Gender-specific growth and hepatic neoplasia in medaka (Oryzias latipes)

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Abstract

Brief exposure of hatchling medaka (Oryzias latipes), to diethylnitrosamine (DEN), resulted in hepatic tumor formation in female medaka at an incidence of 2–3-fold higher than that of their male cohorts. Spontaneous liver tumor incidence was reported in unexposed 3–5 year old medaka. Gender differences were seen; higher incidence was in the females. Aspects of gender-specific growth in hatchling, immature and sexually mature control medaka are reported and studies sought to determine whether growth enhanced tumorigenesis in females. From a pool of 2000 mixed-sex, 3 week old medaka hatchlings, 1350 were exposed to an aqueous bath of 250 ppm DEN for 48 h. Another 650 hatchlings served as controls. For each fish, body- and liver-weights were recorded (BW and LW, respectively) and LW to BW ratio (hepatosomatic index—HSI) was estimated. Next, livers and carcasses were processed for histopathology. BWs of control females were significantly greater than that of males at weeks 8, 20, 32 and 44 (P<0.05). LWs and HSIs were significantly greater in females versus males at all ages (P<0.05). In the DEN-treated medaka, female BWs were significantly more than their male counterparts at weeks 8, 16, 20 and 32 (P<0.05). Female LWs were greater than male values at all weeks except 4 and 6 (P<0.05). Female HSIs were significantly greater than male HSIs at all times (P<0.05). A higher incidence of foci of cellular alteration (40%) distinguished females from males (10%) at week 4 and these values reached 100% incidence (females) and 90% (males) at week 12. Tumor latency periods for adenomas and carcinomas were significantly shorter in females than in males. At week 20, the incidence of tumors was significantly higher in females than in males (P<0.05). Results indicate that gender-specific differences appear in BW, but especially LW and HSI as a function of larval development, ovarian maturation and age in control and DEN-treated medaka. Tumor incidence and time to endpoint (latency period) demonstrate that female growth is a promotional-stimulus, positively modulating DEN hepatocarcinogenesis.

Introduction

The small aquarium fish, medaka (Oryzias latipes), has been shown to be sensitive to a variety of chemical carcinogens in laboratory exposures (Ishikawa et al., 1975, Hatanaka et al., 1982, Hinton et al., 1984, Hawkins et al., 1985). Studies in our laboratory have centered on the pathogenesis of hepatic neoplasia in this model, emphasizing appearance, character (Hinton et al., 1987, Laurén et al., 1990b, Teh and Hinton, 1993) and fate of foci of cellular alteration, hepatic adenomas and their emergence as hepatocellular carcinomas (Teh and Hinton, 1993).

In recent bioassays using large numbers of larvae initiated by a brief, aqueous diethylnitrosamine (DEN) exposure, tumor frequency was consistently 2–3-fold greater in female than in male medaka (unpublished observations, this laboratory). This interesting finding was the impetus for the present study which was undertaken to determine features of developmental growth in control female and male medaka from larvae through sexual maturity and through the time necessary for hepatic tumors to be expressed. Then, a study of similar duration was conducted with medaka being briefly (48 h) exposed as 21-day-old larvae to an aqueous bath of 250 ppm DEN. In this way, the entire time frame for initiation, promotion and progression of hepatic neoplasia was included enabling an evaluation of growth and its effect on the neoplastic process. Results show that preferential hepatic growth of females serves as a promotional stimulus in hepatic neoplasia and results in the female fish being at greater risk.

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Water and diet

Reconstituted water for rearing fish was prepared according to the guidelines of the United States Environmental Protection Agency (USEPA) (Horning and Weber, 1985). All tanks were connected to a partially closed recirculating system equipped with water pump, UV tube, biological and charcoal filter. Water in the recirculating system was maintained at 80–100 mg l−1 CaCO3 (hardness), pH 8.0±0.1, 7.0±1.0 mg l−1 O2 and 25±2.0°C. Ammonia, nitrite and nitrate were kept below detectable limits by

DEN concentration

Concentration of DEN was within 5% of nominal concentration at 0, 24 and 48 h.

DEN toxicity

Acute DEN toxicity was seen during and for 5 days following cessation of exposure (Table 1). Mortality in DEN-treated fish over this period was 14.22% versus control mortality of 3.85% (Table 1). After fish were removed from carcinogen-bath and placed into separate individual tanks with freshly reconstituted water, subsequent mortality did not differ between groups until weeks 20 and 24. At these times, higher

Discussion

Several aspects of medaka growth proved interesting in this study. Medaka, like other fish, apparently grow continuously if food and free space are not limiting (Moyle and Cech, 1982, Weatherley and Gill, 1987, Wieser, 1993). The most rapid medaka growth occurred between week 4 and 12 of this study (Fig. 1, Fig. 2a). This corresponds with reports of maximal growth in other fish (Moyle and Cech, 1982). Although not individually determined, gonad weight, by retrospect, would have been an

Acknowledgements

Funded in part by US Public Health Service Grants CA-45131 from the National Cancer Institute, ES-04699 from the Superfund Basic Science Research Project, CR8191658-010 from the U.C. Davis, US E.P.A. Center for Ecological Health Research and by Grant #R823297 from the USEPA.

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