Mary E. Schultz

Cytochrome P4501A induction and inhibition by 3,3′,4,4′-tetrachlorobiphenyl in an Ah receptor-containing fish hepatoma cell line (PLHC-1)

The induction of cytochrome P4501A1 (CYP1A1) in rat hepatoma cells has been used by some investigators to determine ‘dioxin equivalents’ in environmental samples, including extracts of fish tissues. However, the relative potency of inducing compounds may vary between species, suggesting the need for taxon-specific model systems. In this paper we present an initial characterization of CYP1A induction in one such system, a teleost liver cell line (PLHC-1) derived from a hepatocellular carcinoma of Poeciliopsis lucida (Hightower, L.E. and Renfro, J.L., 1988. J. Exp. Zool. 248, 290). Specific binding of the photoaffinity ligand 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin([125I]N3Br2DD) to proteins in PLHC-1 cytosol indicated the presence of the Ah receptor, which is known to control CYP1A induction in mammals. 3,3′,4,4′-Tetrachlorobiphenyl (TCB) induced a microsomal protein in PLHC-1 cells that was recognized by monoclonal antibody (MAb) 1-12-3 to scup CYP1A1 (P450E) on immunoblots. Immunohistochemical staining of whole cells with MAb 1-12-3 showed specific recognition of CYP1A induced by TCB. No staining was seen in untreated or vehicle-treated cells. There was an excellent quantitative correlation between amounts of CYP1A protein detected immunohistochemically and in immunoblots of cell homogenates. In a dose response experiment, maximal induction of ethoxyresorufin O-deethylase (EROD) activity occurred at 0.1 μM TCB; at higher concentrations (1 and 10 μM), EROD activity was reduced as compared to the activity at 0.1 μM TCB. In contrast, immunoreactive CYP1A protein increased with increasing TCB concentration up to 10 μM. The loss of EROD activity at high concentrations of TCB did not result from changes in cell number or viability. The apparent inhibition or inactivation of CYP1A catalytic activity by the higher concentrations of halogenated biphenyls has been seen, but not generally recognized, both in vivo and in cultured cells from diverse vertebrate species. PLHC-1 cells may be a good model system for studying Ah receptor-mediated regulation of gene expression, for determining the fish-specific toxic or inducing potency of halogenated aromatic hydrocarbon congeners, and for investigating the mechanism of CYP1A inhibition or inactivation by environmental contaminants such as TCB.

Induction of hepatic tumors with 7,12-dimethylbenz(a)anthracene in two species of viviparous fishes (genus Poeciliopsis)

Liver neoplasms were induced in two species of viviparous fishes, Poeciliopsis lucida and P. monacha, by repeated short-term exposures to an aqueous suspension of 5 ppm 7,12-dimethylbenz[a]anthracene (DMBA). Predominantly hepatocellular neoplasms developed in 47 out of 106 fish surviving the period allowed for tumor development, 6–9 months after initial exposure to DMBA. The use of short-term exposure periods of 6 hr in young fish and up to 20 hr in adults reduced stress and mortalities caused by the toxicity of DMBA at 5 ppm. A lower concentration of DMBA, 0.25 ppm, failed to induce tumors in any of the surviving 230 of 295 young fish administered repeated 20-hr exposures. This study shows for the first time that DMBA is carcinogenic to fish.

PRODUCTION OF MALE GAMETES AND AUXOSPORES IN THE CENTRIC DIATOMS CYCLOTELLA MENEGHINIANA AND C. CRYPTICA12

Vegetative cells of Cyclotella meneghiniana Kützing form male gametes and anxospores following transfer from a synthetic freshwater medium to a modified artificial seawater. Both spermatogenesis and auxospore formation are correlated with an increase in the Na+ concentration in the medium. Spermatogenesis can also be induced in C. cryptica Reimann, Lewin, and Guillard in artificial seawater with an adjusted sodium level.

Transplantable chemically-induced liver tumors in the viviparous fish Poeciliopsis

Transplantation of five liver tumors induced with the chemicals diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) in small live-bearing fish of the genus Poeciliopsis is reported. Five permanent strains representing three distinct tumor types were established in isogenic hosts. Histological characterization of hepatocellular carcinoma, hemangiopericytic sarcoma, and cholangiocarcinoma and the development of the neoplasms in host fish is presented. Transplantability of the experimental liver tumors provides evidence of their malignant nature. Metastasis of the hepatocellular carcinoma occurred from tumor implants in the dorsal musculature or peritoneal cavity and from the hemangiopericytic sarcoma implanted in the intraperitoneal cavity.

Initiation of cell proliferation in livers of the viviparous fish Poeciliopsis lucida with 7,12-dimethylbenz[a]anthracene.

Stimulation of liver cell proliferation by sublethal exposures to 7,12-dimethylbenz[a]anthracene (DMBA) is examined in the small, viviparous fish Poeciliopsis lucida. Poeciliopsis is susceptible to induction of liver tumors by repeated short-term exposures, under 24 hr, to waterborne DMBA at 5 ppm. Exposures to 5 ppm for 24 hr was lethal to fish under 1 month old and resulted in 60% mortality of adult females 20-25 mm in length. Response to 16-, 20-, and 22-hr exposures of 5 ppm DMBA, as measured by mitotic index, was similar in females of two size classes, 20-25 mm and 26-30 mm. Differences were observed in the onset of mitosis in livers of fish exposed for 16 hr vs 20 or 22 hr. Hepatocyte proliferation did not begin until 10-11 days after the 16-hr exposure and lasted for only 3 days. When exposure was increased to 20 or 22 hr, mitotic activity was observed earlier, 2 days following treatment, and continued for 6-8 days. The peak period of cell proliferation also varied, occurring 12 days after a 16-hr exposure, 4 days after a 20-hr exposure, and at least 10 days after a 22-hr exposure. The mitotic index was the highest on the final day specimens of the 22-hr treatment were collected. These results suggest that the toxic properties of DMBA in stimulating cell proliferation may function as an important cofactor in hepatocarcinogenesis.

Heat induced liver cell proliferation in the livebearing fish Poeciliopsis

SynopsisLiver regeneration is induced by heat stress in the small viviparous fish, Poeciliopsis. Acute exposure to sublethal temperatures, one to two degrees below their killing temperature, damages tissue and initiates liver cell proliferation in P. lucida, P. monacha, and P. monacha-lucida hybrid clones, SYN-4 and SYN-5. Regeneration of liver cells began within 1–2 days following heat stress and proceeded over 5 days. Peak cell proliferation occurred 2–3 days after treatment in fish of all four genotypes. Cell proliferation was induced in the two all-female clones, SYN-4 and SYN-5, by exposure to 40.5° C for 60 minutes. This treatment imposed mortalities of 17.9% and 16.7%, respectively, whereas reduction of the temperature to 39.5°C and reduction of the time to 30 minutes resulted in no mortalities without significantly lowering the level of cell proliferation (p > 0.05). Liver cell proliferation induced by both heat treatments was significantly higher (p < 0.05) in the SYN-5 hybrids than in SYN-4. The induction of liver cell proliferation with sublethal temperature exposures is discussed as it may relate to chemical carcinogenesis in both feral and laboratory fish. Acute heat exposure may be used experimentally in fish as an independent stimulus for liver cell proliferation in carcinogenesis studies. In poikilothermic animals-heat exposure offers an alternative to surgical removal of approximately two-thirds of the liver, the method most frequently used in rodents to study the process of liver regeneration.