Jerry D. Hendricks

NEOPLASMS IN RAINBOW TROUT, A SENSITIVE ANIMAL MODEL FOR ENVIRONMENTAL CARCINOGENESIS*

The following topics are discussed: susceptibility of rainbow trout (Salmo gairdneri) to aflatoxins and other mycotoxins; the Oregon test diet; carcinogenic effects of cyclopropenoid fatty acids; carcinogenic response of the rainbow trout embryo; metabolism of mycotoxins by rainbow trout; and pathogenesis of rainbow trout liver cancer. (HLW)

Neoplasia in Zebrafish (Danio rerio) Treated with 7,12-Diniethylbenz[a]anthracene by Two Exposure Routes at Different Developmental Stages

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

The Sensitivity of Rainbow Trout and Other Fish to Carcinogens

Systematic design of replacement chemicals with reduced toxicities will require knowledge of mechanisms of action of parent compounds, especially in species which occupy the environment of most likely exposure. For aquatic systems, the rainbow trout has proven a valuable model for studying mechanisms of carcinogenicity. By comparison, small aquarium species show great potential as in situ field monitors of aquatic contamination by toxic chemicals but are less developed for mechanism studies. Fish species, especially rainbow trout, have also proven useful alternatives to traditional rodent models for comparative studies on mechanisms of action of nonaquatic carcinogens. These kinds of comparative studies form an essential basis for extrapolation of animal studies to man. Carcinogenicity testing of individual compounds and their replacements can provide only limited information on the expected impact of such chemicals on natural populations, since these populations are unavoidably exposed to potent modulators of the carcinogenic response. Hence any program which aims at redesign of commercial chemicals with reduced toxicities must have as a prior aim the full understanding of the mechanisms of joint carcinogen-inhibitor-promotor interactions. Because of their high sensitivity, low cost per individual, and low background tumor incidences, fish models such as the rainbow trout may be the only vertebrate models in which it is economically practical to initiate such complex studies.

Mechanisms of anti-carcinogenesis by indole-3-carbinol. Studies of enzyme induction, electrophile-scavenging, and inhibition of aflatoxin B1 activation.

The induction of oxidation and conjugation enzymes, the scavenging of carcinogen electrophiles, and the inhibition of aflatoxin B1 (AFB1) activation were examined as possible mechanisms of anti-carcinogenesis by indole-3-carbinol (I3C). Liver microsomal 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase activities were not induced significantly in rainbow trout fed diets containing 500-2000 ppm I3C for 8 days compared to trout fed the control diet. Furthermore, no detectable changes in the specific contents of cytochrome P-450 isozymes LM2 and LM4b, as measured by Western-blotting and immunoquantitation, were found in liver microsomes following dietary I3C administration. Dietary I3C had no significant effect on liver microsomal uridine diphosphate-glucuronyl-transferase activity, measured using the substrates 1-naphthol and testosterone, or on cytosolic glutathione S-transferase activity, measured using the substrate styrene oxide. The ability of I3C or its acid reaction products (RXM; generated by the reaction of I3C with HCl) to act as scavengers for the direct alkylating agent AFB1-8,9-Cl2 was examined. Addition of I3C or RXM to in vitro incubations did not inhibit the covalent binding of AFB1-8,9-Cl2 to calf thymus DNA. Kinetic analyses of microsome-mediated binding of AFB1 to DNA in vitro indicated that RXM inhibited the metabolic activation of AFB1. RXM increased the apparent Km for the AFB1-DNA binding reaction without changing the associated Vmax; the apparent Km values at 0, 3.5, 35, and 350 microM RXM were 35, 38, 66, and 86 microM for trout liver microsomes. RXM also inhibited the activation of AFB1 by rat liver microsomes, but I3C was not an effective inhibitor against AFB1-DNA binding mediated by either rat or trout liver microsomes. The results of the present study indicate that inhibition of microsome-activated AFB1 binding to DNA by I3C products may be of significant importance in I3C inhibition of hepatocarcinogenesis in trout and other species. The inhibition of carcinogen activation by I3C is contrasted with the mechanism of anti-carcinogenesis by beta-naphthoflavone, which involves induction of xenobiotic metabolizing enzymes.

Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout

Background Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. Objectives In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. Methods We fed aflatoxin B1 or sham-initiated animals 200–1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17β-estradiol (E2, a known tumor promoter) in the diet for 14 days. Results PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal β-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E2 by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E2. Conclusions These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation.

Promotion of aflatoxin B1 hepatocarcinogenesis in rainbow trout by 17β-estradiol

Abstract Four groups of 21-day-old rainbow trout ( Oncorhynchus mykiss ) embryos were exposed to static solutions of water containing 0, 0.005, 0.025, or 0.125 ppm aflatoxin B 1 (AFB 1 ) for 30 min to initiate hepatic neoplasms. Six weeks after AFB 1 exposure, two groups at each dose level were subjected to dietary 17 β-estradiol (E 2 ) treatment, while the two remaining groups received control diet (CD) only. E 2 promoted the hepatic tumor incidence in the fish exposed to the 0.025 ppm subcarcinogenic dose of AFB 1 from 0% to 9%, and enhanced the incidence in the fish exposed to the 0.125 ppm dose of AFB 1 from 5% to 60%. E 2 alone was not carcinogenic under the present experimental conditions. This promotion of AFB 1 carcinogenesis was observed despite significant mortalities and reduction in growth in the E 2 -exposed fish. In a second experiment, 21-day-old embryos were exposed to 0.025 ppm AFB 1 or sham-treated as before. Duplicate groups were started on either CD or CD-containing 5, 10 or 15 ppm E 2 . At each level, E 2 diets were fed every other week, alternating with CD, throughout the experiment. This regimen again produced promotion of AFB 1 carcinogenesis by E 2 . The intermittent exposure to E 2 still resulted in significant growth depression, but E 2 -related mortalities were eliminated. Biochemical studies revealed that E 2 stimulated hepatic ornithine decarboxylase (ODC) activity and DNA synthesis, both indicators of cellular proliferation which may be related to promotional events.

Nonlinear cancer response at ultralow dose: a 40800-animal ED(001) tumor and biomarker study.

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures and by procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well-suited to ultralow-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10000 animals (ED(001)). A total of 40800 trout were fed 0-225 ppm dibenzo[a,l]pyrene (DBP) for 4 weeks, sampled for biomarker analyses, and returned to control diet for 9 months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED(10) linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10000 animals at the lowest dose (that is, an unmodeled ED(0002) measurement). Among nine statistical models explored, three were determined to fit the liver data well-linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED(10) default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 x DBP(1.31)). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED(10)(-6)) that were 500-1500-fold higher than that predicted by the five-order LED(10) extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

Quantitative carcinogenesis and dosimetry in rainbow trout for aflatoxin B1 and aflatoxicol, two aflatoxins that form the same DNA adduct

Two exposure protocols were used to establish complete dose-response relationships for the hepatic carcinogenicity and DNA adduction in vivo of aflatoxin B1 (AFB1) and aflatoxicol (AFL) in rainbow trout. By passive egg exposure, AFL was taken up less well than AFB1, but was more efficiently sequestered into the embryo itself, to produce an embryonic DNA binding curve that was linear with carcinogen dose and with a DNA binding index three-fold greater than AFB1. Both aflatoxins produced the same phenotypic response, predominantly mixed hepatocellular/cholangiocellular carcinoma. Tumor responses as logit [incidence] vs. In [dose] were parallel-offset, non-linear responses showing a three-fold greater carcinogenic potency for AFL at all doses examined (i.e. 3 times more AFB1 than AFL required to produce an equivalent liver tumor incidence). By molecular dosimetry analysis (logit [incidence] vs. In [DNA adducts]), the two data sets were coincident, indicating that, per DNA adduct formed in vivo in total embryonic DNA, these two aflatoxins were equally efficient in tumor initiation. By dietary fry exposure, both carcinogens produced linear DNA binding dose responses in liver, but with an AFL target organ DNA binding index only 1.14 times that of AFB1 by this exposure route. The tumor dose-response curves also did not exhibit the three-fold difference shown by embryo exposure, but were closely positioned non-linear curves. Since the DNA binding indices differed by only 14%, the resulting molecular dosimetry curves for AFL and AFB1 by dietary exposure were similar to the tumor response curves. These results indicate that differing exposure routes produced differing relative carcinogenicity estimates based on doses applied, as a result of protocol-dependent differences in AFL and AFB1 pharmacokinetic behaviors, but that potency comparisons based on molecular dose received were similar for the two protocols. By comparison with standard DNA adducts produced in vitro using the dimethyloxirane-produced 8,9-epoxides of AFB1 and AFL, we conclude that > 99% of AFL-DNA adducts produced in vivo were identical to those produced by AFB1. Thus similar molecular dosimetry responses should be expected under all exposure protocols in which the two parent carcinogens do not exhibit differing toxicities to the target organ.

Metaplastic pancreatic cells in liver tumors induced by diethylnitrosamine

Metaplastic pancreatic cells have been infrequently observed in fish liver tumors induced by chemical carcinogens. An investigation with nitrosamine-exposed trout was undertaken to characterize the relationships of metaplastic pancreatic cells with other cell types. Eight-week-old rainbow trout (Salmo gairdneri) were fed a control diet or diets containing 500 ppm beta-naphthoflavone (BNF), 2000 ppm indole-3-carbinol (13C), or 100 ppm Aroclor 1254 (PCB) for 6 weeks. The fish were then exposed to 250 ppm diethylnitrosamine for 24 hr in an aqueous aquarium bath and reared on control diet for 39 weeks postexposure. Livers were excised, processed to paraffin sections, and stained with hematoxylin and eosin for histological evaluation. Metaplastic pancreatic cells were found only in tumors. Of the tumors with metaplastic pancreatic cells, 100/105 (95.2%) contained neoplastic cholangiolar components. Only 5/105 (4.76%) were hepatocellular carcinomas. 13C pretreatment inhibited the incidence of cholangiolar tumors (cholangioma 3.6% vs 31.3%, cholangiocarcinoma 3.6% vs 13.0%) and metaplastic pancreatic cells (5.1% vs 19.1%), whereas BNF and PCB had no effect. A hepatocellular origin for metaplastic pancreatic cells is supported. Cholangiolar neoplasia is associated with the expression, growth, or survival of metaplastic pancreatic cells in liver tumors. Hepatocarcinogenicity should not be described entirely by hepatocellular events since cholangiolar and metaplastic pancreatic cells can respond associatively to carcinogens and dietary modulators.

The rainbow trout (Oncorhynchus mykiss) tumor model: Recent applications in low-dose exposures to tumor initiators and promoters

The rainbow trout has been utilized as a model for human carcinogenesis for a number of years. Trout are relatively inexpensive to maintain and exhibit (over the 9—12-month tumor assay period) very low spontaneous tumor backgrounds. One of the most powerful applications of this model is the design and conduct of large-scale tumor studies requiring thousands of animals that address statistically challenging questions of dose-response. Two recent examples of such applications include our studies on I3C as a tumor promoter and DBP as a tumor initiator. I3C was shown to promote AFB1- initiated liver cancer at doses near those recommended for supplementation in humans. Further studies are required to determine if the mechanisms responsible for promotion in trout can be extrapolated to humans. In the second example, we report results from the largest animal tumor study ever conducted. A total of 42,000 trout were utilized to measure DBP carcinogenesis down to incidences of 1 in 5,000. The dose response model deviated significantly from linearity although the existence of a threshold could not be statistically established. Extrapolation of the data model predicts a DBP dose producing 1 in 106 cancers that is 1,000-fold higher than predicted by the conservative linear model. If these results can be confirmed with other carcinogens (genotoxic and perhaps nongenotoxic) and other targets, this could have a significant impact on the utilization of animal tumor data in human risk assessment.