Ground-Edgewood Area, Aberdeen Proving Ground, MD) or dechlorinated tap water as the diluent, while concurrent controls were run in the laboratory. Incidental findings included various neoplasms in the nares, ovary, skeletal muscle, skin, swim bladder, testis, thymus, and thyroid. Factors evaluated during statistical analyses of fish neoplasm prevalence included diluent type, groundwater percentage, fish gender, and DEN initiation. Liver neoplasm prevalence was higher in DEN-initiated fish and was frequently higher in males. Concentrations of up to 25% groundwater, by volume, showed no evidence of being a complete carcinogen and showed no consistent, conclusive evidence of being a promoter.

Animals as sentinels of human health hazards of environmental chemicals.

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environment," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and nonmammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Environmental complex mixture toxicity assessment.

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.

The etiology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals progressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion of normal women.

BACKGROUND: The authors previously have shown by gas chromatography-mass spectrometry that the hydroxyl radical (.OH) induces alterations in the DNA base structure of the female breast, which are premalignant markers of breast cancer. Fourier transform-infrared (FT-IR)-spectroscopy also has a high potential for revealing a broad array of structural changes in DNA that may provide important new insight into breast cancer etiology and prediction. METHODS: DNA from normal reduction mammoplasty tissue, invasive ductal carcinoma, and nearby microscopically normal tissue was analyzed by FT-IR spectroscopy. Statistical models based on DNA spectral properties were developed and compared with a statistical model previously used with base modifications. RESULTS: Substantial differences were found in the spectral properties of DNA from women with normal and cancerous breast tissue, indicating an ability to discriminate cancerous tissue from noncancerous tissue with a sensitivity and specificity of 83%. Most importantly, the normal population was divided into subgroups in which a nonrandom progression was identified and a cancer-like DNA phenotype that was highly correlated (r > or = 0.90) with that of the patients with cancer was exhibited in 59% of the women. The spectral data, which also were highly correlated with the base-model data, were used to establish a model for predicting the probability of breast cancer. Consistent with the high cancer reoccurrence rate in the ipsilateral breast, 8 of 10 of the microscopically normal tissue specimens remaining after tumor excision were classified as cancerous using this model. CONCLUSIONS: Progressive structural changes in the DNA of the normal female breast, leading to a premalignant cancer-like phenotype in a high proportion of women, are the basis for a new paradigm for understanding the etiology of breast cancer and predicting its occurrence at early stages of oncogenesis. The results also suggest therapeutic strategies for potentially reversing the extent of DNA damage, which may be useful in disease prevention and treatment.

Oncogenes in hematopoietic and hepatic fish neoplasms.

Neoplastic transformation of cells has often been associated with changes in cellular oncogenes. While much information has been collected in mammalian systems, relatively little is known about the molecular basis of tumor progression in lower vertebrates. For our studies, tumors were collected from feral northern pike (Esox lucius) from Ostego Lake, MI, where the local population exhibited a 15% incidence of large external lymphomas. In laboratory studies, tumors were induced under controlled conditions by known mammalian carcinogens in the Japanese medaka (Oryzias latipes), a small aquarium fish widely used in carcinogenicity studies. DNA isolated from these tumors was assayed for transforming sequences by transfection into NIH3T3 cells. DNAs from the northern pike lymphomas and the chemically induced tumors in the medaka were able to transform NIH3T3 cells and induce tumors in athymic mice. The results of our studies to date are summarized here, together with the current status of oncogene activation in other fish systems.