The Utility of the Guppy (Poecilia reticulata) and Medaka (Oryzias latipes) in Evaluation of Chemicals for Carcinogenicity

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1 TOXICOLOGICAL SCIENCES 92(1), (2006) doi: /toxsci/kfj181 Advance Access publication March 31, 2006 The Utility of the Guppy (Poecilia reticulata) and Medaka (Oryzias latipes) in Evaluation of Chemicals for Carcinogenicity Grace E. Kissling,*,1 Naomi J. Bernheim, William E. Hawkins, Marilyn J. Wolfe, Micheal P. Jokinen, Cynthia S. Smith, Ronald A. Herbert, and Gary A. Boorman *Environmental Medicine and Diseases Program and Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; Gulf Coast Research Laboratory, University of Southern Mississippi, Ocean Springs, Mississippi 39566; Experimental Pathology Laboratories, Inc., Herndon, Virginia 20172; and Pathology Associates, Inc., Cary, North Carolina There has been considerable interest in the use of small fish models for detecting potential environmental carcinogens. In this study, both guppies (Poecilia reticulata) and medaka (Oryzias latipes) were exposed in the aquaria water to three known rodent carcinogens for up to 16 months. Nitromethane, which caused mammary gland tumors by inhalation exposure in female rats, harderian gland and lung tumors in male and female mice, and liver tumors in female mice by inhalation, failed to increase tumors in either guppies or medaka. Propanediol, which when given in the feed was a multisite carcinogen in both sexes of rats and mice, caused increased liver tumors in male guppies and male medaka. There was reduced survival in female guppies and no increased tumors in female medaka. 1,2,3-Trichloropropane, which when administered by oral gavage was a multisite carcinogen in both sexes of rats and mice, caused an increased incidence of tumors in the liver of both male and female guppies and medaka and in the gallbladder of male and female medaka. The results of this study demonstrate that for these three chemicals, under these specific exposure conditions, the fish appear less sensitive and have a narrower spectrum of tissues affected than rodents. These results suggest that fish models are of limited utility in screening unknown chemicals for potential carcinogenicity. Key Words: bioassays; small fish models; medaka; guppy; rodent carcinogens; nitromethane; propanediol; trichloropropane. The existence of a sensitive inexpensive vertebrate model for screening for potential carcinogens would have many advantages where there are more chemicals to evaluate than resources. Small fish models have been suggested as more sensitive, less costly, and quicker than traditional rodent models (Bailey et al., 1984; Ishikawa and Takayama, 1979; Reddy et al., 1999; Simon and Lapis, 1984; Sinnhuber et al., 1978; Walker et al., 1 To whom correspondence should be addressed at Environmental Medicine and Diseases Program, National Institute of Environmental Heath Sciences, MD A3-03, P.O. Box 12233, 111 T. W. Alexander Drive, Research Triangle Park, NC Fax: (919) kissling@niehs.nih.gov. Received February 15, 2006; accepted March 29, ). The National Toxicology Program (NTP) has found that other types of short-term tests have limitations for predicting carcinogenicity when systemically evaluated under controlled conditions in a contract laboratory situation (Zeiger et al., 1990). Most fish cancer studies use potent carcinogens that also cause liver tumors in rodents as well as in the fish models (Brown-Peterson et al., 1999; Hawkins et al., 1998; Law et al., 1998; Liu et al., 2003; Okihiro and Hinton, 1999). However, environmental chemicals may affect a variety of tissues in rodents, and often this spectrum provides clues as to the nature and mechanism of the carcinogenicity of the test chemical. Therefore, we decided to evaluate three chemicals that caused cancer in rodents, but generally not primarily of the liver, using two common small fish models. Two chemicals, 1,2,3-trichloropropane (TCP) and 2,2-bis(bromomethyl)-1,3- propanediol (BMP), are mutagenic and cause a wide spectrum of tumors in rodents. TCP when given by oral gavage in corn oil caused increased incidences of tumors of the oral cavity, forestomach, kidney, harderian gland, Zymbal gland, liver, uterus, pancreas, and other sites in rodents (NTP, 1993). BMP when given in the feed caused increased incidences of tumors of the mammary gland, skin, oral cavity, forestomach, intestines, harderian gland, Zymbal gland, lung, kidney, urinary bladder, and other sites in rodents (NTP, 1996). To determine how these fish models would respond to a less-potent carcinogen, nitromethane (NM), a nonmutagen with a more modest response in rodents was selected. NM did not cause tumors of male rats, but there was clearly a carcinogenic effect in female rats based on increased incidence of mammary gland tumors and clearly a carcinogenic effect in mice based on increased incidences of lung and harderian gland tumors (NTP, 1997). NM exposure was also associated with an increased incidence of liver tumors in female mice. Since chemical evaluations in rodents have benefited from careful attention to study details, we attempted to follow NTP procedures for chemistry, pathology, statistics, and QA evaluations. We also evaluated the data for carcinogenicity determinations as would be done in rodent studies. Thus, the result for each chemical was judged to be Published by Oxford University Press 2006.

2 144 KISSLING ET AL. positive, negative, equivocal, or inadequate for each sex and species combination. Our results indicate that for the three chemicals evaluated under maximally tolerated doses, the medaka and guppy appear less sensitive for a carcinogenic response than did the rodents. Further, while a variety of tissues showed a carcinogenic response in rodents, in the fish model only the liver and biliary system showed increased tumor rates with exposure. This suggests that caution is warranted in using these model systems for evaluating chemicals for which the carcinogenic potential is unknown. MATERIALS AND METHODS Fish. Guppy (Poecilia reticulata) and medaka (Oryzias latipes) fry used in the studies were obtained from established Gulf Coast Research Laboratory (GCRL) cultures and were approximately days old at study start. The fish were maintained in glass aquaria partially submerged in a single water bath to maintain temperature. Fish were approximately equally allocated to each of two duplicate aquaria per dose group. Aquaria were cleaned once weekly except for NM, where the aquaria were cleaned two (guppy) or three (medaka) times per week. Animals were fed flake fish food (Aqua-Tox Flake, Ziegler Brothers, Inc., Gardners, PA) and brine shrimp (Artemia) larvae (Aquarium Products, Glen Burnie, MD) once per day; shrimp were not fed to the fish during the last week of the study. The fish had a 16-h light period/8-h dark period with a 30-min transition period to simulate dawn and dusk. The aquaria temperature was 26 ± 1 C with an aquaria ph between 8.6 and 9.0. More complete details on the fish and animal care procedures are available in the NTP Technical Report (NTP, 2005), which is also available at niehs.nih.gov/. Chemicals. BMP, NM, and TCP were obtained from Aldrich Chemical Company (Milwaukee, WI), and each was found to be 99% pure. All three of the bulk chemicals were stored at approximately 4 C during the study. The stability of the bulk chemical was checked monthly during the study using gas chromatography (GC), and no contaminants were found. More details on the chemical analysis and spectra are available in the NTP Technical Report (NTP, 2005). Exposure solution generation. Exposure was intermittent flow-through and was conducted in a closed chamber similar to that described by Walker et al. (1985). Stock solutions of BMP, NM, and TCP were prepared by adding neat chemical to filtered and ultraviolet-sterilized well water in glass carboys. Dispensing pumps injected the stock solution into glass mixing/splitting boxes prior to the delivery of 2 l of filtered and ultraviolet-sterilized water to produce the required concentrations for the exposure aquaria. A water dispenser was timer regulated to perform at least five volume additions per day to each exposure aquarium. Filtered and ultraviolet-sterilized well water alone was delivered to the control aquaria. Aquaria volumes were maintained by an overflow drain siphon designed to remove water from near the bottom of each aquarium. The exposure delivery system has been described in detail (NTP, 2005). Exposure characterization. Exposure characterizations were performed prior to the start of the 14- and 16-month studies. The generation of the target concentrations of BMP in exposure aquaria by intermittent flow-through was determined with and without fish present. Duplicate aquaria target concentrations of 10, 35, and 100 mg/l were sampled prior to the first injection and 3, 6, 9, 12, 14, and 24 h after the initial injection. Target concentrations were reached by 24 h. Uniformity of BMP concentrations was measured by sampling nine locations at 2 cm below the surface and nine locations within 2 cm of the bottom of the aquaria. GC-analyzed samples demonstrated BMP uniformity. More detailed methods and results are available in the NTP Technical Report (NTP, 2005). NM exposure characterizations were similar to BMP. Duplicate aquaria target concentrations of 8.6, 24.5, and 70 mg/l were sampled prior to the first injection and every 2 h after the initial injection. Target concentrations were reached by 14 h. GC-analyzed samples demonstrated NM uniformity (NTP, 2005). TCP exposure characterizations were similar to BMP using similar concentrations and sampling times. Target concentrations were reached by 24 h. GC-analyzed samples demonstrated TCP uniformity (NTP, 2005). Concentrations of BMP, NM, and TCP in the water of the exposure aquaria were monitored using GC, approximately three times each week. Duplicate samples were analyzed from each aquarium. Table 1 summarizes the doses used for each chemical in the fish studies, as well as in comparative rodent studies. Pathology examination. All animals were observed twice daily. Visual external findings were recorded daily, and body weights and lengths were recorded at sacrifice after overexposure to a lethal concentration of tricaine methanesulfonate (MSZ22). Whole fish with the tails removed were fixed in Bouin s fixative for 96 h and then transferred to 10% neutral buffered formalin, processed, and embedded in paraffin. Five longitudinal step sections and two serials of each step were made, stained with hematoxyl and eosin and examined histologically by a study pathologist. The organs and tissues examined histologically in fish and in comparative rodent studies are listed in Table 2. A second pathologist reviewed all diagnoses in 40% of controls and fish from the high exposure concentration. Subsequently, all neoplasms in the target tissue, liver, were reviewed. Hepatocellular adenomas and hepatocellular carcinomas in fish share many features with rodent liver neoplasia, and similar diagnostic criteria were used for the medaka lesions (Boorman et al., 1997). A pathology working group consisting of pathologists experienced in rodent and/or fish pathology reviewed selected hepatic neoplasms. Details of specimen preparation and examination are included in the NTP Technical Report (NTP, 2005). Statistical analysis. The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Fish that were sacrificed or transferred to another tank were censored; fish found dead before the 9- month and terminal sacrifices were not censored. Possible dose-related trends in survival were tested with Tarone s (1975) life table test; pairwise comparisons with the control group were made with Cox s (1972) method for testing the equality of two groups. For each chemical, data were collected TABLE 1 Doses and Routes of Exposure Used in the Fish and Rodent Studies Fish Rodents Chemical Guppies Medaka Rats Mice BMP 0, 24, 50, 150 mg/l in water 0, 24, 60, 150 mg/l in water 0, 2500, 5000, 10,000 ppm in feed 0, 312, 625, 1250 ppm in feed NM 0, 10, 30, 70 mg/l in water 0, 10, 20, 40 mg/l in water 0, 94, 188, 375 ppm by inhalation 0, 188, 375, 750 ppm by inhalation TCP 0, 4.5, 9, 18 mg/l in water 0, 4.5, 9, 18 mg/l in water 0, 3, 10, 30 mg/kg by gavage 0, 6, 20, 60 mg/kg by gavage

3 USE OF SMALL FISH MODELS IN BIOASSAYS 145 TABLE 2 Tissues and Organs Examined Histologically in Fish and Rodents a Chemical Fish Rodents BMP NM TCP Bone, brain, chromaffin tissue, corpuscle of Stannius, esophagus, eye, gallbladder, gill, heart, hematopoietic tissue, interrenal tissue, intestine, kidney, liver, olfactory tissue, oral cavity, ovary, pancreas, peripheral nerve, pineal gland, pituitary gland, pseudobranch, skeletal muscle, skin, spinal cord, spleen, statoacoustic organ, swim bladder, testes, thymus, thyroid tissue, urinary bladder, gross lesions and tissue masses Bone, brain, chromaffin tissue, corpuscle of Stannius, esophagus, eye, gallbladder, gill, heart, hematopoietic tissue, interrenal tissue, intestine, kidney, liver, olfactory tissue, oral cavity, ovary, pancreas, peripheral nerve, pineal gland, pituitary gland, pseudobranch, skeletal muscle, skin, spinal cord, spleen, statoacoustic organ, swim bladder, testes, thymus, thyroid tissue, urinary bladder, gross lesions and tissue masses Bone, brain, chromaffin tissue, corpuscle of Stannius, esophagus, eye, gallbladder, gill, heart, hematopoietic tissue, interrenal tissue, intestine, kidney, liver, olfactory tissue, oral cavity, ovary, pancreas, peripheral nerve, pineal gland, pituitary gland, pseudobranch, skeletal muscle, skin, spinal cord, spleen, statoacoustic organ, swim bladder, testes, thymus, thyroid tissue, urinary bladder, gross lesions and tissue masses a Organs in bold were examined in both rodents and fish. from the two replicate tanks in the control group and at each dose level. Equality of tumor rates in the replicate tanks was tested with Fisher s exact test. For each combination of chemical, sex, and species, the treatment effects were analyzed separately. Dose-related trends in neoplastic and nonneoplastic lesions were tested with the Cochran-Armitage trend test, and pairwise comparisons with the control group were tested with Fisher s exact test. More statistical details are in the NTP Technical Report (NTP, 2005). Quality assurance methods. The fish studies were conducted in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58). An independent quality assurance contractor also audited the studies retrospectively. Audit procedures and findings are presented in reports that are available from National Institutes of Environmental Health Sciences (NIEHS) (Research Triangle Park, NC). Comparative rodent studies. The NTP conducted 2-year carcinogenicity studies for BMP (NTP, 1996), NM (NTP, 1997), and TCP (NTP, 1993). The detailed pathology results are also available online at nih.gov/. These data for male and female rats and mice were used for comparison with the results from the present studies in two fish species. RESULTS Each study was started with approximately 220 guppies and 340 medaka in each dose group. Twenty fish per dose group were removed after 9 months of exposure for examination. Of the remaining fish, approximately one-third were removed Adrenal gland, bone and marrow, brain, esophagus, gallbladder (mice), heart, kidney, large intestine (cecum, colon, and rectum), liver, lung, lymph nodes (mandibular or mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial or clitoral gland, prostate gland, salivary gland, skin, small intestine (duodenum, jejunum, and ileum), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, gross lesions and tissue masses Adrenal gland, bone and marrow, brain, clitoral gland, epididymis, esophagus, gallbladder (mice), harderian gland (mice), heart, kidney, large intestine (cecum, colon, and rectum), larynx, liver, lung, lymph nodes (bronchial, mandibular, mediastinal, and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, small intestine (duodenum, jejunum, and ileum), spinal cord and sciatic nerve, spleen, stomach (forestomach and glandular stomach), testis, thymus, thyroid gland, trachea, urinary bladder, uterus, gross lesions and tissue masses Adrenal gland, bone and bone marrow, brain, clitoral gland (rats), epididymis, esophagus, gallbladder (mice), heart, kidney, large intestine (cecum, colon, rectum), liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas (islets), parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicles, skin, small intestine (duodenum, jejunum, ileum), spleen, stomach (forestomach, glandular), testes, thymus, thyroid gland, trachea, urinary bladder, uterus, gross lesions and tissue masses from exposure and held in clean water while exposure was continued for the remainder. Fish were collected and examined at 16 months from both the continuous- and stop-exposure groups in the guppies and at months for the medaka. Sex was determined by histological examination, and the tumor incidences were then determined for each sex/species/dose/ chemical combination. Results of BMP Exposure in Guppies and Medaka Exposure of guppies to BMP at 24, 60, and 150 mg/l resulted in a dose-related decrease in survival for fish exposed continuously for 16 months and for fish where exposure was discontinued at 9 months (Table 3). There was no increased tumor incidence at any site in either sex for fish examined at 9 months. At 16 months, there was an increased incidence of hepatocellular adenomas and of hepatocellular carcinomas for male guppies in both the continuous-exposure and stopexposure groups but no increased incidence of tumors in female guppies (Table 4). Medaka exposed to the same doses did not show decreased survival in either the continuous- or stop-exposure groups (Table 3). There was no increase in tumors in male and female medaka at the 9-month interim sacrifice or in medaka exposed

4 TABLE 3 Survival Analyses for BMP, NM, and TCP BMP NM TCP 0 mg/l 24 mg/l 60 mg/l 150 mg/l 0 mg/l 10 mg/l 30 mg/l 70 mg/l 0 mg/l 4.5 mg/l 9 mg/l 18 mg/l Guppies a Number of animals initially in study Month interim evaluation Number of survivors Continuous-exposure study Stop-exposure study Survival analysis p values b Continuous-exposure study < < < < < < < Stop-exposure study < < < < < < < mg/l 10 mg/l 20 mg/l 40 mg/l Medaka a Number of animals initially in study Month interim evaluation Number of survivors Continuous-exposure study Stop-exposure study Survival analysis p values Continuous-exposure study N < < < Stop-exposure study 0.138N c N 0.077N < < a Fish could not be sexed at the beginning of the study, so numbers of fish are not separated by sex. b p Values in the control group column indicate significance of a dose-related trend in survival; p values in the dosed group columns indicate significance of pairwise comparison with the control group. c p Values with an N indicate negative trends or differences. ownloaded from by guest on 03 October KISSLING ET AL.

5 USE OF SMALL FISH MODELS IN BIOASSAYS 147 TABLE 4 Dose-Related Neoplasms in Guppies and Medaka Exposed to BMP 0 mg/l 24 mg/l 60 mg/l 150 mg/l guppies 9-Month interim evaluation Number of livers examined Hepatocellular adenoma Continuous-exposure study Number of livers examined Hepatocellular adenoma 3** 4 4 8* Hepatocellular adenoma or carcinoma 4** ** Stop-exposure study Number of livers examined Hepatocellular adenoma 0** 3 3 6** Hepatocellular carcinoma 0* Hepatocellular adenoma or carcinoma 0** 3 3 9** guppies Continuous-exposure study Number of livers examined Hepatocellular adenoma 2* Stop-exposure study Number of livers examined Cholangioma or cholangiocarcinoma 0* medaka Continuous-exposure study Number of livers examined Hepatocellular adenoma 1** Hepatocellular adenoma or carcinoma 1** 0 0 8* Stop-exposure study Number of livers examined Hepatocellular adenoma or carcinoma 1* medaka Continuous-exposure study Number of livers examined No dose-related neoplasms Stop-exposure study Number of livers examined Hepatocellular adenoma 1* Hepatocellular adenoma or carcinoma 1* *In the control column indicates a significant trend at p<0.05; * in the dose group columns indicate differences from control group at p<0.05. **In the control column indicate a significant trend at p<0.01; ** in the dose group columns indicate differences from control group at p<0.01. to BMP for 9 months and held to 14 months. In medaka continuously exposed for 14 months, only male medaka at 150 mg/l had tumors, which were restricted to the liver (Table 4). Comparison with Results of BMP Exposure in Rats and Mice BMP was given in the feed at 0, 2500, 5000, or 10,000 ppm for 2 years to male and female F344/N rats (NTP, 1996). Increased incidences of tumors of the skin, mammary gland, Zymbal s gland, oral cavity, esophagus, forestomach, small and large intestines, mesothelium, urinary bladder, lung, thyroid gland, hematopoietic system, and seminal vesicle were found in male rats with BMP exposure. In female rats, increased incidences of tumors of oral cavity, esophagus, mammary gland, and thyroid gland were found with BMP exposure (Table 5). BMP was given in the feed at 0, 312, 624, or 2500 ppm for 2 years to male and female B6C3F1 mice (NTP, 1996). Increased incidences of tumors of the harderian gland, lung, and kidney were seen in male mice while increased incidences of tumors of the harderian gland, lung, and skin were seen in female mice (Table 5). Results of NM Exposure in Guppies and Medaka Exposure of guppies to NM at 10, 30, and 70 mg/l resulted in decreased survival at 70 mg/l for both continuous- and stop-exposure groups (NTP, 2005) (Table 3). There was no increased incidence of tumors at the 9-month interim sacrifice or at 16 months for both the continuous-exposure and the stop-exposure groups in male guppies, but decreased survival at 70 mg/l may have limited the sensitivity of the study to detect a carcinogenic response. There was no increase in tumors at any site in female guppies (Table 6). At 13 months, medaka exposed to NM at 10, 20, and 40 mg/l did not show decreased survival in either the continuousexposure or the stop-exposure groups (Table 3). Increased tumors were not found at the 9-month interim sacrifice. At 13 months, there were occasional liver tumors in exposed males but no significantly increased incidence at any site. medaka exposed to NM failed to show an increase in tumors at any site in either the continuous-exposure or the stopexposure groups (Table 6).

6 Sex/species guppies guppies TABLE 5 Bases of Carcinogenicity Calls in Rodent and Fish Studies of BMP Fish Rodents (NTP, 1996) medaka Neoplastic effects Liver a,b None Liver a,b None Skin c ; subcutaneous skin tissue d ; mammary gland e ; Zymbal s gland f ; oral cavity g ; esophagus h ; forestomach h ; large intestine f ; small intestine f ; malignant mesothelioma; urinary bladder i ; lung j,k ; thyroid gland l ; seminal vesicle f ; hematopoeitic system m medaka rats rats mice mice Oral cavity g ; esophagus h ; mammary gland n ; thyroid gland l Harderian gland f ; lung j ; kidney (renal tubule) o Harderian gland f ; lung j ; subcutaneous skin tissue p Uncertain effects None None None None Kidney (renal tubule) o ; pancreas q None Forestomach g Mammary gland r ; forestomach h ; circulatory system s Level of evidence of carcinogenic activity Positive Inadequate Positive Negative Clear evidence Clear evidence Clear evidence Clear evidence a Hepatocellular adenoma. b Hepatocellular adenoma or carcinoma. c Squamous cell papilloma, keratoacanthoma, trichoepithelioma, basal cell adenoma, basal cell carcinoma, or squamous cell carcinoma. d Fibroma, fibrosarcoma, or sarcoma. e Fibroadenoma or adenoma. f Adenoma or carcinoma. g Squamous cell papilloma or carcinoma. h Squamous cell papilloma. i Transitional cell papilloma or carcinoma. j Alveolar/bronchiolar adenoma or carcinoma. k Squamous cell carcinoma. l Follicular cell adenoma or carcinoma. m Mononuclear cell leukemia. n Fibroadenoma. o Adenoma. p Sarcoma. q Acinar adenoma. r Carcinoma. s Hemangioma and hemangiosarcoma. ownloaded from by guest on 03 October KISSLING ET AL.

7 USE OF SMALL FISH MODELS IN BIOASSAYS 149 TABLE 6 Dose-Related Neoplasms in Guppies and Medaka Exposed to NM 0 mg/l 10 mg/l 30 mg/l 70 mg/l guppies Core study Number of livers examined Hepatocellular adenoma 4* or carcinoma Stop-exposure study Number of livers examined Hepatocellular adenoma 5* N Hepatocellular adenoma 5* N or carcinoma guppies Core study Number of livers examined; no dose-related neoplasms Stop-exposure study Number of livers examined; no dose-related neoplasms medaka 0 mg/l 10 mg/l 20 mg/l 40 mg/l 9-Month interim evaluation Number of livers examined Cholangiocarcinoma Hepatocellular adenoma Core study Number of livers examined; no dose-related neoplasms Stop-exposure study Number of livers examined; no dose-related neoplasms medaka 9-Month interim evaluation Number of livers examined Hepatocellular adenoma Core study Number of livers examined; no dose-related neoplasms Stop-exposure Study Number of livers examined; no dose-related neoplasms *In the control column indicates a significant trend at p<0.05; * in the dose group columns indicate differences from control group at p<0.05. *N indicates a negative trend at p<0.05. Comparison with Results of NM Exposure in Rats and Mice NM was given by inhalation at 0, 188, 375, or 750 ppm 6 h/ day 5 days/week for 2 years to male and female F344/N rats (NTP, 1997). There were no increased incidences of tumors in male rats with NM exposure. In female rats, increased incidences of tumors of the mammary gland were found with NM exposure (Table 7). NM was given by inhalation at 0, 188, 375, or 750 ppm 6 h/ day 5 days/week for 2 years to male and female B6C3F1 mice (NTP, 1997). Increased incidences of tumors of the harderian gland and lung were seen in male mice while increased incidences of tumors of the harderian gland, lung, and liver were seen in female mice (Table 7). both males and females (Table 8). Tumors were of both hepatocellular and cholangiolar origin. Medaka exposed to the same doses showed decreased survival in both the continuous-exposure and the stop-exposure groups (Table 3). At the 9-month interim sacrifice, there was an increased incidence of cholangiocarcinomas in male medaka exposed to TCP and a marginal increase for the females (Table 8). At 13 months, male medaka exposed to TCP had increased cholangioma or cholangiocarcinoma in both the continuousand the stop-exposure groups (Table 8). medaka at 13 months had increased incidence of both hepatocellular and cholangiolar carcinomas in both the continuous- and the stopexposure groups (Table 8). Results of TCP Exposure in Guppies and Medaka Exposure of guppies to TCP at 0, 4.5, 9, and 18 mg/l resulted in a dose-related decrease in survival in both the continuousexposure and the stop-exposure groups (Table 3). There was no increased incidence of liver tumors at the 9-month interim sacrifice, but at 16 months, increased liver tumors were seen in both the continuous-exposure and the stop-exposure groups in Comparison with Results of TCP Exposure in Rats and Mice TCP was given at 0, 3, 10, or 30 mg/kg body weight by oral gavage 5 days/week for 2 years to male and female F344/N rats (NTP, 1993). Increased incidences of tumors of the oral cavity, forestomach, pancreas, kidney, preputial gland, and Zymbal s gland were found in male rats with TCP exposure. In female rats, increased incidences of tumors of oral cavity,

8 150 KISSLING ET AL. TABLE 7 Bases of Carcinogenicity Calls in Rodents and Fish Exposed to NM Fish Rodents (NTP, 1997) Sex/species guppies guppies medaka medaka rats rats mice mice Harderian gland b,d,e ; Harderian gland d,e ; Neoplastic effects None None None None None Mammary gland a,b,c lung f,g liver h,i ; lung f,g,j Equivocal findings None None Liver k,l None None None None None Level of evidence of carcinogenic activity Inadequate Negative Equivocal Negative No evidence Clear evidence Clear evidence Clear evidence a Fibroadenoma. b Carcinoma. c Fibroadenoma, adenoma, or carcinoma. d Adenoma. e Adenoma or carcinoma. f Alveolar/bronchiolar carcinoma. g Alveolar/bronchiolar adenoma or carcinoma. h Hepatocellular adenoma. i Hepatocellular adenoma or carcinoma. j Alveolar/bronchiolar adenoma. k Cholangioma. l Cholangioma or cholangiocarcinoma. forestomach, clitoral gland, mammary gland, and Zymbal s gland were found with TCP exposure (Table 9). TCP was given at 0, 6, 20, or 60 mg/kg body weight by oral gavage 5 days/week for 2 years to male and female B6C3F1 mice (NTP, 1993). Increased incidences of tumors of the forestomach, liver, and harderian gland were found in male and female mice with TCP exposure. mice also had increased incidences of tumors of the oral cavity and uterus with TCP exposure. DISCUSSION Long-term rodent studies are one of the major tools for determining potential carcinogenicity of environmental chemicals. Rodent studies are costly and time consuming; however, their strengths and weaknesses are fairly well understood (Bennett and Davis, 2002; Haseman, 2000). Limitations in current models (Alden et al., 1996; Eastin, 1998) and the desire to use fewer and lower phylogenetic animals (Salem and Katz, 1998) stimulated the NTP to evaluate two small fish models. Assays using fish species are reported to be more sensitive to carcinogens (Ishikawa et al., 1984; Liu et al., 2003; Zimmerer, 1984), less expensive, and faster to perform than rodent studies in carcinogenicity evaluations (Bailey et al., 1984; Ishikawa and Takayama, 1979; Sinnhuber et al., 1978; Walker et al., 1985). During the past 50 years, housing conditions, rodent chow, disease surveillance procedures, genetics, and standards for study conduct have been established for rodent studies. In contrast, for this study we used laboratory-raised (GCRL) medaka and guppies fed a combination of commercial fish flake food and brine shrimp. We used 16-h light and 8-h dark cycle, a photoperiod used by other investigators for long-term studies (Davis et al., 2002; Zimmerer, 1984). In spite of care to minimize disease, the fish in this study had evidence of bacterial, fungal, and parasitic disease. This may reduce the sensitivity of the fish model, particularly if it results in increased mortality. In rodent studies, dose selection is usually based on toxicokinetic studies, clinical pathology findings, in-life observations, and histopathology using 14-day and 90-day exposures. For the current fish studies, doses were determined using 2-day static exposures and 7-day and 28-day flowthrough range finding studies (NTP, 2005). Mortality and lethargy were the endpoints used in dose selection. The goal was to select a top dose that did not cause increased mortality by 16 months plus two lower doses. While the guppy could tolerate 16-month exposures, increased mortality was seen in the NM-exposed medaka, and the medaka studies were terminated at 13 or 14 months. This may not be surprising since guppies are considered to have a longer life span and to be less sensitive to carcinogens than the medaka (Hawkins et al., 2003). An oft-stated attribute of small fish models is that they are very sensitive to detecting potential carcinogens (Hawkins et al., 1985, 2003). All three chemicals selected for study were considered clearly carcinogenic for rodents, causing increased cancer incidence at multiple sites in all four sex/species combinations except for NM which was positive only in female rats and male and female mice (NTP, 1993, 1996, 1997). BMP and

9 USE OF SMALL FISH MODELS IN BIOASSAYS 151 TABLE 8 Dose-Related Neoplasms in Guppies and Medaka Exposed to TCP 0 mg/l 4.5 mg/l 9 mg/l 18 mg/l guppies 9-Month interim evaluation Number of livers examined Hepatocellular adenoma Continuous-exposure study Number of livers examined Cholangiocarcinoma 0** 0 0 3* Cholangioma or cholangiocarcinoma 0** 0 1 3* Hepatocellular adenoma 3** 8* 17** 9** Hepatocellular carcinoma 0** 1 0 7** Hepatocellular adenoma or carcinoma 3** 9* 17** 15** Hepatocholangiocarcinoma 0** Stop-exposure study Number of livers examined Hepatocellular adenoma 3* 4 5 7* Hepatocellular carcinoma 0** 0 1 4* Hepatocellular adenoma or carcinoma 3** ** guppies Continuous-exposure study Number of livers examined Cholangioma 0 3 4* 2 Cholangiocarcinoma 0** 0 0 6** Cholangioma or cholangiocarcinoma 0** 3 4* 8** Hepatocellular adenoma 5* Hepatocellular carcinoma 0* Hepatocellular adenoma or carcinoma 5** 4 4 8* Hepatocholangiocarcinoma 0* Stop-exposure study Number of livers examined Cholangiocarcinoma 0** 0 0 5* Cholangioma or cholangiocarcinoma 2** 2 0 7* medaka 9-Month interim evaluation Number of livers examined Cholangiocarcinoma 0** 2 5* 8** Hepatocellular adenoma Hepatocholangiocarcinoma Number of gallbladders examined Papillary adenoma Continuous-exposure study Number of livers examined Cholangioma 0 6* 2 1 Cholangiocarcinoma 0** 32** 43** 45** Cholangioma or cholangiocarcinoma 0** 38** 45** 46** Hepatocellular adenoma 0* Hepatocellular carcinoma 0* Hepatocellular adenoma or carcinoma 0** 2 1 6** Hepatocholangiocarcinoma 0** 0 2 6** Number of gallbladders examined Papillary adenoma 0** 0 4* 9** Stop-exposure study Number of livers examined Cholangiocarcinoma 0** 19** 16** 26** Cholangioma or cholangiocarcinoma 0** 19** 19** 27** Number of gallbladders examined Papillary adenoma 0** 0 3 4* medaka 9-Month interim evaluation Number of livers examined Cholangiocarcinoma 0* Cholangioma Hepatocellular carcinoma Continuous-exposure study Number of livers examined Cholangiocarcinoma 0** 29** 53** 41** Cholangioma or cholangiocarcinoma 0** 30** 55** 42** Hepatocellular adenoma 1** 1 5 8** Hepatocellular carcinoma 1** 1 2 5* Hepatocellular adenoma or carcinoma 2** **

10 152 KISSLING ET AL. TABLE 8 Continued 0 mg/l 4.5 mg/l 9 mg/l 18 mg/l Hepatocholangiocarcinoma 0** 0 4 7** Number of gallbladders examined Papillary adenoma 0** 1 5* 6** Stop-exposure study Number of livers examined Cholangiocarcinoma 1** 15** 31** 21** Cholangioma or cholangiocarcinoma 1** 16** 33** 21** Hepatocellular carcinoma 0** 2 1 4* *In the control column indicate a significant trend at p<0.05; * in the dose group columns indicate differences from control group at p<0.05. **In the control column indicate a significant trend at p<0.01; ** in the dose group columns indicate differences from control group at p<0.01. TCP were shown to be mutagenic (NTP, 1993, 1996) while NM was selected as a nonmutagenic chemical (NTP, 1997). The length of exposure that would be necessary for detection of carcinogenicity of these three chemicals was not known. The time for tumor induction in guppies has been reported to be from 3 to 11 months (Zimmerer, 1984), while medaka exposed to potent carcinogens may show liver tumors in 2 3 months (Hinton et al., 1984). Since animals that die early are often lost for examination, an interim sacrifice was included at 9 months for the detection of microscopic tumors and preneoplastic lesions. At 9 months, significantly increased incidences of tumors were seen only in TCP-exposed medaka; increased tumors were not seen in either species for the other chemicals by 9 months, including TCP-exposed guppies where increased tumors were seen by 16 months. This suggests that at least for these three chemicals, exposures less than 1 year are likely to be insensitive. For the medaka, where reduced survival limited the studies to 13 or 14 months, this could pose a problem for many chemical evaluations. A hypothesis tested during this study was that for toxic chemicals, the fish would live longer, and neoplasms might develop more rapidly if the chemical was withdrawn during the last few study months. For about one-third of the fish, the chemical exposure was stopped at 9 months, and the fish were allowed to live until 16 months (guppy) or months (medaka). A chemical-free observation period has been used in other fish studies (Simon and Lapis, 1984). We generally found a higher percentage of tumors and/or a greater spectrum of liver tumors in the groups continuously exposed versus those for which exposures were stopped at 9 months. In one case, BMPexposed male medaka, the stop-exposure group did not have any significant increase in liver tumors, but the continuousexposure group had increased hepatocellular adenomas or carcinomas at 14 months. This suggests that, at least under the conditions of this study, there is no advantage to stopping exposure and holding the fish. BMP waterborne exposure in medaka and guppies resulted in an increased incidence of tumors only in the males and only at the 150 mg/l concentration, which also resulted in decreased survival. The next lowest concentration at 60 mg/l failed to cause an increased tumor incidence in any of the four sex/ species combination. Ten out of 38 (26%) high-dose BMP male guppies had hepatocellular tumors compared to four out of 61 (7%) in the controls; medaka were similar with 8/59 (14%) high-dose BMP male medaka with hepatocellular tumors compared with 1/47 (2%) in controls. Thus, for BMP, medaka and guppy provided a very narrow dose window in which to detect an effect, and the response was not very robust. In contrast, male and female rats exposed to BMP at 0.25, 0.5, and 1% (rats) or 0.03, 0.06, or 0.12% (mice) in the feed had significantly increased tumor rates in 15 different tissues among the sex/species combinations (NTP, 1996). Several tissues showed an increase in tumors even at the lowest dose evaluated. In some cases, the increase was sufficient to suggest a carcinogenic response based on the lowest dose alone. For example, in female rats, 25/50 (50%) controls were diagnosed with fibroadenomas increasing to 45/51 (88%) at the lowest dose, and in female mice harderian gland tumors were 3/52 (6%) in controls versus 12/50 (24%) for the low dose (NTP, 1996). Similar, but not statistically significant, increases in tumors in the target tissues were also seen in males. This suggests that at least for BMP under the exposure conditions evaluated, rodents showed a wider spectrum of tumors with a greater range of doses giving a positive carcinogenic response. A similar pattern was seen for NM, a nonmutagenic chemical that caused increased tumors in female rats and male and female mice (NTP, 1997). NM evaluated at 10, 30, and 70 mg/l in guppies and 10, 20, and 40 mg/l in medaka did not cause increased tumors at any site in any sex/species combination. There was decreased survival at 70 mg/l, and because of early deaths, the male guppy study was judged to be inadequate. NM when given to F344/N rats at 94, 188, or 375 ppm by inhalation caused increased mammary gland tumors in females with combined tumors of 21/50 (42%) in controls versus 41/50 (82%) in the highest concentration. There were no tumors that were considered related to exposure in male rats. NM when given to B6C3F1 mice at 188, 375, or 750 ppm by inhalation caused increased harderian gland tumors and pulmonary tumors in males and females with increased liver tumors in female mice only (NTP, 1997). The harderian gland tumors

11 Sex/species guppies guppies TABLE 9 Bases of Carcinogenicity Calls in Rodents and Fish Exposed to TCP Fish Rodents (NTP, 1993) medaka Neoplastic effects Liver a,b,c,d Liver b,c,e Liver a,b,c,e,f Gallbladder g medaka rats rats mice mice Liver a,b,c,d,f Oral cavity h,i ; forestomach h,i ; Gallbladder g Pancreas j ; kidney (renal tubule) k ; preputial gland k,l ; Zymbal s gland l ; Oral cavity h,i ; forestomach h,i ; clitoral gland k,l ; mammary gland m ; Zymbal s gland l Forestomach h,i ; liver a,b ; harderian gland k Oral cavity i ; forestomach h,i ; liver a,b ; harderian gland k ; uterus k,m,n Uncertain findings None None None None Intestine m,o Intestine m Oral cavity h None Level of evidence of Positive Positive Positive Positive Clear evidence Clear evidence Clear evidence Clear evidence carcinogenic activity a Hepatocellular adenoma. b Hepatocellular adenoma or carcinoma. c Cholangiocarcinoma. d Hepatocellular carcinoma. e Cholangioma. f Hepatocholangiocarcinoma. g Papillary adenoma. h Squamous cell papilloma. i Squamous cell carcinoma. j Acinar adenoma. k Adenoma. l Carcinoma. m Adenocarcinoma. n Stromal polyp. o Adenomatous polyp. ownloaded from by guest on 03 October 2018 USE OF SMALL FISH MODELS IN BIOASSAYS 153

12 154 KISSLING ET AL. were significantly increased at both the 375 and 750 ppm concentrations for both male and female mice (NTP, 1997). This suggests that at least for NM under the exposure conditions evaluated, rodents showed a wider spectrum of tumors with a greater range of doses giving a positive carcinogenic response than fish in which no increase in neoplasia was seen in any tissue. The fish studies were further compromised by the nitrogeneous nature of NM that promoted bacterial growth in the aquaria, resulting in the need for frequent and aggressive cleaning that may have caused stress in the fish and increased mortality. TCP waterborne exposure at 4.5, 9, and 18 mg/l in medaka and guppies resulted in an increased incidence of tumors of hepatocellular and cholangiolar origin in both guppies and medaka. The combined hepatocellular tumors were 3/61 (5%) in control male guppies versus 15/27 (56%) in top dose males. The hepatocellular response was more modest in female guppies being 5/64 (8%) in controls versus 8/33 (24%) in the top dose. In the medaka, a dramatic increase in cholangiocarcinoma was seen with no tumors in the unexposed fish and with 45/78 (58%) males and 41/67 (61%) female medaka exposed to 18 mg/l. TCP when given to F344/N rats at 3, 10, or 30 mg/kg by oral gavage caused increased tumors at six sites in male and five tissues in females with increased incidences for most tumors at the lowest exposure. TCP when given to B6C3F1 mice at 6, 20, or 60 mg/kg body weight by oral gavage caused increased incidence of harderian gland tumors, forestomach tumors, and liver tumors in males and females with increased oral cavity and uterine tumors in female mice only (NTP, 1993). As with rats, tumor incidences were often increased at all three exposure concentrations. This suggests that at least for TCP under the exposure conditions evaluated, rodents showed a wider spectrum of tumors with a greater range of doses giving a positive carcinogenic response than fish where tumors response was restricted to the liver and gallbladder. Thus, for the three chemicals evaluated, the rodents in each case showed a wider variety of tissues and a broader range of doses with increased tumor incidences. It is important to note, however, that rodents treated with BMP, NM, and TCP exhibited neoplasms in a number of tissues that are not found in the fish species tested. These tissues include stomach, uterus, lungs, mammary glands, harderian glands, Zymbal s glands, preputial glands, clitoral glands, and seminal vesicles. In the BMP study, seven of the 15 target tissues in rodents were present and evaluated in fish; in the NM study, one of the four target tissues in rodents was present and evaluated in fish; and in the TCP study, three of the 11 target tissues in rodents were present and examined in fish. Another important consideration in comparing the fish studies to mammalian studies must be the differences in routes of exposure. The fish were exposed to chemicals in the water. In rodents, BMP was administered in the feed, TCP was administered by oral gavage, and NM was administered via inhalation. It needs to be cautioned that it is difficult to compare feed studies, oral gavage studies or inhalation studies in rodents with waterborne studies in medaka and guppies. Toxicokinetic studies and 90-day evaluations are very useful in dose selection. Certainly with more experience, one may be better able to select doses for small fish models. It would be difficult to argue that higher exposure levels would have resulted in a greater spectrum of neoplasia since there was increased mortality in the high dose for nearly all sex/species combinations. Increased mortality also had a confounding effect on body weight and length. In the high-dose aquaria having a lower density of animals due to decreased survival, fish were sometimes larger in both body weight and body length than controls. Other rodent carcinogens have also been reported not to cause increased tumors in medaka. Bromodichloromethane exposure at up to 1.4 mg/l for 9 months did not cause increased replication in the liver and was not hepatocarcinogenic (Toussaint et al., 2001). 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX), a potent mutagenic furanone that is found in drinking water, failed to cause hepatic mutations in the liver of transgenic medaka (Geter et al., 2004). The significance of this finding is unknown because while MX is a potent rat liver carcinogen (Komulainen et al., 1997), no consistent pattern of mutations is found in the induced tumors (Komulainen et al., 2000). The failure of trichlorethylene to promote liver tumors in medaka that had been initiated with diethylnitrosamine (DEN) is another example where a known carcinogen failed to cause cancer in a small fish model (Gardner et al., 1998). It is possible that a critical factor is the partition coefficient for the chemical. TCP was the most lipid soluble and gave the greatest carcinogenic response of the three chemicals we evaluated. In another study where DEN was used as an initiator, exposing medaka to groundwater that contained five heavy metals plus multiple chlorinated aliphatic hydrocarbons showed no evidence of being a complete carcinogen (Toussaint et al., 1999). In that study there was no consistent evidence of the complex water mixture acting as a tumor promoter (Toussaint et al., 1999). The perception that small fish models are exquisitely sensitive to waterborne containments may need to be revisited. Dibenzo[a]pyrene when given in the feed causes a high incidence of liver tumors in medaka (Reddy et al., 1999), suggesting that oral exposure, if palatable, may be an alternative route for chemicals where waterborne exposure is not appropriate. Low costs have been another attribute that have created considerable interest in small fish models. We were surprised to find that the savings were considerably less than anticipated. For example, having the entire fish on two or three slides would appear to engender considerable savings in pathology evaluations. However, trying to search for and evaluate nearly 30 tissues in several step sections was fairly time consuming and costly. In future studies, examination of only the tissues for which a carcinogenic response is expected could save considerable time, but potential information could be lost. We also found that attempting to follow Good Laboratory Practice procedures also added considerable costs to these studies.

13 USE OF SMALL FISH MODELS IN BIOASSAYS 155 The chemicals were selected because they caused cancer in a variety of tissues and not just the liver. We were surprised that the cancer response in fish was restricted to the liver except for TCP that also caused a modest increase in tumors of the bile duct. The limited fish cancer response appears less informative than the wide spectrum of tumors found in rodent studies. The loss of specimens during the study limited the information that was available for analysis. For example, time to tumor and presence of preneoplastic lesions could not be analyzed with the present study design. Multiple interim sacrifices may have been helpful. However, the 9-month sacrifice showed few or no tumors with these three chemicals, and the medaka had to be terminated at 13 or 14 months (16 months for guppy), providing a very small window of time in which to follow tumor development. One concern that was difficult to address was the possibility that some fish that developed tumors died early and were lost to the study. For example, all three guppy studies had reduced survival in the high-dose groups after 9 months when the groups were split into continued exposure and stop-exposure groups. The percentage of fish examined at final sacrifice varied between 55% in the NM study and 71% in the BMP study. It is not known if this resulted in an underestimation of the true tumor incidence or whether this affected the sensitivity of the model for detecting carcinogenicity. This study based on a limited number of chemicals suggests to us that routine use of small fish models in waterborne assays is likely to underestimate the number of chemicals that would demonstrate carcinogenic activity if evaluated in standard rodent studies. Small fish species continue to be excellent research models to address mechanistic questions for a variety of disease processes including carcinogenicity. This is especially true for zebra fish (Danio rerio) where the genome has been sequenced (Jekosch, 2004), and resources such as the Zebrafish Information Network (Rasooly et al., 2003) are available to support investigators who address mechanistic questions. ACKNOWLEDGMENTS This research was supported in part by the Intramural Research Program of the National Institutes of Health and National Institutes of Environmemtal Health Sciences. The in-life portion of the study was conducted under NIEHS contract NO1-ES to Gulf Coast Research Laboratories. The authors thank Drs Michael Cunningham and Robert Maronpot for their helpful suggestions. REFERENCES Alden, C. L., Smith, P. F., Piper, C. E., and Brej, L. (1996). A critical appraisal of the value of the mouse cancer bioassay in safety assessment. Toxicol. Pathol. 24, Bailey, G. 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