Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas.

We have cloned a medaka homolog of the human retinoblastoma (Rb) susceptibility gene. The medaka Rb cDNA encodes a predicted protein of 909 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the medaka Rb cDNA is highly conserved in regions of functional importance. An antibody raised against an epitope of the human pRb recognizes the protein product of the medaka Rb gene, detecting a 105 kDa protein in all tissues examined and at differential levels for the stages of embryonic development studied. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated a preliminary investigation of the molecular etiology of chemically-induced retinoblastoma. The mutational alterations characterized suggest that medaka may provide a novel model and, thus, provide additional insight into the human retinoblastoma condition.

Four resource centers for fishes: specifies, stocks, and services.

A conference on "Aquaria Fish Models of Human Disease" was held September 20-23, 2000, at Southwest Texas State University, San Marcos, Texas, USA. The meeting was sponsored by the National Cancer Institute (National Institutes of Health), the Roy and Joan Mitte Foundation, and Southwest Texas State University, home of the Xiphophorus Genetic Stock Center. In conjunction with the meeting, the conference organizers asked several participants to describe those components of their research programs that provide services and information to other researchers. This article summarizes their responses.

The factor structure of the Minnesota Satisfaction Questionnaire and participants of vocational rehabilitation.

This study investigated the factor structure of the Minnesota Satisfaction Questionnaire when used by individuals with psychiatric disabilities who are participating in vocational rehabilitation. The sample consisted of 87 respondents recruited from Columbus, Ohio, in 1996 who worked in noncompetitive employment. Factor analyses with varimax rotation conducted on the short-form of the questionnaire indicated three factors, an intrinsic factor and an extrinsic factor (as proposed by the Herzberg two-factor theory) as well as another pertaining to satisfaction derived from participating in vocational rehabilitation.

Life cycle of Calyptospora funduli (Apicomplexa: Calyptosporidae).

The taxonomic status of the extraintestinal piscine coccidium Calyptospora funduli is based in part on its requirement of an intermediate host (the daggerblade grass shrimp Palaemonetes pugio). In the present study, grass shrimp fed livers of Gulf killifish (Fundulus grandis) infected with sporulated oocysts of C. funduli exhibited numerous sporozoites suspended in the intestinal contents when fresh squash preparations were examined by light microscopy. Using this method, sporozoites were not seen in intestinal epithelial cells of the grass shrimp or in any other cell types. Ultrastructural examination, however, revealed sporozoites in the cytoplasm of the gut basal cells. Cross-sections of 1-13 sporozoites were seen within a single cell, and those sporozoites each appeared to be situated in individual membrane-bound vesicles, rather than in a single parasitophorous vacuole. These ultrastructural observations indicate that in the grass shrimp intermediate host, sporozoites that develop into an infective stage probably undergo that development in gut mucosal basal cells. Prior studies revealed that these sporozoites modified their structure over 4-5 days and that before that time, they were not infective to the fish host. Following ingestion of an infected shrimp by a killifish, the infective sporozoites apparently reach the liver of their killifish definitive hosts through the bloodstream. Sporozoites were seen in blood smears from the longnose killifish, Fundulus similis, 4 hr after fish were fed experimentally infected grass shrimp. Additionally, coccidian trophozoites and early meronts were seen in hepatocytes from several longnose killifish at 48, 72, and 96 hr postinfection. This study, in conjunction with previous findings, clearly confirms that a true intermediate host is required in the life cycle of C. funduli, that a developmental period of about 5 days in grass shrimp is necessary for sporozoites to become infective to killifishes, and that sporozoites do occur intracellularly in gut basal cells of the grass shrimp.

Proliferative lesions in swimbladder of Japanese medaka Oryzias latipes and guppy Poecilia reticulata.

Thirteen cases of proliferative lesions of the swimbladder were encountered in Japanese medaka Oryzias latipes and guppy Poecilia reticulata from about 10,000 medaka and 5000 guppies used in carcinogenicity tests and histologically examined. Two of the 4 cases from medaka and 8 of the 9 from guppies occurred in untreated control specimens. The lesions affected the gas gland epithelium and included hyperplasia, adenoma, and adenocarcinoma. One medaka had hyperplasia of the gas gland epithelium and in 1 guppy the gland was enlarged with an increase in the number of epithelial layers. Gas gland adenomas, 3 cases in medaka and 1 in the guppy, were typically larger than the hyperplastic lesions, formed expansive masses up to 1 mm in greatest dimension, and exhibited a solid or glandular growth pattern and mild cellular pleomorphism. Adenocarcinoma was the most advanced lesion and all 7 cases occurred in guppies. Adenocarcinomas sometimes filled the entire swimbladder and measured up to 2.5 mm in diameter. Cells of adenocarcinomas were highly pleomorphic, with atypical nuclei, and an elevated mitotic activity. Because most of these tumors occurred in fish from control groups or in tests with noncarcinogenic compounds, the lesions observed here are probably spontaneous rather than chemically induced. Their rare occurrence, however, makes swimbladder proliferative lesions in small-fish carcinogenesis models sensitive indicators of compounds that might target cells of the gas gland.

N-nitrosodiethylamine initiation of carcinogenesis in Japanese medaka (Oryzias latipes): hepatocellular proliferation, toxicity, and neoplastic lesions resulting from short term, low level exposure.

To investigate relationships among carcinogen exposure, cell proliferation, and carcinogenesis, 14-day post-hatch Japanese medaka (Oryzias latipes) were exposed to 0, 10, 25, 50, or 100 ppm N-nitrosodiethylamine (DEN) for 48 h under static renewal conditions. They were then held in clean water until sampling at 3 and 6 months. The frequencies of hepatic lesions and neoplasms were determined from hematoxylin/eosin-stained paraffin sections. A significant (p < 0.0001) concentration-related increase in hepatic vacuolated foci occurred in 3- and 6-month samples, with males having a significantly (p = 0.02) higher incidence than females. Concentration-related increases in degenerative lesions were documented for spongiosis hepatis at 3 months (p = 0.053) and hepatic vacuoles at 6 months (p = 0.005). There was a significant (p = 0.0001) concentration-related increase in macrophage aggregates at 6 months. Basophilic foci were significantly related (p < 0.0001) to DEN concentration at 3 months post-exposure and were unaffected by gender or age. At both 3 and 6 months, there were significant concentration-related increases in hepatocellular carcinoma (p < or = 0.02). Hepatocyte proliferation in 3-month whole specimens was quantified using an immunohistochemical assay for proliferating-cell nuclear antigen. Trend tests and a probit dose-response model showed a significantly positive correlation (p = 0.015) between proliferating hepatocytes and DEN concentrations. These results confirm that short-term exposure to low and moderate levels of DEN initiates concentration-dependent carcinogenic effects in medaka that are apparent at 3 months postexposure. DEN could be an effective initiator in an initiation/promotion assay for medaka using a 48-h exposure period, DEN concentrations < or = 10 ppm, and a 6-month sampling period.

Depressive symptomatology and specificity of social support.

Psychosocial interventions should be based on research which reflects the multidimensionality of psychosocial constructs. This study investigated six categories of social support which were associated with depressive symptomatology in a sample of 531 college students. The Social Provisions Scale was used to measure social support while the Center for Epidemiological Studies-Depression Scale was used to measure depressive symptomatology. Standard multiple regression analysis was used to analyze the variance contributed by each of the six categories of social support found on the Social Provisions Scale to depressive symptomatology. Specifically, the following categories of social support were significantly associated with depressive symptomatology: Reassurance of Worth (for male and female respondents) and Attachment (for females only). Implications for practice and research are discussed.

Do actions speak louder than words? Perceived peer influences on needle sharing and cleaning in a sample of injection drug users.

Past research (Dielman, Butchart, Shope, & Miller, 1990; Kandel, 1980) has found peer influence, perceived peer norms and perceived peer behavior as the strongest predictors of drug use in adolescent and young adult populations. The purpose of this study was to investigate whether verbal persuasion (peer norms) and/or observation of peer behavior (modeling) were significantly associated with the injection practices of unclean needle sharing and needle cleaning of 642 high risk for HIV infection active injection drug users in Baltimore, Maryland, in 1991 and 1992. Using regression analyses to examine interview reports of infection practices, it was determined that subjects who reported observing more peer protective HIV-related behavior were also more likely to report lower frequencies of HIV risk behavior (unclean needle sharing) and increased frequencies of HIV protective behavior (always cleaning needles). Reports of verbalizations of peer norms about reducing risk were not associated with decreased HIV risk behavior. Reports of "encouragement by peers to engage in cleaning needles" was paradoxically related to increased risk of sharing unclean needles. In conclusion, peer behavior rather than verbal persuasion appears to influence injection practices.

Job satisfaction and intent to continue working among individuals with serious mental illness.

This study investigated the relationship between job satisfaction and the intention to continue working in a sample of 87 individuals with psychiatric disabilities who worked in supported or supervised employment. Data were collected from respondents in Columbus, Ohio in 1996. After controlling for selected correlates (education, length of employment, contract type, social support, and attitudes concerning pay and toward work), standard multiple regression analyses indicated that job satisfaction was a significant predictor of intent to continue working. Analyses also indicated that respondents who completed high school and who said that they worked primarily for the money were less likely to want to continue working at current jobs. These findings may be used to improve the vocational success of individuals with psychiatric disabilities who work in supported and supervised employment.

Carcinogenic effects of 1,2-dibromoethane (ethylene dibromide; EDB) in Japanese medaka (Oryzias latipes).

The carcinogenicity of 1,2-dibromoethane (ethylene dibromide; EDB) was investigated in the Japanese medaka (Oryzias latipes), a small fish species. EDB was administered in water continuously for 97 days to a low concentration group, for 73 days to an intermediate concentration group, and intermittently for 24 h once each week over 97 days to a high concentration group. Medaka were 7 days old at the beginning of the tests. Mean measured EDB concentrations in the ambient water were 0.13 mg l-1, 6.20 mg l-1, and 18.58 mg l-1 in the low, intermediate, and high concentration groups, respectively. Two control groups, one inside and one outside the exposure apparatus, were used. Samples were examined histologically at 24, 36, and 58 weeks from the beginning of the tests. EDB was clearly carcinogenic to medaka in the intermediate and high concentration groups causing (1) hepatocellular adenomas and carcinomas, (2) cholangiomas, (3) chloangiocarcinomas, and (4) gall bladder papillary adenomas and adenocarcinomas. In separate studies, medaka exposed to 1.0 mg l-1 EDB for 2 to 5 weeks had elevated hepatic glutathione S-transferase activities, possibly indicating induction of a pathway that forms the reactive metabolite of EDB in mammals. SDS-PAGE of hepatic cytosolic fractions of EDB-exposed medaka showed a pronounced increase in a band at 26,000 Da, the expected position for GSH-S-transferase. Although little is known about EDB's mechanisms of action, medaka appear exceptionally sensitive to the carcinogenic effects of EDB and could serve as a model test species for studying similar compounds.

Depressive symptoms and HIV-risk behavior in inner-city users of drug injections.

The association of HIV risk-taking behavior with frequency of depressive symptoms varied by specific risk behavior and amount of harm reduction of behavior of men and women (N = 642), 80% of whom were African-American.

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.

Effect of concentration problems on the malleability of children's aggressive and shy behaviors.

OBJECTIVE: Previous research has demonstrated the central role of early childhood concentration problems in the development of aggression and other maladaptive behaviors. The present study investigated the moderating effect of concentration problems on the impact of a classroom-based preventive intervention directed at aggressive and shy behaviors in an epidemiologically defined sample of 1,084 urban first-grade children. METHOD: Concentration problems, aggressive behavior, and shy behavior were assessed by a structured teacher interview (the Teacher Observation of Classroom Adaptation-Revised) in the fall and spring of first grade. RESULTS: Children with high ratings on concentration problems in the fall had higher levels of teacher-rated aggressive and shy behavior in the spring than did children without such problems. The intervention reduced aggressive and shy behavior in children regardless of fall concentration level. Boys, but not girls, in the intervention condition with high concentration problems had higher levels of spring aggression than those without such problems, but they also showed the greatest reductions in aggressive behavior from fall to spring. CONCLUSIONS: These results suggest that aggressive behavior is malleable in children with concentration problems, provide further evidence on the etiological significance of concentration problems for the development of maladaptive behavior, and highlight the importance of directly targeting concentration problems to maximize preventive intervention impact.

Gender differences of reported safer sex behaviors within a random sample of college students.

This study investigated the frequency of safer sex behaviors with a random sample of sexually active college students (N = 315) at a university in the Northwest. The most frequent safer sex behaviors were discussion of contraceptives (58.6%), being more selective (46.5%), and reducing the number of sexual partners (43.6%). The least frequent safer sex behaviors included discussion of partner's sexual health prior to sexual behavior (26.1%), using condoms or dental dams (24.4%), one sexual partner (22.6%), and abstaining from sex as a safer sex practice (12.3%). The only two behaviors which indicated gender differences were (a) if they were being more selective as a safer sex practice and (b) reducing number of sexual partners as a safer sex practice. Women were more likely to state that they were " almost always" more selective than their male peers. Findings from this study indicated that a substantial number of students reported "risk factors" sexual practices. These findings indicated a need for HIV-prevention efforts.

Relationships among petroleum refining, water and sediment contamination, and fish health.

Water, sediment, and fish were sampled from three streams that were receiving or had received effluents from oil refineries. Water and sediment samples were analyzed by gas chromatography/mass spectrometry. Each stream contained aromatic carbons including substituted benzenes and naphthalenes, which are related to oil refinery operations. Fish were identified, counted, and examined for external lesions. Lengths and weights were recorded for older bullhead catfish, and their livers were examined histologically. Differences were seen in the diversity and abundance of fish among the upstream, impacted (effluent-receiving), and downstream stations. In one stream, differences in liver pathology were observed between reference bullhead, collected from an upstream station, and those collected at impacted stations with more than 50% of the bullheads taken from impacted stations having some sort of pathological change, including one with a liver clear-cell focus, which is considered a preneoplastic lesion in rodents. These data suggest a correlation between contamination of water and sediments with aromatic hydrocarbons, presumably from refinery effluents, and compromised fish health.

Pigmented subcutaneous spindle cell tumors in native gizzard shad (Dorosoma cepedianum).

An epizootic of pigmented subcutaneous spindle cell tumors affected nearly 25% of the adult gizzard shad (Dorosoma cepedianum) sampled from Lake of the Arbuckles in central Oklahoma over a 2 year period. Grossly, the tumors were primarily distributed over the head, trunk and fins as superficial raised masses that were almost always darkly pigmented. Histologically, they were located in the dermis, had a variable amount of connective tissue, and consisted of cells in a variety of forms and arrangements. Most tumors were composed of fusiform or spindle cells arranged in wavy bundles, whirling patterns or interwoven fascicles. Pigmentation was attributed to large dense deposits of melanin or to scattered individual melanin-containing cells. Immunohistochemical detection of proliferating cell nuclear antigen revealed a high proliferative activity in the spindle cells. Electron microscopy showed that the tumors were composed of several cell types, including host reactive cells, melanocytes in stages of maturity, and fibroblast-like cells. Tumor cells had neither cell-to-cell junctions nor an external lamina. Although the cell of origin of the tumors was not identified, evidence points toward melanocytes or, possibly, nerve sheath cells. However, an origin from fibroblasts or some other poorly differentiated cell cannot be ruled out. The etiology of the tumors was not determined. Fractionation of lake water and sediment samples followed by GC-MS analysis revealed no carcinogenic compounds. A retroviral etiology is unlikely because assays for reverse transcriptase in tumor homogenates were negative, and no evidence of viral particles was found in specimens examined by electron microscopy.

Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.

Long-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.

Detection of proliferating cell nuclear antigen in tissues of three small fish species.

An immunohistochemical assay for proliferating cell nuclear antigen (PCNA) identifies cells in all active phases of the cell cycle. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and western mosquitofish (Gambusia affinis) that are used in carcinogenesis bioassays and environmental sentinel studies. Our study showed that PCNA can be identified in routinely processed, paraffin embedded specimens of these fishes. Optimum staining conditions were dependent on fixative, primary antibody, antigen retrieval processing, and protein blocking reagent. Best results were achieved using 10% neutral buffered formalin as the fixative, clone PC10 as the primary antibody, and a combination of powdered milk and bovine serum albumin as a protein block. Except for medaka specimens, antigen retrieval was not required for specimens preserved in 10% neutral buffered formalin, but was required for the other fixatives tested. In whole fish specimens, PCNA marked cells in normally proliferating tissues such as testis, ovary, primary filament epithelium of the gill, hematopoietic tissues, thymus, retina and alimentary tract. The study demonstrated the successful application of mammalian-based PCNA technology to these aquatic species. Further applications of the assay will aid in understanding the role of cell proliferation in normal, diseased, and toxicant-affected tissues of aquatic animals.

Hepatocarcinogenesis in western mosquitofish (Gambusia affinis) exposed to methylazoxymethanol acetate.

To assess the potential of western mosquitofish (Gambusia affinis) for the detection of environmental carcinogens, laboratory-reared specimens were exposed to methylazoxymethanol acetate 10 mg per 1 for 2 h and then examined periodically for the onset of neoplastic lesions. Approximately 33 per cent of the exposed fish developed liver neoplasms within 25 weeks of exposure and 52 per cent within 40 weeks. The lesions were mostly hepatocellular carcinomas and cholangiocarcinomas. No neoplastic lesions were detected in other organs. The carcinogen sensitivity and the widespread distribution of the mosquitofish suggest that this species would be useful as a warm water sentinel for environmental contamination.