Manuscript received 9 August 1995; manuscript accepted
26 September 1995.
Technical paper number 10,831 from the Oregon Agricultural
Experiment Station. This work was partially supported by U.S. Public Health
Service grants ES00210, ES03850, and ES04766 from the National Institute
of Environmental Health Sciences.
Address correspondence to Dr. George S. Bailey, Department
of Food Science and Technology, Marine/Freshwater Biomedical Sciences Center,
Oregon State University, Corvallis, OR 97331. Telephone: (503) 737-3164.
Fax: (503) 737-1877. E-mail: baileyg@bcc.orst.edu
Abbreviations used: 2-AAF, 2-acetylaminofluorene; o-AAT,
ortho-aminoazotoluene; AC, adenocarcinoma; ACC, acinar cell carcinoma;
AFB1, aflatoxin B1; AFG1, aflatoxin G1;
AFM1, aflatoxin M1; AFP1, aflatoxin
P1; AFQ1, aflatoxin Q1; ANF, 
-naphthoflavone; Ah, aryl hydrocarbon; B[a]P, benzo[a]pyrene;
BeP, benzoyl peroxide; BNF, ß-naphthoflavone; CCC, cholangiocellular
carcinoma; CYP, cytochrome P450; DBP, dibenzo[a,l]pyrene; DEN, N-nitrosodiethylamine;
DHEA, dehydroepiandrosterone; DMAB, dimethylaminoazobenzene; DMBA, 7,12-dimethylbenz[a]anthracene;
DMN, N-nitrosodimethylamine; ER, ethoxyresorufin; GST, glutathione-S-transferase;
HA, hepatic adenoma; HCC, hepatocellular carcinoma; HMBA, hydroxymethylbenz(a)anthracene;
I3C, indole-3-carbinol; I33´, 3,3´diindolylmethane; K, kidney;
LA, lauric acid; LV, liver; MAMA, methylazoxymethanol acetate; MMA, 3´-primer
mismatch polymerase chain reaction analysis; MNNG, N-methyl-N´-nitro-N-nitrosoguanidine;
MNU, N-methylnitrosourea; NM, N-nitrosomorpholine; P, progesterone; PAH,
polycyclic aromatic hydrocarbon; PCB, polychlorinated biphenyl; PCR, polymerase
chain reaction; PFOA, perfluorooctanoic acid; RB, rhabdomyosarcoma; RXM,
I3C reaction mixture formed in vitro upon acid treatment; SB, swim bladder;
ST, glandular stomach; T, testosterone; tBuOOH, t-butyl hydroperoxide;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; 3-MC, 3-methylcholanthrene.
Introduction
Fish have gained increasing attention over the past three decades as
valuable models for environmental carcinogenesis research. Various flsh
species have been investigated as nonmammalian vertebrate models for carcinogen
testing, as surrogates for understanding mechanisms of human cancer and
its prevention, as feral species indicators of ecologic contamination, as
indicators of potential human exposure to carcinogens in the water column
or aquatic food chain, and for application as in situ fleld monitors of
integrated carcinogenic hazard in groundwaters near toxic waste sites. Interest
in the use of small aquarium flsh species for cancer research arose from
the pioneering work of Stanton (1), who in 1965 demonstrated the hepatocarcinogenicity
of N-nitrosodiethylamine (DEN) to the zebra danio. Table 1 presents a partial
list of species used and carcinogens examined since that time. Exposures
in these studies have included continuous or acute water bath treatment,
dietary intake, or direct injection of embryos or later life stages. Additional
species and carcinogens have been explored (23-25), and comprehensive testing
of 30 National Toxicology Program carcinogens in one species, the medaka,
is in progress at the Duluth laboratory of the U.S. Environmental Protection
Agency (R Johnson, personal communication).
Through work with various aquarium flsh species, many attributes have
been identified: their low cost, portability, and ease of laboratory culture;
their potential for in situ fleld monitoring; and their potential for lifetime
bioassay, short reproductive cycle, and ease of genetic studies. However,
while aquarium flsh models have distinct appeal, knowledge of mechanisms
of carcinogenesis (e.g., procarcinogen metabolism, DNA adduction and repair,
targeted oncogenes) and its modulation (inhibition, promotion-progression)
by environmental and dietary factors is at present more advanced in the
rainbow trout (Oncorhynchus mykiss). Attention was drawn to this species
in the early 1960s when epizootics of liver cancer in Paciflc Northwest
trout hatcheries ultimately led to the identiflcation of aflatoxin B1
(AFB1) as a potential human hepatocarcinogen (26-28). This review
focuses on the use of the rainbow trout in environmental carcinogenesis
research. This model shares some attributes with aquarium species but also
has unique features such as wide-ranging body size and target organ tissue
availability not applicable to small flsh models.
Carcinogen Bioassay, Response, and Pathology in Rainbow
Trout
Rainbow trout occupy an important niche in the history of carcinogenesis.
It was in this species that the carcinogenicity of the aflatoxins was first
recognized. Based on this original discovery, two major and several minor
research efforts using rainbow trout for cancer research were launched.
One of the major programs was centered at the U.S. Fish and Wildlife Service's
Western Fish Nutrition Laboratory at Cook, Washington, under the direction
of Dr. John Halver. This program started in the late 1950s and was phased
out in the early 1970s. The other major program was started in 1963 at Oregon
State University under the direction of Russell O. Sinnhuber and continues
to date. Carcinogenesis research with rainbow trout in this program has
followed multidisciplinary mechanistic guidelines for many years, with an
in vivo whole-animal response to carcinogens as its foundation. The remainder
of this section will discuss various aspects of this whole-animal research,
including routes of exposure, experimental protocols, carcinogens tested,
and pathology.
Routes of Exposure
Dietary. The original discovery of the carcinogenicity of aflatoxins
in rainbow trout was the result of aflatoxin contamination of dietary foodstuffs,
primarily cottonseed meal. Thus the route of exposure was clearly dietary.
All of the early experimental work used the dietary route of exposure, usually
by incorporating aflatoxin into a semipurilied diet such as the Oregon Test
Diet developed at Oregon State University (29,30). Doses in the low (1-20
µg/kg parts per billion [ppb]) range fed continuously for 9 to 18
months (28,31,32) or higher doses (10-80 ppb) fed continuously for shorter
periods of time (1-30 days) (33-35) were found to be carcinogenic.
The dietary route of exposure was, and continues to be, a useful procedure
for certain desired end points and for specilic carcinogens, particularly
those with low or negligible water solubility. Its primary weakness is that
it is voluntary and inevitably results in unequal exposures within experimental
groups of fish housed in the same tank.
Embryo Waterbath. For this reason, alternative and passive routes
of exposure have been developed. Wales et al. (36) showed that a brief exposure
(0.5-1.0 hr) of trout embryos to a static solution (0.5 ppm or less) of
AFB1 was an effective way to initiate neoplasms. They also demonstrated
that embryo sensitivity was a function of age, being very low before liver
organogenesis but increasing steadily from that time through and after hatching.
This exposure method is especially useful for initiation/promotion protocols
in which a lengthy period of time between initiation and subsequent dietary
promotion is desirable. Subsequent experiments revealed that this method
was effective for a number of carcinogens and that the incidences were dose
responsive (Table 2). The subject of embryo initiation of carcinogenesis
in rainbow trout has been extensively reviewed (37-39).
Fry Waterbath. Continuous or acute waterbath exposures of free-swimming
fish to carcinogens have been used extensively with
aquarium fish. This exposure method was not used for
many years with rainbow trout, but recently the acute or short-term version
of this method has been used with much success (40-43,63-65). Its greatest
utility is for inhibition experiments in which the protocol calls for a
pre-initiation dietary exposure to a potential inhibitor, followed by a
short-term pulse initiation. It works best with small trout, which requires
relatively less water and less carcinogen and results in less splashing,
an inevitable occurrence with larger fish. This route
of exposure is more effective (requiring lower doses to achieve the same
response) than embryo waterbath because the protective chorion of the egg
is gone. It can also result in different target organ specificity when compared
to embryo waterbath exposure. For example, embryo exposure to N-methyl-N´-nitro-N-nitrosoguanidine
(MNNG) results in the following organ tumor response (liver>stomach>kidney),
but fry waterbath exposure produces a different response (stomach>kidney>liver)
[JD Hendricks, unpublished results (57)].
Embryo/Sac-fry Microinjection. Microinjection of rainbow trout
embryos was first reported by Metcalfe and Sonstegard (44). The technique
was improved by Black et al. (45) and semiautomated at Oregon State University
(46). These improvements permit one person to microinject up to 4000 embryos
in an 8-hr day. Our current procedure uses a Hamilton Microlab 900 pump
(Hamilton Company; Reno, Nevada) interfaced with a computer. The computer
is programmed to automatically fill a microsyringe from a reservoir of injectant
and accurately dispense 1-µl doses when a footswitch is pressed. Fine
teflon tubing connects the microsyringe to a 31-gauge stainless steel needle
mounted on a micromanipulator. The needle is inserted through the chorion
and into the yolk sac where the droplet is released. We routinely use a
carrier of 25% acetone/75% vegetable oil for AFB1 and experience
a low mortality of 5 to 10% from carrier-only injections. The following
is an example of results obtained with this technique: AFB1 doses
of 0.5, 1.0, 2.0, and 4.0 ng/µl/egg produced hepatic tumor incidences
of 26, 34, 45, and 48% nine months later (GS Bailey, unpublished results).
The obvious advantages of this procedure include the extremely small doses
required to initiate neoplasia and the ability to expose embryos to highly
water-insoluble carcinogens.
Sac-fry microinjection was first reported by Metcalfe et al. (66). The
procedure is similar to embryo microinjection except the sac-fry are anesthetized
in CO2-saturated water before injection. This allows for more
accurate placement of the injection droplet within the yolk sac, less trauma
to the immobilized sac-fry, fewer injection-related mortalities, and in
general a greater sensitivity to carcinogens because the older organisms
may be more metabolically competent than younger embryos.
Intraperitoneal Injection. Intraperitoneal injection (ip) is rarely
used as an initiating protocol for trout, with only two such studies known
to be in the literature (47,58). However, such injections are used routinely
for short-term metabolism experiments or for DNA-binding studies.
Gavage. Gavaging or stomach tubing is a problematic route of carcinogen
exposure for rainbow trout due to their strong tendency to regurgitate anything
that is irritating to the stomach. This reaction was reported by Bauer et
al. (67) and has been personally observed repeatedly.
Carcinogens Tested
Table 2 is a compilation of all the carcinogens that have produced neoplasms
in rainbow trout. Several interesting features of carcinogenesis in rainbow
trout emerge from the data in this table. a) The trout liver is the primary
organ responding to almost all carcinogens, regardless of the route of exposure.
Only the dietary exposure of trout to MNNG, a direct-acting carcinogen,
failed to produce liver tumors in all of the experiments where a positive
neoplastic response was seen. b) A carcinogen that produces only pancreatic
neoplasms in Syrian golden hamsters, 2,2´-dioxo-di-n-propylnitrosamine
(68), produces only hepatocellular neoplasms in rainbow trout. c) Only four
target organs of the trout have been shown to respond to the carcinogenic
stimulus of a wide variety of chemical carcinogens: the liver, glandular
stomach, kidney, and swim bladder (SB); and d) different routes of exposure
may change the primary target organ but usually not the spectrum of responding
organs.
Pathology of Neoplasms in Rainbow Trout
Liver. The subject of the pathology of hepatocellular neoplasms
in rainbow trout has been thoroughly described and reviewed (69,70). Here
we will only refer to the types of tumors observed and provide corresponding
illustrations. The predominant tumor, occurring in response to all the carcinogens
tested to date, is a mixed hepatocellular/cholangiocellular carcinoma (59,60,71)
(Figure 1). These tumors consist of peripheral hepatic tubules filled
with basophilic hepatocytes and centrally located biliary cells together
with their connective tissue stroma. The biliary compartment can be either
well differentiated into ducts or poorly differentiated and occur as broad
sheets of cells. Typically, over 60% of the tumors examined from a termination
necropsy will be of this general type. One variant of the mixed carcinoma
contains an additional cellular component, pancreatic acinar cells, usually
in close association with the biliary portion of the tumor (64) (Figure
2). Another type of mixed carcinoma contains just biliary and pancreatic
components.
Figure 1. A small
mixed carcinoma in a rainbow trout initiated by 20 ppb AFB1 in
the diet for 1 month. Note the deeply basophilic peripheral hepatocellular
portion and the central biliary ducts. H & E; x 14.
Figure 2. A mixed
carcinoma in a rainbow trout initiated by embryo exposure to a 0.05 ppm
solution of AFB1 for 1 hr. The tumor is composed of centrally
located biliary ducts (right), adjacent pancreatic acinar units (middle),
and peripheral hepatocellular tubules. On the far left is normal liver tissue.
H & E; x 56.
The second most abundant tumor type (25-30%) is the pure
hepatocellular carcinoma (Figure 3). These tumors are composed of
broad tubules of basophilic hepatocytes, with many cells between adjacent
sinusoids and frequent mitotic figures. Cholangiocellular carcinomas are
rare, usually less than 1%, but consist of ducts or sheets of cells, have
minimal stroma, and are invasive into surrounding liver tissue (Figure
4). All these malignant tumor types are capable of distant metastasis
or direct growth into surrounding visceral tissues, but we rarely see this
occur within the 9- to 12-months time frame of most of our experiments.
Metastases are rather common if the fish are held for
2 years or longer.
Figure 3. A portion
of a large hepatocellular carcinoma in a rainbow trout initiated by dietary
exposure to 20 ppb AFB1 for 1 month. Note the broad tubules of
basophilic hepatocytes between adjacent sinusoids and the numerous mitotic
figures. H & E; x 140.
Figure 4. The
advancing invasive edge of a cholangiocellular carcinoma initiated by dietary
exposure of rainbow trout to 800 ppm N-nitrosodimethylamine for 12 months.
The tumor is composed of neoplastic bile ducts and minimal connective tissue
stroma. H & E; x 56.
Several nonmalignant tumors are also observed. Hepatocellular adenomas
tend to be small and noninvasive, with cells that are basophilic but occur
within normal-appearing hepatic tubules (Figure 5). This is usually
not an end-stage neoplasm but appears to progress to hepatocellular carcinoma.
The prevalence varies depending on the time of termination, but in most
cases, it is between 5 and 10%. Very rarely we observe an adenoma that is
composed of eosinophilic hepatocytes, but the vast majority are basophilic.
Very small foci of basophilic hepatocytes are interpreted to be the beginning
stages of either hepatocellular adenomas or carcinomas and not a separate
preneoplastic lesion. Cholangioma is an infrequent tumor type consisting
of mostly normal-appearing bile ducts and abundant stroma that usually encapsulate
the structure (Figure 6). Usually 1 to 2% of the total incidence
are cholangiomas. Mixed hepatocellular/cholangiocellular adenomas are tumors
having the cellular features of adenomas and cholangiomas together in the
same cellular mass (Figure 7). These tumor types are seldom seen.
Figure 5. A hepatocellular
carcinoma (left) growing within a hepatocellular adenoma (middle), with
normal rainbow trout liver tissue on the far right. Contrast the deeply
basophilic broad tubules of the carcinoma with the less basophilic two-cell-wide
tubules of the adenoma. This occurrence supports the hypothesis that carcinomas
develop from adenomas. The tumor was initiated by embryo exposure to aqueous
AFB1 (0.5 ppm) for 1 hr. H & E; x 90.
Figure 6. A small
cholangioma in a rainbow trout initiated by 20 ppb dietary AFB1
for 1 month. Note the mostly normal appearance of the ducts and encapsulation
by connective tissue stroma. H & E; x 200.
Figure 7. A small
mixed adenoma initiated by a 2-week feeding of 800 ppb aflatoxicol-M1
to rainbow trout. The neoplasm consists of basophilic hepatocytes similar
to those observed in hepatocellular adenomas and a beginning proliferation
of bile ducts in the central region. H & E; x 224.
A final tumor type that has been observed only twice in over 30 years
of histopathologic examinations of tens of thousands of liver sections is
what we interpret to be a hepatoblastoma. These two tumors consist of deeply
basophilic, highly undifferentiated cells with an extremely high rate of
mitosis (Figure 8). The cells palisade around vascular channels and
outstrip the vascular supply to the rapidly expanding cellular mass. This
leads to extensive necrosis that extends into veins and causes serious hemorrhaging
(Figure 9).
Figure 8. A portion
of a presumptive hepatoblastoma initiated by continuous dietary exposure
of rainbow trout to 20 ppb AFB1. Note the poorly differentiated
nature of the cells, scanty cytoplasm, numerous mitotic figures, and palisading
of cells around a large vein. H & E; x 224.
Figure 9. A portion
of a presumptive hepatoblastoma initiated by continuous dietary exposure
of rainbow trout to 20 ppb AFB1 (the same tumor shown in Figure
8). Extensive necrosis in the broad bands of cells (upper right) has progressed
into a large vein causing hemorrhaging. H & E; x 358.
Kidney. The nephroblastoma is an almost exclusive chemically inducible
neoplasm of trout kidneys. It consists of deeply basophilic, highly mitotic
blastema cells; abortive, poorly differentiated glomerular structures; incompletely
differentiated tubules; and abundant connective tissue stroma (Figure
10). Six carcinogens, listed in Table 2, have produced nephroblastomas,
but the treatment of choice for producing a high incidence of these tumors
is a static waterbath exposure of rainbow trout fry to a solution of 50
ppm MNNG for 30 min. This will result in about 50% of the fish
having one or more large nephroblastomas 6 to 9 months later. These tumors
grow rapidly, become very large, and kill the fish
through destruction of normal kidney tissue and obstruction of urine flow.
To our knowledge, the rainbow trout is the only animal model in which nephroblastoma
can be routinely initiated in a high incidence by a chemical carcinogen
(72).
Figure 10. A large
nephroblastoma initiated by exposure of rainbow trout embryos to aqueous
10 ppm methylazoxymethanol acetate (MAMA) for 24 hr. Note the deeply basophilic
undifferentiated mass of blastema cells (upper right); incompletely differentiated
tubules with numerous mitotic figures; abortive, poorly formed glomerularlike
structures (lower left); and abundant connective tissue stroma. H &
E; x 90.
Stomach. All the stomach tumors that we have observed in rainbow
trout have been benign papillary adenomas of the mucosal lining of the glandular
stomach. Typically, they grow upward into the luminal space. Some tumors
produced by dietary exposure to MNNG (56) also exhibited downward growth
but never penetrated the basement membrane (Figure 11).
Figure 11. A papillary
adenoma of the glandular stomach of a rainbow trout initiated by 500 ppm
dietary MNNG for 18 months. Primary proliferation of mucosa cells is upward
into the lumen of the stomach, although some neoplastic cells occur in the
gastric pits as well. H & E; x 56.
Swim Bladder. As with the stomach tumors described above, the
swim bladder adenomas are benign papillary overgrowths of epithelial cells
that protrude into the lumen of the swim bladder. The unique feature of
the cells of this lesion is the marked increase in size of the tumor cells
compared with the normal mucosal cells. The columnar height of these cells
is often several times greater than the normal cells (Figure 12).
Figure 12. A papillary
adenoma in the swim bladder of a rainbow trout initiated by embryo exposure
to aqueous, methylazoxymethanol acetate (MAMA) 10 ppm for 24 hr. Note the
tremendous proliferation of cells and their increased size compared to normal
swim bladder mucosal epithelium at the right. H & E; x 56.
This brief review of the procedures involved in the initiation and identification
of neoplasms in rainbow trout is intended to portray this model as a viable
alternative for many aspects of cancer research. The fish
are easy and economical to rear, and they respond to classical carcinogens
in a predictable, dose-responsive manner.
Pathways of Procarcinogen Metabolism, DNA Adduction,
and Repair
Cytochromes P450
As is the case in humans (73), trout cytochromes P450 (CYPs) play a crucial
role in the bioactivation of procarcinogens to electrophilic metabolites
capable of covalently binding to DNA. The study of properties of the trout
CYP-dependent mixed-function oxidase system, response to inducers, and metabolism
of xenobiotics was pioneered by a number of laboratories including DR Buhler
(Oregon State University), J Lech (Medical College of Wisconsin), and L
Forlin and T Andersson (University of Goteborg) [reviewed in (74-79)]. Although
relatively few trout CYPs have been sequenced and assigned to a CYP subfamily,
a number of trout CYPs have been purified and partially characterized with
respect to bioactivation of procarcinogens (Table 3). Much of this
work has been performed by the laboratory of D.R. Buhler; the reader is
referred to an excellent recent review (97) for more detailed information.
Table 3 does not include information on the trout CYPs responsible for steroid
synthesis, P450scc (CYP11A1), P450C17 (CYP17) or P450arom
(CYP19), as these display little or no activity toward procarcinogens.
Role of Trout CYPs in the Bioactivation of Procarcinogens
Aflatoxin B1. AFB1 is metabolized by CYP
to a number of monohydroxylated metabolites including aflatoxins M1,
Q1, and P1 [see (98) for an excellent recent review],
all of which are less mutagenic and carcinogenic than AFB1 and
therefore represent detoxication reactions. These monohydroxylated metabolites
are substrates for conjugation by uridine diphosphate-(UDP) glucuronosyltransferase
and can be eliminated in the bile. CYP epoxygenation at the 8,9-position
results in production of the electrophilic ultimate carcinogen AFB1-8,9-epoxide.
The major human CYPs involved in the bioactivation of AFB1 to
AFB1-8,9-epoxide are 1A2 and 3A4 (98-101). The relative contribution
of these two isoenzymes toward bioactivation of AFB1 probably
varies markedly between individuals due to large interindividual variations
caused by genetic and environmental factors (102-104), which along with
interindividual differences in DNA repair rates may account for some of
the variation between humans with respect to susceptibility to some cancers
(105). In trout, the majority of AFB1 8,9-epoxygenation is catalyzed
by CYP2K1 (89). The covalent adduct produced is the same as in mammals,
8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-GUA)
(106,107).
As is the case in mammals, trout CYP1A is active in the hydroxylation
at the 10 position to produce AFM1 (108). The induction of CYP1A
by compounds such as ß-naphthoflavone (BNF), indole-3-carbinol (I3C),
and polychlorinated biphenyls (PCBs) was thought to be the mechanism by
which these modulators acted as chemopreventors of AFB1-initiated
hepatocarcinogenesis in trout (109,110). However, recently our laboratory
has determined that inhibition of CYP activity may be a more important mechanism
of action than CYP1A induction, especially with I3C (111-114). In studies
examining the time course and dose response of dietary I3C, trout responded
only weakly and transiently to this compound as an CYP1A inducer, and the
degree of reduction in covalent binding of AFB1 to DNA in vivo
did not correlate to CYP1A induction (111,112). Similarly, BNF was found
to significantly reduce AFB1 covalent binding to DNA at dietary
doses too low for CYP1A induction (114). In the course of these studies,
we found that both BNF and various acid condensation products of I3C were
potent inhibitors of a number of trout and mammalian CYPs, with inhibition
contents well below the levels known to be attained in liver after administration
of anticarcinogenic doses (112-115).
In addition to possessing a CYP (2K1) with high activity toward bioactivation
of AFB1, the remarkable sensitivity of trout toward AFB1-initiated
carcinogenesis can be explained by their lack of a constitutive or inducible
glutathione S-transferase (GST) with appreciable activity toward AFB1-8,9-epoxide
(116). The high activity displayed by constitutive mouse Yc GST, compared
to rat, is thought to be the major factor for the remarkable resistance
of mice (98,117-121). The importance of this enhanced phase II reaction
is confirmed by the observation that mice are actually more prolific at
production of AFB1-8,9-epoxide than rats. Administration of I3C
to rats induces a form of GST (Yc2) with high activity toward conjugation
of the exo-AFB1-8,9-epoxide and presumably contributes to chemoprevention
in this animal model (122). Trout, however, appear refractory toward induction
of this type of GST (116).
Polycyclic Aromatic Hydrocarbons. Environmental exposures to polycyclic
aromatic hydrocarbons (PAHs) (possibly in conjunction with PCBs, dioxins,
and dibenzofurans) are thought to be related to epizootic outbreaks of liver
neoplasia in feral fish from various regions of the
country (123-127). Benzo[a]pyrene (B[a]P) is hepatocarcinogenic in rainbow
trout, but long-term exposures through the diet or intraperitoneally are
required (58). The racemic (±)-trans-B[a]P-7,8-dihydrodiol is a much
more potent carcinogen in trout (59). As is the case in mammalian models,
the (-) enantiomer is roughly an order of magnitude more potent that the
(+) enantiomer (59). Reconstitution studies with purified enzyme and liver
microsomes from BNF-treated trout indicate that CYP1A is the predominant
subfamily involved in B[a]P and B[a]P-7,8-dihydrodiol bioactivation to the
ultimate carcinogen 7S-trans-7,8-dihydrobenzo[a]pyrene-7,8-diol-anti-9,
10-epoxide (59,80,128).
7,12-Dimethylbenz[a]anthracene (DMBA) is a much more potent hepatocarcinogen
in trout than B[a]P and produces tumors in kidney, swim bladder, and stomach
as well (Table 2) (60). Trout embryos metabolize DMBA to 12-hydroxymethymethyl-7-methylbenz[a]anthracene
(12-HMBA) and 3,4-dihydroxy-3,4-dihydro-DMBA (DMBA-3,4-diol) (60). In addition
to these metabolites, juvenile and adult trout metabolize DMBA to the 8,9-dihydrodiol,
7-HMBA, and 2- and 3- hydroxy-DMBA (DR Buhler et al., unpublished data).
DMBA is metabolized by both constitutive and induced CYPs (Table 3). Pretreatment
of trout with inducers of CYP1A markedly enhances the metabolism, covalent
binding, and carcinogenic potency of DMBA, suggesting that this subfamily
is very efficient at bioactivation of this PAH (Hendricks et al., unpublished
observations). The phenolic and dihydrodiol metabolites of DMBA are substrates
for phase II conjugation reactions by UDP-glucuronosyltransferases and perhaps
sulfotransferases (129). The ultimate carcinogenic metabolite of DMBA has
previously been identified as the bay region DMBA-3,4-diol-1,2-epoxide (130),
although recent evidence suggests the possible contribution of other metabolites
(131-133). The DMBA-DNA adducts produced and the resulting mutational spectrum
may be different in trout than in mammals. A high percentage of liver tumors
in trout treated with DMBA carried activated Ki-ras, mostly G
A transitions
and GT transversions in the first and second G, respectively,
of the 12th codon, as opposed to the major mutation seen in mouse
hepatic tumors (GC transversion in the first G of
codon 13) (60).
We are currently investigating the potential for dibenzo(a,l)pyrene (DBP)
as a model PAH carcinogen in trout. The advantage of DBP replacing DMBA
in studies on PAH carcinogenesis lies mainly in the fact that DBA is a potent
environmental contaminant whereas DMBA is not (134). Preliminary evidence
indicates that DBP resembles DMBA with respect to trout target tissues but
is a more potent carcinogen, especially for liver and swim bladder (GS Bailey
et al., unpublished observations).
N-Nitrosodiethylamine (DEN). DEN is a potent hepatocarcinogen
in trout (53,63,135,136). The major adducts produced are 7-ethylguanine
and O6-ethylguanine (63,136), indicating that, as in mammals,
metabolic activation occurs through N-deethylation. In rats, mice and humans,
the major CYP catalzying O-deethylation of DEN is CYP2E1, with some contribution
from CYP2A6 (137-139). Little information (140) is available on which trout
CYPs may be responsible for N-dealkylation of DEN or other dialkylnitrosamines.
To our knowledge, no ortholog of CYP2E1 has been identified in any fish. Pretreatment of trout with BNF enhances the hepatocarcinogenesis
of DEN, suggesting a role for CYP1A (63).
DNA Adduction and Repair
As discussed above, the major initial covalent AFB1-DNA adduct
produced is the same in trout and mammals, trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin
B1, which is spontaneously converted to the more persistant ring-opened
formaminopyrimidine. A dose-dependent linear increase in liver AFB1-covalent
adduction is observed upon feeding trout dietary carcinogenic doses of AFB1
over a period of 2 to 4 weeks (141). A similar dose-dependent, steady-state
linear increase in AFB1-DNA adduction has been observed with
chronic dosing in the rat model (142). The doses used in the trout study
(141) were relevant to human AFB1 consumption and, importantly,
provided no evidence of a threshold dose below which AFB1 was
not genotoxic. The rate of repair of AFB1-DNA adducts in trout
is much slower than in mammals. The pseudo half-life for loss of the initial
adduct is 7.5 hr in rats. In contrast, the pseudo half-life for AFB1-DNA
adducts in trout is on the order of 21 days (107). (Note that neither of
these values is a true half-life since loss is due to chemical conversion,
depurination, and enzymatic removal and is not a first-order process.) The
remarkable sensitivity of rainbow trout to AFB1 hepatocarcinogenesis
may be due in large part to this reduced ability to repair bulky DNA adducts.
Linear regression analysis of the relative tumor risk versus steady state
AFB1-DNA adducts yields the identical line for both rat and trout
(143), indicating that those adducts that form and persist lead to tumors
with equivalent efficiency in the two species (51,144-146). These results
strengthen the reliance on the molecular dosimetry concept for risk assessment
for AFB1 exposure to humans (147,148).
Relative to AFB1, little information is available on the identity,
kinetics of formation and repair, and relationship to final tumor response
for PAHs in trout. Induction of trout CYP1A enhances the covalent adduction
of either B[a]P or B[a]P-diol to liver DNA (128,149). Consistent with tumor
data, trout are relatively resistant to the formation of appreciable levels
of DNA adduction following exposure to B[a]P (150). 32P-Postlabeling
analysis indicates that the major adduct in trout liver DNA following administration
of (+) 7S-trans-7,8-dihydrobenzo[a]pyrene-7,8-diol is (+)-syn-7S-trans-7,8-dihydrobenzo[a]pyrene-7,8-diol-9,10
epoxide-dG (128). Bath exposure of trout embryos or embryo-derived cells
with DMBA produces a concentration-dependent increase in DNA adduction,
but the adducts have yet to be identified. Interestingly, a high percentage
of liver tumors from trout treated with DMBA as embryos carry activated
Ki-ras with alleles distinctly different from DMBA-induced liver tumors
in mice (60,151). Thus in species as different as trout and mice, biochemical
differences in procarcinogen metabolism and adduction can exist, which nonetheless
lead to similar oncogenic pathways involving prevalent ras activation.
Treatment of trout with DEN produces dose-dependent increases in DNA
adduction, primarily 7-ethylguanine and O6-ethylguanine (63,136).
The formation of the latter adduct correlates with tumor incidence and is
consistent with the dominance of a GA transition mutation
in Ki-ras isolated from these tumors (53,63). This mutagenic specificity
probably derives from ineffective alkyltransferase removal of O6-ethylguanine
in these animals (53,63). Based on total tumorigenic dosage required, however,
Shasta trout and F344 rats show comparable sensitivity to hepatocarcinogenesis
by DEN (53).
Protooncogene Activation in Trout Tumors
An understanding of the molecular basis for cancer initiation, promotion,
and progression is necessary to more readily relate cancer studies in fish to those in mammals including humans. Mutational inactivation
of the p53 tumor suppressor gene (152) and activation of the ras protooncogene
(153) represent two of the most frequently observed and thoroughly studied
molecular events in human cancer. Initial studies in our laboratory have
not detected p53 codon 248 or 249 mutations as common events in AFB1-initiated
hepatic tumors in trout (GS Bailey, unpublished results); however, we have
yet to determine if mutations may occur at other p53 sites or with other
carcinogens or protocols, including chronic treatment that may more closely
resemble human AFB1 exposure. In this regard, mutations in p53
have been observed relatively infrequently in rats and mice. By comparison,
we have provided partial sequences for several ras genes in rainbow trout
(154) and have shown that mutational activation of an expressed Ki-ras gene
is a frequent occurrence in hepatic tumors initiated by the prototypical
mycotoxin AFB1 (155), the polycyclic aromatic hydrocarbon DMBA
(60), and the N-nitroso compound DEN (53). Table 4 summarizes current
knowledge regarding Ki-ras mutational activation among the various tumor
types elicited by these and additional carcinogens in trout. Overall, the
data demonstrate that Ki-ras mutagenic activation can be a frequent event
in the initiation of liver, stomach, and swim bladder neoplasms in the trout
model. We have yet to establish full trout sequences for N-and Ha-ras homologues
and to determine if mutations may also occur in these genes.
The Ki-ras mutational data have been accumulated by allele-specilic hybridization,
3´-primer mismatch polymerase chain reaction analysis (MMA) and direct
sequencing of polymerase chain reaction (PCR) products. Though sequencing
provides a more direct identification of any mutant that may exist within
the PCR product, it has one disadvantage: it fails to detect mutants carried
by less than 10 to 20% of the cells in the tumor isolate. Thus, data generated
by this method provide only a minimal estimate of the percent of trout tumors
bearing Ki-ras mutations. For example, MMA detected mutant Ki-ras alleles
in 100% of stomach tumors elicited by DMBA fry bath exposure, whereas direct
sequencing detected only 11% incidence (Table 4). As seen in the table,
each carcinogen appears to generate a specilic spectrum of Ki-ras mutant
alleles. AFB1-initiated liver tumors contain primarily Ki-ras
codon 12 GGAGTA and codon 13 GGTGTT transversions,
whereas MNNG elicits entirely GGA GAA and GGAAGA transitions. These are compatible with the well-known mutagenic
properties of the major DNA adducts elicited by these carcinogens in bacterial
systems. Spontaneous liver tumors occur only rarely in trout and few have
been available for analysis; of the limited number examined to date (Table
4), we have been unable to detect mutant Ki-ras alleles by MMA. These data
taken together provide evidence that the mutant alleles that we have observed
in carcinogen-treated fish occur as a direct result
of carcinogen-DNA adduction in the ras gene in vivo rather than from amplilication
of background mutational events in this model.
Most of the data we have generated to date involve liver tumors. The
hepatocarcinogens listed here all induce primarily mixed cholangiocellular/hepatocellular
carcinomas, with relatively few pure hepatocellular carcinomas induced.
AFB1, MNNG, DEN, and DBP induce hepatic tumors with a high incidence
(71-100%) of activated Ki-ras alleles. An interesting exception is dehydroepiandrosterone
(DHEA), an endogenous steroid that is also hepatocarcinogenic in the rat
but not previously known to be genotoxic. The mutant ras incidence of 32%
(8/25) is low, but the 12(1)GA mutation observed
is not compatible with indirect damage such as 8-hydroxydeoxyguanosine.
The precise origin of the observed ras mutations remains to be established.
For DMBA, the incidence of Ki-ras mutations in liver tumors varied from
44% (7/16) to 100% (9/9) among the various experiments. The number of mutant
alleles observed is too small at present to know if the DMBA mutational
spectrum is protocol dependent. Among the liver tumors examined, codon 61
AT transversions have been rarely detected, with codon
12 and 13 guanine-based mutations (GA, GT, GC) more frequently observed. Of 24 liver tumors
elicited by the environmental PAH DBP, comparable numbers of codon 12 GA and GT mutations were observed and three tumors
showed evidence of double Ki-ras mutations. Experiments are in progress
to establish if these mutations reside on the same or separate ras sequences
and to establish the specilic DBP-DNA adducts that give rise to ras mutations
in the trout model.
Hepatic tumors from AFB1-treated trout (Table 4) and rats
(156) show frequent ras mutation, yet this has not been reported to occur
in hepatocellular carcinoma from AFB1-exposed humans. Mutant
Ha-ras alleles have, however, been reported in human cholangiocellular carcinoma
(157). We are attempting to establish if ras mutation in the trout may also
be restricted to neoplasms having cholangiocellular involvement. An alternative
hypothesis is that the trout and rat laboratory models do not completely
mimic AFB1-related human hepatocarcinogenesis, which may frequently
involve tumor progression under the combined inliuence of chronic carcinogen
intake and hepatitis infection.
Tumor Promotion and Inhibition in Trout
Use of the trout tumor model to study modulation of carcinogenesis has
been reviewed elsewhere (158-160). The historically low spontaneous liver
tumor incidence (0.1%) is a signilicant advantage in the statistical design
of multidose tumor inhibition and promotion experiments. In this overview,
we will summarize some past results and also present some recent unpublished
lindings.
The majority of studies on anticarcinogenesis has focused on inhibition
of AFB1-initiation of hepatocarcinogenesis (Table 5),
but we are currently investigating multiple target tissues and combinations
of inhibitors. For example, we recently conducted a preliminary experiment
examining the chemoprevention potential of two anticarcinogenic chemicals
that have different mechanisms of action. Dietary I3C at 1,500 ppm reduced
AFB1-initiated hepatocarcinogenesis from 31% to 16%, 1,500 ppm
chlorophyllin gave 24% incidence (not signilicant), and the combination
of I3C plus chlorophyllin was synergistic (4.5% incidence).
In another recent preliminary collaborative study with Gary Stoner of
Ohio State University, postinitiation feeding with ellagic acid suppressed
DMBA-dependent stomach carcinogenesis, which represents the first example
of postinitiation suppression by any agent in the trout model. By comparison,
postinitiation chlorophyllin was without effect. Compounds that have proven
negative to date as anticarcinogens in the trout model include BHA, BHT,
d-limonene, Oltipraz, menthol, green tea extract, vitamin E, freeze-dried
onion or garlic, and mint oil.
A number of environmental agents have been demonstrated to function as
postinitiation tumor promoters or enhancers in the trout model (Table
6). Compounds proven ineffective at promotion to date include transitions
metals (with or without H2O2 as prooxidant) and the
peroxisome proliferators clolibrate and WY14,643. In addition, some promoters
appear to have initiator or tissue specificity. For example, postinitiation
dietary Aroclor 1254 promotes hepatocarcinogenesis with DMBA as initiator
but not with AFB1 (160). Postinitiation I3C and BNF enhance carcinogenesis
intitiated by AFB1, DMBA, or MNNG in liver but not in other target
organs such as kidney and, in fact, they usually reduce the incidence in
stomach. We have also tested a number of compounds including vitamin E,
green tea extract, nicotinic acid, d-limonene, chlorophyllin, and menthol
as potential antipromoters with no success except for one compound, ellagic
acid, which was discussed earlier.
Very little is known about the mechanism of action of tumor promoters.
In trout, prooxidants such as peroxides, CCl4, and choline deliciency
are effective promoters (Table 6), yet to date, co-treatment with antioxidants
has not provided any protection. Our laboratory is currently studying the
mechanism of I3C dietary modulation of cancer using trout and murine models.
If given before and during initiator exposure, I3C functions in trout as
an anticarcinogen, but chronic postinitiation exposure enhances tumorigenesis.
These opposing actions have similar potencies (EC50 [median effective
concentration] =1,000-1,500 ppm) in trout (42). The mechanism of I3C inhibition
in trout appears to be largely due to inhibition of CYP bioactivation by
I3C acid condensation products rather than aryl hydrocarbon (Ah) receptor-dependent
induction of CYP1A (111-113).
We are currently investigating the role of the Ah receptor in tumor promotion.
Previous studies have documented that a number of I3C acid condensation
products have high aflinity for the mammalian Ah receptor (167). Inititial
attempts to block trout Ah receptor-dependent promotion with -naphtholiavone
(ANF) were unsuccessful. In fact, ANF alone in trout was a promoter of hepatocarcinogenesis,
and the combination of ANF and BNF was additive (Table 6). Further work
has documented that ANF is an Ah receptor agonist in trout. We had hoped
to use congenic mice to directly address the role of the Ah receptor in
I3C promotion; however, preliminary studies indicated that I3C fails to
promote hepatocarcinogenesis in mice (DEN) as it does in rats (AFB1)
(DE Williams, unpublished observations).
Our laboratory has recently found DHEA to be a potent promoter in the
trout model (Table 6), and in fact it is a complete carcinogen (165). DHEA
has received much attention recently with respect to its chemopreventive
properties in humans and animal models with respect to a number of diseases
including atherosclerosis, diabetes, obesity, lupus, trauma injury, AIDS,
and in aging (168). Currently, a number of clincial trials are being conducted
with DHEA, an agent that is also being marketed in health food stores. DHEA
is hepatocarcinogenic in rats under protocols requiring high doses (>=4,500
ppm) over long exposure periods (>=1 year). The carcinogenicity of DHEA
in rodents is thought to be caused by its actions as a peroxisome proliferator
(169). Humans are relatively insensitive to peroxisome proliferators; therefore,
the rodent lindings are thought not applicable to human risk assessment
(170). Our lindings with the trout model are the first to document the carcinogenicity
of DHEA in the absence of peroxisome proliferation. Signilicant promotion
in trout is observed in as little as 8 weeks of feeding (5 days per week)
at levels that approximate doses previously used in some human clinical
trials. The mechanism of DHEA promotion in trout may be hormonal. DHEA is
a precursor for both estrogens (already known to promote hepatocarcinogenesis
in trout) and androgens, and the plasma levels of both steroids increase
markedly in trout fed DHEA. The estrogenic potency of DHEA in trout can
be observed by following vitellogenin levels in plasma. The DHEA analog
(171) developed by Arthur Schwartz (Temple University), 16-liuoro-5-androsten-17-one
(8354), was much weaker as a precursor for androgens and estrogens in trout,
was not a complete carcinogen, and did not significantly promote AFB1
hepatocarcinogenesis at a dietary level (444 ppm) for which DHEA was very
effective (165). The 8354 analogue is currently undergoing human clinical
trials. Based on the lindings in rodents and trout, it may be prudent to
proceed cautiously with high DHEA supplementation over prolonged periods
and to continue developing safer analogues.
Strengths and Limitations of Environmental Carcinogenesis
Research in the Trout Model
There should be no expectation that trout will supplant traditional rodent
models in carcinogen bioassays or as surrogates for human cancer research.
A most evident limitation common to all lower vertebrates is the lack of
complete organ homology needed to study cancers of the lung, colon, breast,
and prostate, the leading cancers in the United States. While carcinogens
that initiate these tumors in rodents can be carcinogenic in trout, organospecilicity
is lost and liver is the most common target organ. An additional limitation
is that trout have late sexual maturity (2-3 years) and a long life span
during which the animal continues somatic growth. These limit the potential
for genetic studies and preclude lifetime bioassay protocols in carcinogen
testing. Under the latter limitation, a negative carcinogen bioassay result
would not be considered delinitive. (This species instead assesses cancer
risk through the embryonic and juvenile lifestages, which are not unimportant.
Moreover, any chemical that is positive in a trout carcinogen bioassay should
reasonably be considered a potential human carcinogen, barring mechanism
information to the contrary.) A linal limitation of the trout model is the
continuing lack of knowledge in this species of fundamentals in the genetics
and cell biology of cancer; owing to the relatively few investigators involved
in its development, this is likely to remain a chronic problem.
Even with these limitations, there are many attributes of trout and other
fish models that afford unique approaches in the study
of cancer. Where mechanistically reasonable, the use of trout and other
fish models can substantially reduce our dependence
on small mammals for health research. Cost is a considerable advantage,
especially for investigating statistically challenging issues that can be
addressed only with large numbers of animals. Our molecular dosimetry studies
using 8,000 to 10,000 animals to quantify the interrelationships among carcinogen
dose, anticarcinogen dose, DNA adduct formation, and linal tumor outcome
exemplify this (35,162). Similarly, the low husbandry costs for fish
permit tumor studies designed to deline the shape of cancer dose-response
curves down to 0.1% incidence. The design of any such experiment requires
at least 31,000 animals to provide adequate statistical power, given the
expectation of only 10 tumors among 10,000 animals at the targeted lowest
dose. One such experiment is in progress testing DEN in the medaka model.
DBP and DEN will be similarly tested in the next 2 years in the trout model,
together with biochemical studies of metabolism, DNA adduction and repair,
Ki-ras activation, target organ toxicity, and proliferation that aim to
deline mechanisms accompanying any departure from linearity. An advantage
of trout inherent in this study is its historically zero background tumor
rate in two of the target organs, which assures that all observed tumors
in these organs can be ascribed to carcinogen treatment. In the third organ,
liver, the historic background tumor rate of 0.1% will become important
at the lowest carcinogen dose only. Even here, given the high incidence
of activated Ki-ras in carcinogen-initiated tumors only, it may be possible
to separate almost all carcinogen-related and spontaneous tumors. The entire
tumor study, including in-house pathology as well as the proposed mechanism
studies, is to be carried out with trout for a total budget that is only
5 to 10% of the per-diem costs alone for a comparable 40-week single-sex,
single species rat or mouse experiment. Given current budgetary restrictions,
it seems unlikely that the 24,000-animal megamouse study of 2-acetylaminofluorene
dose-response will be extended to additional carcinogens; the more affordable
fish models can help in addressing at least some important
mechanistic questions in dose-response.
Sensitivity of fish models can be an important attribute.
Trout embryos, which are readily available in the thousands at any specilied
stage of development, provide a highly sensitive early life stage model
enabling nanogram to microgram bioassay of scarce materials (e.g., high-performance
liquid chromatography [HPLC] fractions). For example, we have been able
to bioassay scarce HPLC-purified metabolites of the phytochemical indole-3-carbinol
to identify the anticarcinogenic intermediates by co-injection with AFB1
(46), and we have generated entire tumor dose-response curves with 2,000
individuals, requiring less than 100 µg total of AFM1 and
related aflatoxins (GS Bailey, unpublished data). We have determined that
a measured dose of 0.5 ng AFB1 per trout embryo will induce 26% incidence
of hepatic tumors 9 months after embryo injection; this is nine orders of
magnitude lower than the dose of AFB1 required to elicit a similar
response in monkeys. No other established cancer model offers this sensitivity.
Finally, the nonmammalian status of trout also has its inherent advantages
in that a comparative approach in cancer research is no less useful than
in other lields of biology. Thus the establishment of ras activation as
a common oncogenic pathway in trout strongly supports a commonality in molecular
mechanisms of cancer in lower and higher vertebrates. The fact that trout
are more humanlike than rats in their resistance to the phenomenon of hepatic
peroxisome proliferation makes them an attractive species in which to establish
if peroxisome proliferators might pose carcinogenic risk by mechanisms other
than peroxisome proliferation itself--as discussed above, we now know that
some of these compounds are complete carcinogens and are possible genotoxins
in trout. AFB1 hepatocarcinogenesis can be blocked in rats by
co-treating animals with the antioxidant ethoxyquin (172) or the antischistosomal
drug Oltipraz (173), apparently through induction of GST Yc2 with high specificity
for AFB1-8,9-oxide. The importance of this mechanism to planned
human intervention trials in China with Oltipraz is underscored by our determination
that neither Oltipraz nor antioxidants induce AFB1-glutathione
detoxication in the trout model, and hence both fail to provide protection
against AFB1 hepatocarcinogenesis [GS Bailey, unpublished results;
(108)]. There is notable uncertainty that such an enzyme is inducible in
human liver in vivo. The demonstration, first in trout (34) and later confirmed
in rats (174), that the candidate anticarcinogen indole-3-carbinol can behave
as a potent tumor promoter under some exposure protocols has raised as yet
unresolved safety concerns over its proposed use in human breast cancer
chemoprevention.
In summary, it is evident that there are many experimental situations
in which trout and other fish models are inadequately
developed or inappropriate to address certain issues in cancer research.
It is equally apparent that these species can serve as highly useful adjuncts
to the traditional rodent models in the study of human environmental cancer
risks and in some situations can provide wholly unique approaches.
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