Review
The rainbow trout liver cancer model: Response to environmental chemicals and studies on promotion and chemoprevention

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Abstract

Rainbow trout (Oncorhynchus mykiss) are an outstanding model of liver cancer induction by environmental chemicals and development of strategies for chemoprevention. Trout have critical and unique advantages allowing for cancer studies with 40,000 animals to determine dose–response at levels orders of magnitude lower than possible in rodents. Examples of two promoters in this model, the dietary supplement dehydroepiandrosterone (DHEA) and industrial chemical perfluorooctanoic acid (PFOA), are presented. In addition, indole-3-carbinol (I3C) and chlorophyllin (CHL) inhibit initiation following exposure to potent human chemical carcinogens (e.g., aflatoxin B1 (AFB1)). Two “ED001” cancer studies have been conducted, utilizing approximately 40,000 trout, by dietary exposure to AFB1 and dibenzo[d,e,f,p]chrysene (DBC). These studies represent the two largest cancer studies ever performed and expand the dose–response dataset generated by the 25,000 mouse “ED01” study over an order of magnitude. With DBC, the liver tumor response fell well below the LED10 line, often used for risk assessment, even though the biomarker (liver DBC–DNA adducts) remained linear. Conversely, the response with AFB1 remained relatively linear throughout the entire dose range. These contributions to elucidation of mechanisms of liver cancer, induced by environmental chemicals and the remarkable datasets generated with ED001 studies, make important contributions to carcinogenesis and chemoprevention.

Introduction

Rainbow trout have been utilized as a model for human cancer, especially liver cancer, at Oregon State University (OSU) for over 40 years (reviewed in Bailey et al., 1996, Walter et al., 2008). This species demonstrates a remarkable sensitivity to the mycotoxin and potent human liver carcinogen, aflatoxin B1 (AFB1). Our group has demonstrated that the mechanism of cytochrome P450 bioactivation to AFB1-8,9-exo-epoxide, and production of the potent mutagenic and carcinogenic DNA adduct 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1, are similar in trout and mammals (Nunez et al., 1990, Bailey, 1994, Bailey et al., 1996). In addition, there are other similarities to human AFB1-initiated liver cancer including Ki-ras mutations, histopathology and alterations in gene expression (Hendricks et al., 1984a, Fong et al., 1993, Tilton et al., 2005). Although a great deal of the focus has been on AFB1 as the initiator, other classes of human carcinogens requiring metabolic activation including nitrosamines, 1,2-dibromoethane, 2-acetylaminofluorene and polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP), dibenzo[a,l]pyrene (DBP) and 7,12 dimethylbenz[a]anthracene (DMBA) are effective in the trout model, as are direct acting carcinogens such as N-methyl-N′-nitrosoguanidine (MNNG) (Hendricks et al., 1985, Hendricks et al., 1995, Kelly et al., 1992, Bailey et al., 1996, Walter et al., 2008).

Over the years liver cancer, initiated by environmental chemicals, has been demonstrated in this model to be subject to either inhibition or promotion by the addition of dietary supplements to our specially developed semi-synthetic diet, the Oregon Test Diet (OTD) (Lee et al., 1991). Among the environmental chemicals that promote liver cancer in trout are, other mycotoxins such as fumonisin, polyhalogenated biphenyls (PCBs) and other aryl hydrocarbon receptor (AHR) ligands such as β-naphthoflavone (BNF), promoters of oxidative stress (e.g., t-butylhydroperoxide, hydrogen peroxide, carbon tetrachloride and choline deficiency) and even elevated rearing temperature (Bailey and Hendricks, 1988, Fong et al., 1988, Curtis et al., 1995, Bailey et al., 1996, Carlson et al., 2001, Williams et al., 2003, Walter et al., 2008). Estrogens, xenoestrogens, and phytoestrogens are liver tumor promotors in this model (Nunez et al., 1988, Nunez et al., 1989, Williams et al., 1998). The examples presented here, dehydroepiandrosterone (DHEA) and perfluorooctanoic acid (PFOA), both appear to function as liver tumor promoters via estrogenicity (Orner et al., 1995, Orner et al., 1996a, Orner et al., 1996b, Orner et al., 1998, Benninghoff et al., 2011, Benninghoff et al., in press). With respect to chemoprevention of AFB1-initiated liver cancer, it is interesting to note that BNF, PCBs, and indole-3-carbinol (I3C), when administered in the diet prior to and concurrent with carcinogen exposure, inhibit tumorigenesis in contrast to promotion when given long-term post-initiation (Nixon et al., 1984, Shelton et al., 1986, Bailey and Hendricks, 1988, Goeger et al., 1988, Bailey et al., 1996, Orner et al., 1998).

I3C, a major component of cruciferous vegetables and a popular dietary supplement, is chemopreventive against cancer in a number of animal models (Aggarwal and Ichikawa, 2005). Owing to the power of statistical analysis when using large numbers of animals, we have documented the relative potency of I3C as an inhibitor and promoter of cancer across a wide dose-range of carcinogen doses (Dashwood et al., 1988, Dashwood et al., 1989a, Dashwood et al., 1990, Bailey et al., 1991). Further analysis of alterations in gene expression using custom microarrays (Tilton et al., 2006) has documented that I3C is a phytoestrogen in the trout and this appears to partially explain its promotion of hepatocarcinogenesis when given post-initiation (Oganesian et al., 1999, Tilton et al., 2006). I3C acts, at least in part, as a blocking agent when given prior to and concurrent with the carcinogen by induction of phase I and phase II enzymes that metabolize and detoxify the procarcinogen (Nixon et al., 1984, Dashwood et al., 1988, Dashwood et al., 1989a).

Trout were the first animal model in which chlorophyllin (CHL) and natural chlorophylls (Chl) were demonstrated to be anticarcinogens (Breinholt et al., 1995a). Further study of the mechanism of CHL and Chl chemoprevention showed that complexation with carcinogens containing planar aromatic structure reduced bioavailability (Breinholt et al., 1995b, Hayashi et al., 1999). CHL was shown to be effective in a human clinical trial in Qidong, China where dietary exposure to AFB1 is high (Egner et al., 2001). CHL, given over a 4 month period, reduced the urinary biomarker of AFB1 exposure, aflatoxin-N7-guanine, by 55% (Egner et al., 2001). Subsequently, a study led by Dr. George Bailey at OSU, in partnership with Dr. Kenneth Turteltaub at Lawrence Livermore National Laboratory, examined the impact of CHL and Chl on [14C]-AFB1 bioavailability in humans (Jubert et al., 2009).

Finally, results from two “ED001” studies will be presented. These studies each employed more than 40,000 animals and determined the shape of the dose–response curve down to levels less than 1 tumor per 1000 animals. These studies were done with two different classes of environmental chemicals classified as human carcinogens, the PAH, DBC (previously referred to in the literature as dibenzo[a,l]pyrene or DBP) and AFB1. These studies represent the two largest chemical cancer datasets ever produced and have the potential to be very valuable in risk assessment. The DBC study has been published in its entirety (Bailey et al., 2009) as have partial results from the AFB1 study (Williams et al., 2009) which, although complete, has not yet been published.

Section snippets

Materials and methods

As this paper represents a synopsis of a number of studies, this section will describe how trout cancer studies employed various methods of carcinogen exposure and dietary administration of tumor modulators. The experimental design of the ED001 studies will also be described.

The DHEA story

DHEA is found in most animals at high levels. In humans, it is the steroid found in blood at the second highest concentration (mostly as the 3β-sulfate). Interestingly, levels of DHEA decline with age in humans which, in part, explains the public interest in marketing this supplement as an anti-aging compound (Bauleiu, 1996). DHEA can be converted in one enzymatic reaction to an androgen and then, following the action of aromatase, estrogen. This also explains, in part, why it has been marketed

Conclusions

Rainbow trout have proven versatile in studying the mechanisms of hepatocarcinogenesis, the response to dietary modulators and, owing to the capabilities of our unique facility at OSU, chemoprevention and tumor promotion with thousands of animals. The two ED001 studies with DBC and AFB1 represent the largest tumor studies ever conducted and the richest data set available for risk assessment with these human carcinogens. The results from both studies eclipsed the mouse ED01 study with

Acknowledgments

There are too many outstanding individuals that have worked on the trout model over the 40 plus years and too many individual NIH grants to list them all here (although this author would like to acknowledge the grant that supported the AFB1 ED001 study, ES013534). I would like to acknowledge Dr. Russell Sinnhuber and his co-workers who first pioneered the use of this model. A great deal of the research listed here was directed by Dr. George Bailey who has been a tireless champion and spokesman

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