Promotion of hepatocarcinogenesis by perfluoroalkyl acids in rainbow trout.

Previously, we reported that perfluorooctanoic acid (PFOA) promotes liver cancer in a manner similar to that of 17ß-estradiol (E2) in rainbow trout. Also, other perfluoroalkyl acids (PFAAs) are weakly estrogenic in trout and bind the trout liver estrogen receptor. The primary objective of this study was to determine whether multiple PFAAs enhance hepatic tumorigenesis in trout, an animal model that represents human insensitivity to peroxisome proliferation. A two-stage chemical carcinogenesis model was employed in trout to evaluate PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS), and 8:2 fluorotelomer alcohol (8:2FtOH) as complete carcinogens or promoters of aflatoxin B(1) (AFB(1))- and/or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced liver cancer. A custom trout DNA microarray was used to assess hepatic transcriptional response to these dietary treatments in comparison with E2 and the classic peroxisome proliferator, clofibrate (CLOF). Incidence, multiplicity, and size of liver tumors in trout fed diets containing E2, PFOA, PFNA, and PFDA were significantly higher compared with AFB(1)-initiated animals fed control diet, whereas PFOS caused a minor increase in liver tumor incidence. E2 and PFOA also enhanced MNNG-initiated hepatocarcinogenesis. Pearson correlation analyses, unsupervised hierarchical clustering, and principal components analyses showed that the hepatic gene expression profiles for E2 and PFOA, PFNA, PFDA, and PFOS were overall highly similar, though distinct patterns of gene expression were evident for each treatment, particularly for PFNA. Overall, these data suggest that multiple PFAAs can promote liver cancer and that the mechanism of promotion may be similar to that of E2.

Cancer chemoprevention by dietary chlorophylls: a 12,000-animal dose-dose matrix biomarker and tumor study.

Recent pilot studies found natural chlorophyll (Chl) to inhibit carcinogen uptake and tumorigenesis in rodent and fish models, and to alter uptake and biodistribution of trace (14)C-aflatoxin B1 in human volunteers. The present study extends these promising findings, using a dose-dose matrix design to examine Chl-mediated effects on dibenzo(def,p)chrysene (DBC)-induced DNA adduct formation, tumor incidence, tumor multiplicity, and changes in gene regulation in the trout. The dose-dose matrix design employed an initial 12,360 rainbow trout, which were treated with 0-4000ppm dietary Chl along with 0-225ppm DBC for up to 4weeks. Dietary DBC was found to induce dose-responsive changes in gene expression that were abolished by Chl co-treatment, whereas Chl alone had no effect on the same genes. Chl co-treatment provided a dose-responsive reduction in total DBC-DNA adducts without altering relative adduct intensities along the chromatographic profile. In animals receiving DBC alone, liver tumor incidence (as logit) and tumor multiplicity were linear in DBC dose (as log) up to their maximum-effect dose, and declined thereafter. Chl co-treatment substantially inhibited incidence and multiplicity at DBC doses up to their maximum-effect dose. These results show that Chl concentrations encountered in Chl-rich green vegetables can provide substantial cancer chemoprotection, and suggest that they do so by reducing carcinogen bioavailability. However, at DBC doses above the optima, Chl co-treatments failed to inhibit tumor incidence and significantly enhanced multiplicity. This finding questions the human relevance of chemoprevention studies carried out at high carcinogen doses that are not proven to lie within a linear, or at least monotonic, endpoint dose-response range.

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.

Rainbow trout (Oncorhynchus mykiss) and ultra-low dose cancer studies.

Cancer risk assessment utilizing rodents requires extrapolation across five orders of magnitude to estimate the Virtually Safe Dose (VSD). Regulatory agencies rely upon the Linear Extrapolated Dose (LED) except when sufficient information on mechanism of action justifies alternative models. Rainbow trout (Oncorhynchus mykiss) has been utilized at Oregon State University as a model for human cancer for forty years. Low cost and high capacity, made possible by our unique facility, along with low spontaneous background and high sensitivity, allow design and conduct of statistically challenging studies not possible in rodents. Utilization of custom microarrays demonstrates similarities in gene expression in trout and human hepatocellular carcinoma (HCC). We have completed one study employing over 42,000 trout with dibenzo[a,l]pyrene (DBP) and determined the dose resulting in 1 additional cancer in 5000 animals, a 50-fold enhancement over the mouse ED(01) study. Liver tumor incidence at low dose deviated significantly from linearity (concave down), whereas, DBP-DNA adductions deviated slightly (convex up). A second study is underway with aflatoxin B(1) (AFB(1)). Results to date indicate AFB(1) at low dose, in contrast to DBP, elicits a linear dose-response function on the log-log scale which falls below the LED with a slope slightly greater than 1.0. Such studies demonstrate the statistical power of the trout cancer model and strengthen the case for incorporation of these data-sets into risk assessment for these environmental human carcinogens.

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 B(1) 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 17beta-estradiol (E(2), 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 beta-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E(2) by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E(2). 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.

Low-dose dietary chlorophyll inhibits multi-organ carcinogenesis in the rainbow trout.

We recently reported that chlorophyll (Chl) strongly inhibits aflatoxin B(1) preneoplasia biomarkers in rats when administered by co-gavage (Simonich et al., 2007. Natural chlorophyll inhibits aflatoxin B1-induced multi-organ carcinogenesis in the rat. Carcinogenesis 28, 1294-1302.). The present study extends this by examining the effects of dietary Chl on tumor development, using rainbow trout to explore ubiquity of mechanism. Duplicate groups of 140 trout were fed diet containing 224 ppm dibenzo[a,l]pyrene (DBP) alone, or with 1000-6000 ppm Chl, for 4 weeks. DBP induced high tumor incidences in liver (51%) and stomach (56%), whereas Chl co-fed at 2000, 4000 or 6000 ppm reduced incidences in stomach (to 29%, 23% and 19%, resp., P<0.005) and liver (to 21%, 28% and 26%, resp., P<0.0005). Chlorophyllin (CHL) at 2000 ppm gave similar protection. Chl complexed with DBP in vitro (2Chl:DBP, K(d1)=4.44+/-0.46 microM, K(d2)=3.30+/-0.18 microM), as did CHL (K(d1)=1.38+/-0.32 microM, K(d2)=1.17+/-0.05 microM), possibly explaining their ability to inhibit DBP uptake into the liver by 61-63% (P<0.001). This is the first demonstration that dietary Chl can reduce tumorigenesis in any whole animal model, and that it may do so by a simple, species-independent mechanism.

Protective effect of dietary tomatine against dibenzo[a,l]pyrene (DBP)-induced liver and stomach tumors in rainbow trout.

The potential anti-carcinogenic effects of tomatine, a mixture of commercial tomato glycoalkaloids alpha-tomatine and dehydrotomatine (10:1), were examined in the rainbow trout chemoprevention model. Prior to the chemoprevention study, a preliminary toxicity study revealed that tomatine in the diet fed daily at doses from 100 to 2000 parts per million (ppm) for 4 weeks was not toxic to trout. For the tumor study, replicate groups of 105 trout were fed diets containing dibenzo[a,l]pyrene (DBP) alone (224 ppm), (N = 3), DBP plus tomatine at 2000 ppm (N = 2), tomatine alone (N = 2), or control diet (N = 2) for 4 weeks. The fish were then returned to control diet for 8 months and necropsied for histopathology. Dietary tomatine was found to reduce DBP-initiated liver tumor incidence from 37.0 to 19.0% and stomach tumor incidence from 46.4 to 29.4%. Tomatine also reduced stomach tumor multiplicity. The tomatine-containing diets did not induce mortality, change in fish weights, or liver weights. No adverse pathological effects in the tissues of the fish on the tomatine diets were observed. Dose-response and chemopreventive mechanisms for tomatine protection remain to be examined. This is the first report on the anticarcinogenic effects of tomatine in vivo.

Gene expression analysis during tumor enhancement by the dietary phytochemical, 3,3'-diindolylmethane, in rainbow trout.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM), a primary I3C derivative, are known dietary chemopreventive agents also available as supplements. However, I3C has been found to act as a tumor promoter in rat (multi-organ) and trout (liver) models. I3C and DIM were previously found to be estrogenic in trout liver based on toxicogenomic profiles. In this study, we compare the post-initiation effects of DIM and 17beta-estradiol (E2) on aflatoxin B(1) (AFB(1))-induced hepatocarcinogenesis in trout. Trout were initiated as embryos with AFB(1) and juvenile fish were fed diets containing 0, 120 or 400 p.p.m. DIM or 5 p.p.m. E2 for 18 weeks. Tumor incidence was determined at 13 months and found to be significantly elevated in AFB(1)-initiated trout fed either 400 p.p.m. DIM or 5 p.p.m. E2 compared with control animals. To evaluate the mechanism of tumor enhancement, hepatic gene expression profiles were examined in animals fed promotional diets during the course of tumorigenesis and in hepatocellular carcinomas (HCCs) of initiated animals. We demonstrate that DIM alters gene expression profiles similar to E2 in liver samples during tumorigenesis and in HCC tumors. Further, HCCs from animals on DIM and E2 promotional diets had a transcriptional signature indicating decreased invasive or metastatic potential compared with HCCs from control animals. Overall, these findings are the first to demonstrate tumor promotion by DIM. They confirm the importance of estrogenic signaling in the mechanism of promotion by dietary indoles in trout liver and indicate a possible dual effect that enhances tumor incidence and decreases potential for metastasis.

The importance of carcinogen dose in chemoprevention studies: quantitative interrelationships between, dibenzo[a,l]pyrene dose, chlorophyllin dose, target organ DNA adduct biomarkers and final tumor outcome.

Chlorophyllin (CHL) is a potent antimutagen in vitro, an effective anti-carcinogen in several animal models, and significantly reduced urinary biomarkers of aflatoxin B(1) (AFB(1)) exposure in a human population. Here we report an expanded analysis of CHL chemoprevention using the potent environmental hydrocarbon dibenzo[a,l]pyrene (DBP). A dose-dose matrix design employed over 12 000 rainbow trout to evaluate the interrelationships among dietary carcinogen dose, anti-carcinogen dose, carcinogen-DNA adduct levels at exposure and eventual tumor outcome in two target organs. Included was an evaluation of the pharmaceutical CHL preparation (Derifil), used previously in a study of individuals chronically exposed to AFB(1). CHL was pre-, co- and post-fed at doses of 0-6000 p.p.m. and co-fed with DBP at doses of 0-371.5 p.p.m. for 4 weeks. This protocol generated a total of 21 dose-dose treatment groups, each evaluated with three or more replicates of 100 animals. The DBP-only treatment produced dose-responsive increases in liver and stomach DBP-DNA adducts, whereas increasing CHL co-treatment doses produced successive inhibition in liver (49-83%) and stomach (47-75%) adduct levels at each DBP dose examined. The remaining 8711 trout were necropsied, 10 months later. DBP treatment alone produced a logit incidence versus log [DBP] dose-response curve in stomach that was linear; CHL co-treatment provided dose-dependent tumor inhibition which ranged from 30 to 68% and was predictable from the adduct response. The Derifil CHL preparation was also found to effectively reduce DNA adduction and final tumor incidence in stomach (as well as liver), with a potency compatible with its total chlorin content. Liver tumor incidence in the DBP-only groups appeared to plateau near 60%. At DBP doses of

Use of a rainbow trout oligonucleotide microarray to determine transcriptional patterns in aflatoxin B1-induced hepatocellular carcinoma compared to adjacent liver.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, and its occurrence is associated with a number of environmental factors including ingestion of the dietary contaminant aflatoxin B(1) (AFB(1)). Research over the last 40 years has revealed rainbow trout (Oncorhynchus mykiss) to be an excellent research model for study of AFB(1)-induced hepatocarcinogenesis; however, little is known about changes at the molecular level in trout tumors. We have developed a rainbow trout oligonucleotide array containing 1672 elements representing over 1400 genes of known or probable relevance to toxicology, comparative immunology, carcinogenesis, endocrinology, and stress physiology. In this study, we applied microarray technology to examine gene expression of AFB(1)-induced HCC in the rainbow trout tumor model. Carcinogenesis was initiated in trout embryos with 50 ppb AFB(1), and after 13 months control livers, tumors, and tumor-adjacent liver tissues were isolated from juvenile fish. Global gene expression was determined in histologically confirmed HCCs compared to noncancerous adjacent tissue and sham-initiated control liver. We observed distinct gene regulation patterns in HCC compared to noncancerous tissue including upregulation of genes important for cell cycle control, transcription, cytoskeletal formation, and the acute phase response and downregulation of genes involved in drug metabolism, lipid metabolism, and retinol metabolism. Interestingly, the expression profiles observed in trout HCC are similar to the transcriptional signatures found in human and rodent HCC, further supporting the validity of the model. Overall, these findings contribute to a better understanding of the mechanism of AFB(1)-induced hepatocarcinogenesis in trout and identify conserved genes important for carcinogenesis in species separated evolutionarily by more than 400 million years.

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 10(6) 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.

Temperature-modulated carcinogenicity of 7,12-dimethylbenz[a]anthracene in rainbow trout.

Temperature-modulated hepatic disposition, covalent binding of radiolabeled genotoxin to hepatic DNA, and cancer incidence in rainbow trout (Oncorhyncus mykiss) were assessed after a single exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Fish (2 g) were acclimated at 10, 14, or 18 degrees C for 1 mo and then exposed to 1 ppm DMBA in their water for 20 h. Exposures were at respective acclimation temperatures, or 10 and 18 degrees C acclimated fish were shifted to 14 degrees C for DMBA exposures. After 4 but not 20 h of exposure, hepatic [(3)H]DMBA equivalents increased with temperature for fish exposed at their respective acclimation temperatures (10 or 18 degrees C). Covalent binding of [(3)H]DMBA to hepatic DNA was similar after 3 d in fish exposed at their respective acclimation temperatures. However, in fish exposed at 14 degrees C, after 3 d the concentration of [(3)H]DMBA covalently bound to hepatic DNA was higher in 10 degrees C than 18 degrees C acclimated fish. After 21 d, covalent binding of [(3)H]DMBA to hepatic DNA was less persistent in 18 degrees C than 10 degrees C acclimated, exposed, and reared fish. There were no differences between temperature-shifted groups at that time. Temperature effects on tumor incidence were assessed 9 mo after DMBA waterborne exposures in fish that were reared at (1) their respective acclimation and exposure temperatures, (2) 14 degrees C after exposure at their respective acclimation temperature, and (3) 14 degrees C after 14 degrees C exposures. Incidence of stomach, liver, and swimbladder cancer increased dramatically with rearing temperature. Differences in tumor incidence were less marked in fish reared at the same temperature (14 degrees C). A strong negative correlation between liver tumor incidence and persistence of [(3)H]DMBA equivalents covalently bound to hepatic DNA suggested increased error-prone DNA repair at warmer temperature played an important role in increased tumor incidence.