William E. Hawkins

Diagnostic Criteria for Degenerative, Inflammatory, Proliferative Nonneoplastic and Neoplastic Liver Lesions in Medaka (Oryzias latipes): Consensus of a National Toxicology Program Pathology Working Group

Diagnostic criteria are presented for degenerative, inflammatory, nonneoplastic proliferative, and neoplastic lesions in the liver of medaka (Oryzias latipes), a small fish species frequently used in carcinogenesis studies. The criteria are the consensus of a Pathology Working Group (PWG) convened by the National Toxicology Program. The material examined by the PWG was from medaka exposed to N-nitrosodiethylamine for 28 days, removed to clean water, and sacrificed 4, 6, or 9 mo after initiation of exposure. Degenerative lesions included hepatocellular intracytoplasmic vacuolation, hepatocellular necrosis, spongiosis hepatis, hepatic cysts, and hepatocellular hyalinization. Inflammatory lesions consisted of granulomas, chronic inflammation, macrophage aggregates, and focal lymphocytic infiltration. Nonneoplastic proliferative lesions comprised foci of cellular alteration (basophilic focus, eosinophilic focus, vacuolated focus, and clear cell focus) and bile duct hyperplasia. Neoplastic lesions included hepatocellular adenoma, hepatocellular carcinoma, cholangioma, and cholangiocarcinoma. Two lesions composed mainly of spindle cells were noted, hemangiopericytoma and spindle cell proliferation. Rather than being an exhaustive treatment of medaka liver lesions, this report draws from the published literature on carcinogen-induced liver lesions in medaka and other fish species and attempts to consolidate lesion criteria into a simplified scheme that might be useful to pathologists and other researchers using medaka lesions for risk assessment or regulatory purposes.

Carcinogenicity tests with small fish species

Abstract Development of carcinogenesis bioassays that utilize small fish species is important principally for investigating the causes of neoplasms in wild fishes and for providing alternative or supplementary models to rodent carcinogenicity tests. Availability, economy, latency of tumorigenic response, and ease of maintenance and exposure are commonly cited advantages of small fish species in carcinogenesis bioassays. Carcinogen metabolism and mechanisms of carcinogenesis in several small fish species appear similar to those processes in the more thoroughly studied large fish species as well as in rodents. Recent studies suggest that small fish species are appropriate test models for waterborne carcinogens having a variety of mechanisms. Several small fish species readily develop hepatic and non-hepatic neoplasms following brief static exposures to direct-acting genotoxic compounds such as methylazoxymethanol acetate (MAM-Ac) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Tumorigenic responses appear to be related to species and to various exposure factors. Indirect-acting genotoxic carcinogens such as nitrosamines and polynuclear aromatic hydrocarbons have not been thoroughly tested in small fish species but appear to require longer exposures than direct-acting ones to induce hepatic neoplasms. An especially important potential use of small fish carcinogenesis bioassays is in testing carcinogens that might have epigenetic mechanisms, especially complex mixtures that might affect man or the environment but are difficult to test in rodent models. Preliminary studies indicate that prolonged exposures of up to 6 months to a mixture of halogenated organic compounds result in hepatic neoplasms in two small fish species. To improve their usefulness and exploit small fish carcinogenesis models better, gaps in several areas need to be filled. These include (1) a better understanding of biological and nutritional requirements of test species as related to carcinogenesis, (2) a broader database on neoplastic responses of various species to various chemicals, and (3) development of special exposure methods and standardization of test protocols.

Carcinogenic effects of some polycyclic aromatic hydrocarbons on the Japanese medaka and guppy in waterborne exposures

In this paper, we review studies on the carcinogenic effects of two polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP) and 7,12-dimethylbenz(a)anthracene (DMBA), on the Japanese medaka (Oryzias latipes) and the guppy (Poecilia reticulata). Exposure media were prepared by adding PAHs to water, with and without dimethylformamide (DMF) as a carrier, and passing this solution through various sized filters. The low exposure medium was a 0.45 micron filtrate without DMF that contained less than 5 micrograms L-1 PAH. The intermediate medium was a DMF-mediated 0.45 micron filtrate which contained 30-50 micrograms L-1 of PAH. The high medium was a DMF-mediated glass-fibre filtrate which contained 150-250 micrograms L-1 of PAH. Young fish specimens (6-10 d old) were given a 6 h exposure once weekly for 2 to 4 wk. Both BaP and DMBA induced hepatic neoplasms in the two species, with the medaka being more sensitive than the guppy, and DMBA being a stronger carcinogen than BaP. Liver neoplasms almost exclusively developed after exposure to BaP and the neoplasms were limited to the high concentration exposure, whereas DMBA caused a substantial number of extrahepatic neoplasms as well as hepatic ones, especially in the medaka. Furthermore, all three concentrations of DMBA induced hepatic neoplasms in the medaka and all but the low concentration induced neoplasms in the guppy. These studies demonstrate the carcinogenic effects of two waterborne PAHs on two small fish species following brief exposures to very low concentrations, and support the contention that environmental PAHs can contribute to the occurrence of cancer in wild fishes.

Dose-related carcinogenic effects of water-borne benzo [a] pyrene on livers of two small fish species

Benzo[a]pyrene (BaP) administered by water-borne exposures caused dose-related carcinogenic effects in livers of two small fish species, the Japanese medaka (Oryzias latipes) and the guppy (Poecilia reticulata). Medaka and guppies each were given two 6-h exposures. The first exposure was conducted on 6- to 10-day-old specimens. The second exposure was given 7 days later. The tests incorporated five treatment groups: (1) control, (2) carrier (dimethylformamide) control, (3) low BaP dose (not detectable--4 ppb), (4) intermediate BaP dose (about 8-47 ppb BaP), and (5) high BaP dose (200-270 ppb). Following the high-dose exposure, hepatocellular lesions classified as foci of cellular alteration (altered foci), adenomas, and hepatocellular carcinomas occurred in both species. In medaka, the lesions appeared to develop sequentially with the appearance of altered foci followed by adenomas and then hepatocellular carcinomas. Most lesions in guppies, however, were classified as altered foci although a few adenomas occurred in the early (24-week) sample and hepatocellular carcinomas occurred in the late (52-week) sample. When total lesions were combined, medaka had an 11% incidence at 24 weeks after the initial exposure and 36% incidence at 36 weeks. In guppies, 8% had liver lesions at 24 weeks, 19% at 36 weeks, and 20% at 52 weeks. A single extrahepatic neoplasm, a capillary hemangioma in a gill filament, occurred in a medaka from the 36-week high-dose sample. The results suggest that the medaka and guppy are capable of metabolizing water-borne BaP to carcinogenic metabolites which initiate hepatic tumor development.

Effect of 3-methylcholanthrene on monooxygenase, epoxide hydrolase, and glutathione S-transferase activities in small estuarine and freshwater fish

Abstract Monooxygenase activities, epoxide hydrolase, and glutathione S -transferase activities were measured in fractions prepared from pooled livers of three small estuarine fish species, Fundulus grandis, Cyprinodon variegatus , and Poecilia latipinna , and three freshwater species, Oryzias latipes, Peocilia reticulata , and Pimephales promelas . Interspecies differences in xenobiotic metabolizing activities of up to 10-fold were observed, with the smaller freshwater species generally having lower activities than the estuarine species. Groups of each species were injected with microliter volumes of corn oil or a solution of 3-methylcholanthrene (3MC) in corn oil, and enzyme activities measured 4–5 days later in pooled liver fractions from control or 3MC-treated fish. Although statistical analysis of induction effects was not possible, monooxygenase activities with ethoxyresorufin and benzo( a )pyrene were 2.5–15 times higher in 3MC-treated fish than in corn oil or untreated control groups. In 3MC-treated C. variegatus and F. grandis , microsomal cytochrome P-450 content was doubled and the spectral maximum of the CO-reduced microsomes was shifted from 449.6 to 448.5 nm. Microsomes from 3MC-treated C. variegatus and F. grandis metabolized benzo( a )pyrene 5–6 times faster than controls (per mg protein), but the proportion of 9,10- and 7,8-dihydrodiol metabolites was lower in 3MC-treated fish, suggesting that epoxide hydrolase may be rate limiting for production of dihydrodiols. 3MC treatment did not alter styrene oxide hydrolase or glutathione S -transferase activities at the time point tested.

Use of the Japanese medaka (Oryzias latipes) and guppy (Poecilia reticulata) in carcinogenesis testing under national toxicology program protocols.

A need exists for whole animal toxicity, mutagenesis, and carcinogenesis models that are alternative to the traditional rodent test models and that are economical, sensitive, and scientifically acceptable. Among small fish models, the Japanese medaka (Oryzias latipes) is preeminent for investigating effects of carcinogenic and/or toxic waterborne hazards to humans. The guppy (Poecilia reticulata ), although less widely used, is valuable as a comparison species. Both species are easy to maintain and handle in the laboratory and there is a large body of background information on their responsiveness to a range of classes of carcinogens. There are considerable data on the occurrence of background diseases and on spontaneous neoplastic lesions, both of which occur relatively rarely. With few modifications, the medaka and guppy are amenable to carcinogenicity testing under the rigid standards established by the National Toxicology Program (NTP) for rodent tests. The advantages of the small fish in carcinogenesis studies are best realized in long-term studies that involve environmentally realistic exposures. Studies to identify chronic effects can be conducted in about 12 months, near the life span of medaka in our laboratory. Practically, 9-month studies are optimal but shorter study cycles and a variety of exposure/growout and initiation/promotion scenarios are available. Studies on 3 compounds tested in medaka under NTP protocols are under review and preliminary analysis indicates that chronic carcinogenicity bioassays with medaka, guppy, and potentially with other small fish species are feasible and scientifically valid.

Rickettsial and mollicute infections in hepatopancreatic cells of cultured Pacific white shrimp (Penaeus vannamei)

Infections by multiple species of bacteria occurred in hepatopancreatic epithelial cells of cultured Pacific white shrimp (Penaeus vannamei). Grossly, hepatopancreases of moribund shrimp were pale white. Light microscopically, hepatopancreatic tubules appeared atrophied and were associated with granulomas. Examination by scanning and transmission electron microscopy revealed heavy cytoplasmic infections by three forms of microorganisms: (1) a rickettsia-like bacterium, (2) a helical form of a mollicute-like bacterium, and (3) a filamentous mollicute-like bacterium. The rod-shaped rickettsia (900 nm long by 300 nm wide) appeared to be free in the cytoplasm and had both a plasma membrane and a cell wall. Neither form of mollicute possessed a cell wall. The helical mollicute was blunt at its wide end (about 260 nm in diameter) where it contained electron-lucent bodies. Helical turns along its tapered axis resembled those of a spiroplasma (the only helical form of mycoplasma in the class Mollicutes) or a spirochete. The helical bacterium did not possess periplasmic flagella characteristic of spirochetes, which lends support to its being a type of spiroplasma. The filamentous mollicute consisted of masses of short, branched filaments 60 nm wide with intermittent spherical dilations and terminal blebs on the branches. The presumed mollicutes have not been reported previously in crustaceans. Each bacterium, or concurrent infections of the bacteria, are pathogenic to cultured shrimp, could impact culture operations and thus deserve more study.

Morphological Deformities as Biomarkers in Fish from Contaminated Rivers in Taiwan

Tilapia (Oreochromis spp.) were collected seasonally from four contaminated rivers in southwestern Taiwan for studies of morphological deformities that could be used as biomarkers of contamination. Morphological deformities found in tilapia were separated into 15 categories. Overall, the prevalence of deformities such as split fins, lower lip extension and gill deformities were significantly related to various water quality parameters, including low DO and high ammonium, lead and zinc concentrations. The persistence of tilapia in polluted waters and the development of a suite of morphological deformities suggest that tilapia can be used as sentinels of non-point source pollution in rivers.

Carcinogenicity Tests Using Aquarium Fish

In this chapter, the rationale, guidelines, and approaches for conducting small-fish carcinogenicity tests were presented. Goals were to demonstrate that the tests are relatively easy to conduct, that they require little in equipment or expertise in addition to that already available in a modern aquatic toxicology laboratory, and that the tests can approach the power and relevance of rodent carcinogenicity bioassays. Because the development of neoplasms following exposure to a carcinogenic substance is a latent or chronic response, a whole-animal carcinogenicity test using a fish model can be considered an elaboration or extension of routine aquatic toxicity tests that might otherwise have mortality or physiological perturbations as the principal end points.

LONG-TERM PRIMARY CULTURE OF EPITHELIAL CELLS FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) LIVER

SummaryLong-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged.The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.