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

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 B(1) (AFB(1))). Two "ED(001)" cancer studies have been conducted, utilizing approximately 40,000 trout, by dietary exposure to AFB(1) 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 "ED(01)" study over an order of magnitude. With DBC, the liver tumor response fell well below the LED(10) line, often used for risk assessment, even though the biomarker (liver DBC-DNA adducts) remained linear. Conversely, the response with AFB(1) 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 ED(001) studies, make important contributions to carcinogenesis and chemoprevention.     

Interactions between hepatitis B virus infection and exposure to aflatoxins in the development of hepatocellular carcinoma: a molecular epidemiological approach.

Aflatoxins and hepatitis B virus (HBV) are major risk factors for hepatocellular carcinoma (HCC) in high incidence areas for this cancer, namely southeast Asia and parts of Africa. There is evidence from both epidemiological studies and animal models that the two factors can act synergistically to increase the risk of HCC. The cellular and molecular mechanism of the interaction between these two factors is as yet undefined. However, one possible mechanism attested to by studies in HBV transgenic mice is that chronic liver injury alters the expression of specific carcinogen metabolising enzymes thus modulating the binding of aflatoxin to DNA in hepatocytes. The high levels of aflatoxin exposure which occur in many areas of the world where chronic HBV infection is endemic indicate that measures to reduce aflatoxin exposure would contribute to reducing HCC incidence. In preliminary studies, Guinea-Conakry have established baseline data for the implementation of a community-based intervention study to evaluate the effectiveness of improved post-harvest processing and storage of the groundnut crop, a major source of aflatoxins. Aflatoxin-albumin adducts were measured in 423 sera from individuals living in the four natural geographic zones of Guinea. More than 95% of the serum samples were positive for this biomarker and highest exposures were found in Lower Guinea where groundnuts are consumed as a dietary staple. Variations in mean levels between villages within a geographic region did not vary greatly. HBV infection was endemic in all regions with an overall prevalence of 16.7% chronic carriers. Thus in this population both HBV vaccination and reduction in aflatoxin exposure would be beneficial in decreasing morbidity and mortality from liver disease.     

The anticarcinogen 3,3′-diindolylmethane is an inhibitor of cytochrome P-450

Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3′-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with K_i values in the low micromolar range. We now report a similar potent inhibition by 3,3′-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3′-Diindolylmethane also inhibited in vitro CYP-mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B₁. There was no inhibition of cytochrome c reductase. In addition, we found 3,3′-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3′-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans. © 1995 John Wiley & Sons, Inc.     

Activation of aflatoxin B1 by primary cultures of adult rat hepatocytes: effects of hepatocyte density

Primary monolayer cultures of adult rat hepatocytes readily activate aflatoxin B1 as determined by bacterial mutagenesis (Ames test) and the extent of apparent covalent binding of aflatoxin B1 residues to hepatocyte macromolecules. For intact cultures inoculated with 3 X 10(5)-3 X 10(6) cells/dish, the efficiency of activation decreases with increasing cell density whereas permeabilized hepatocytes prepared from similarly-handled monolayer cultures show with increasing protein proportional increases in the capacity to activate aflatoxin B1. The density effect observed with intact cultured hepatocytes appears not to be due to substrate (aflatoxin B1) or oxygen depletion. These findings have apparent relevance to studies of carcinogen metabolism and in the design of carcinogen/mutagen testing protocols which utilize cultured hepatocytes.     

Effect of hepatic cytochrome P-450 inducing agents on mutagen activity in the host-mediated assay.

Intraperitoneal treatment of female BALB/c mice with either phenobarbitone or beta-naphthoflavone led to the induction of various hepatic enzymes associated with xenobiotic metabolism and to increased abilities of hepatic S9 fractions to convert the dietary carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to an active bacterial mutagen. In the case of another carcinogen, aflatoxin B1 an increase in in vitro hepatic activation was seen only in mice treated with phenobarbitone. In contrast, pretreatment with either phenobarbitone or beta-naphthoflavone reduced the in vivo activity of both aflatoxin B1 and MeIQx in the host mediated bacterial mutation assay. These data indicate that, for some carcinogens at least, the host-mediated assay may be used to predict the carcinogenic consequences of hepatic enzyme induction.     

Role of Oxidative Stress in Sclerotial Differentiation and Aflatoxin B1 Biosynthesis in Aspergillus flavus

We show here that oxidative stress is involved in both sclerotial differentiation (SD) and aflatoxin B1 biosynthesis in Aspergillus flavus. Specifically, we observed that (i) oxidative stress regulates SD, as implied by its inhibition by antioxidant modulators of reactive oxygen species and thiol redox state, and that (ii) aflatoxin B1 biosynthesis and SD are comodulated by oxidative stress. However, aflatoxin B1 biosynthesis is inhibited by lower stress levels compared to SD, as shown by comparison to undifferentiated A. flavus. These same oxidative stress levels also characterize a mutant A. flavus strain, lacking the global regulatory gene veA. This mutant is unable to produce sclerotia and aflatoxin B1. (iii) Further, we show that hydrogen peroxide is the main modulator of A. flavus SD, as shown by its inhibition by both an irreversible inhibitor of catalase activity and a mimetic of superoxide dismutase activity. On the other hand, aflatoxin B1 biosynthesis is controlled by a wider array of oxidative stress factors, such as lipid hydroperoxide, superoxide, and hydroxyl and thiyl radicals.     

Carcinogen aflatoxin B1 is located preferentially in internucleosomal deoxyribonucleic acid following exposure in vivo in rainbow trout

The purpose of this work was to investigate the distribution in chromatin of deoxyribonucleic acid (DNA) adducts of aflatoxin B1, following exposure in vivo. Rainbow trout were injected intraperitoneally with radiolabeled aflatoxin B1, a potent procarcinogen known to readily induced hepatocellular carcinomas in these fish. After maximum incorporation, liver nuclei were prepared and digested with micrococcal nuclease. Mono-, di-, and trinucleosomal fractions were purified from several stages of nuclease digestion, and the lengths and specific activities of their DNA were determined. The results indicate that aflatoxin B1 is approximately 5 times as likely on a per nucleotide basis to localize on internucleosomal (linker) DNA as on nucleosomal core DNA in this system.     

DNA damage by mycotoxins

Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B1 (AFB1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB1-DNA adduct was identified as 8, 9-dihydro-8-(N7-guanyl)-9-hydroxy-AFB1 (AFB1-N7-Gua), which derives from covalent bond formation between C8 of AFB1-8,9-epoxides and N7 of guanine bases in DNA. Initial AFB1-N7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB1-FAPY. The formation of AFB1-N7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB1-N7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB1-N7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB1 and excretion of AFB1-N7-guanine in urine. Replication of DNA containing AFB1-N7-guanine adduct-induced G-->T mutations in an experimental model. Activation of ras protooncogene has been found in AFB1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G-->T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms.     

Expression of rainbow trout apolipoprotein A-I genes in liver and hepatocellular carcinoma.

Screening for genes overexpressed in trout aflatoxin B1-induced hepatocellular carcinomas resulted in the isolation of cDNA sequences of two apolipoprotein A-Is, apoA-I-1 and apoA-I-2. The levels of apoA-I-1 and -2 mRNAs of liver and tumor were quite different. ApoA-I-1 mRNA was the major species in liver, while apoA-I-1 and -2 mRNAs were present at similar levels in several tumors. This elevated level of apoA-I-2 mRNA was observed in seven different tumors, suggesting that the overexpression of apoA-I-2 was a general feature of aflatoxin B1-induced liver tumors. Hybridization to genomic DNA demonstrated that trout has two different apoA-I genes which is in contrast to other vertebrates which have one gene coding for apoA-I. Liver apoA-I-1 and -2 cDNA clones specified the same amino acid sequence as the tumor apoA-I-1 and -2 cDNA clones. Analysis of the cDNA-derived amino acid sequences showed that trout apoA-I-1 and -2, like human apoA-I, consist largely of multiple 22 amino acid repeats having the potential to generate an amphipathic alpha-helix. The similarity of the repeat pattern in trout and human apoA-Is suggests that all the internal repeats in these sequences arose before the fish-mammal split, some 400 million years ago.     

Effects of hydrogen ion and fatty acid concentrations on the binding of aflatoxin B1 to human albumin

The influence of pH and long-chain fatty acids on the interaction between aflatoxin B1 and human albumin was investigated by fluorescence spectroscopy. Both the binding of aflatoxin B1 to albumin and the fluorescence of albumin-bound aflatoxin are pH-dependent over the pH range of 6-9.5. The data indicates that the carcinogen has a higher affinity for the basic(B) than for the neutral(N) conformation of human albumin. Palmitic, stearic and oleic acids up to a molar ratio of 2 over albumin, increases the binding strength of aflatoxin B1 by means of an allosteric mechanism. Furthermore, the pH-dependence of the aflatoxin-albumin interaction is affected by the presence of oleic acid by narrowing the pH range over which the dependence occurs. At molar ratios of oleic acid to albumin in excess of 4.25 at pH6, 3.1 at pH7.4 and 2.4 at pH9 cause a decrease in aflatoxin B1 fluorescence as a result of reduced binding to albumin.     

Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[a]pyrene (BP), 2-aminofluorene, and aflatoxin B1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied [3H]BP and [3H]DMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti-tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity.     

Benzo[a]pyrene-induced cytochrome P450 1A and DNA binding in cultured trout hepatocytes - inhibition by plant polyphenols

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP) mainly induce lung cancer in humans, but induce liver cancer in fishes. The chemoprevention of cancers through inhibition of molecular events via phytochemicals is a potentially beneficial area of research, and has been carried out in human cell cultures in the past. Carcinogenesis initiation events are thought to occur in similar ways in fish and humans. Our study investigated the feasibility of using cultured rainbow trout CRL-2301 liver cells as a model for BaP-induced carcinogenesis and its prevention by dietary phytochemicals. Treatment with 1 microM BaP resulted in extensive time-dependent covalent binding to cellular DNA and marked cytochrome P450 (CYP) 1A induction, for both about a 20-fold increase, which is similar to what has been observed in cultured human cells. A surprisingly high expression of epoxide hydrolase (EH) activity in these cells likely contributed substantially to the bioactivation of BaP. Two methoxylated flavones and the stilbene resveratrol were effective inhibitors of both the BaP-DNA binding and CYP 1A induction, in particular 5,7-dimethoxyflavone (5,7-DMF), supporting a role for these dietary compounds as cancer chemopreventive agents. Unlike in human liver or bronchial cells, the main mechanism of inhibition of BaP-induced CYP 1A activity in trout liver cells appears to be direct competition at the protein level. Different cellular responses in any particular model used can be expected and the effect of cell context on the biological responses to xenobiotics, including carcinogens as well as polyphenols, must be considered. The trout CRL-2301 cells' sensitivity to BaP treatment is a clear advantage when contemplating a model system for studies of PAH-induced carcinogenesis and cancer chemoprevention. However, extrapolation to human organs should be done cautiously.     

Carcinogen-nucleic acid interactions: equilibrium binding studies of aflatoxins B1 and B2 with DNA and the oligodeoxynucleotide d(ATGCAT)2

Equilibrium binding is believed to play an important role in directing the subsequent covalent attachment of many carcinogens to DNA. We have utilized UV spectroscopy to examine the non-covalent interactions of aflatoxin B1 and B2 with calf thymus DNA, poly(dAdT):poly(dAdT), and poly(dGdC):poly(dGdC), and have utilized NMR spectroscopy to examine non-covalent interactions of aflatoxin B2 with the oligodeoxynucleotide d(ATGCAT)2. UV-VIS binding isotherms suggest a greater binding affinity for calf thymus DNA and poly(dAdT):poly(dAdT) than for poly(dGdC):poly(dGdC). Scatchard analysis of aflatoxin B1 binding to calf thymus DNA in 0.1 M NaCl buffer indicates that binding of the carcinogen at levels of bound aflatoxin less than 1 carcinogen per 200 base pairs occurs with positive cooperativity. The cooperative binding effect is dependent on the ionic strength of the medium; when the NaCl concentration is reduced to 0.01 M, positive cooperativity is observed at carcinogen levels less than 1 carcinogen per 500 base pairs. The Scatchard data may be fit using a "two-site" binding model [L.S. Rosenberg, M.J. Carvlin, and T.R. Krugh, Biochemistry 25, 1002-1008 (1986)]. This model assumes two independent sets of binding sites on the DNA lattice, one a high affinity site which binds the carcinogen with positive cooperativity, the second consisting of lower affinity binding sites to which non-specific binding occurs. NMR analysis of aflatoxin B2 binding to d(ATGCAT)2 indicates that the aflatoxin B2/oligodeoxynucleotide complex is in fast exchange on the NMR time scale. Upfield chemical shifts of 0.1-0.5 ppm are observed for the aflatoxin B2 4-OCH3, H5, and H6a protons. Much smaller chemical shift changes (less than or equal to 0.06 ppm) are observed for the oligodeoxynucleotide protons. The greatest effect for the oligodeoxynucleotide protons is observed for the adenine H2 protons, located in the minor groove. Nonselective T1 experiments demonstrate a 15-25% decrease in the relaxation time for the adenine H2 protons when aflatoxin B2 is added to the solution. This result suggests that aflatoxin B2 protons in the bound state may be in close proximity to these protons, providing a source of dipolar relaxation. Further experiments are in progress to probe the nature of the aflatoxin B1 and B2 complexes with polymeric DNA and oligodeoxynucleotides, and to establish the relationship between the non-covalent DNA-carcinogen complexes observed in these experiments, and covalent aflatoxin B1-guanine N7 DNA adducts.     

Effects of anthocyanins and anthocyanin-rich extracts on the risk for cancers of the gastrointestinal tract

Anthocyanins are the largest group of water-soluble pigments in the plant kingdom. Anthocyanins are responsible for most of the red, blue, and purple colors of fruits, vegetables, flowers, and other plant tissues or products. In recent years, numerous studies have shown that anthocyanins display a wide range of biological activities. This review summarises recent literature evidence on the association of anthocyanins and anthocyanin-rich extracts consumption with the risk for gastrointestinal tract cancer, concentrating on the results from in vivo animal model tumor systems, as well as data from human epidemiological studies. Potential cancer chemopreventive activities of anthocyanins were revealed from in vitro studies. In vivo animal model tumor systems showed that dietary anthocyanins inhibit cancers of the gastrointestinal tract. Some epidemiological studies have revealed protective effects of anthocyanins consumption on gastrointestinal cancer risk in humans. Pharmacokinetic data indicate that absorption of anthocyanins into the bloodstream of rodents and humans is minimal, suggesting that they may have little efficacy in tissues other than the gastrointestinal tract and skin. Future studies should be undertaken to determine if the anticancer effects of anthocyanins are due to the parent compounds and/or to their metabolites.     

Mutagenicity of TCDD in Big Blue transgenic rats

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a significant environmental contaminant resulting from such industrial processes as pulp and paper production. TCDD is a suspected human carcinogen and its ability to induce cancer in laboratory rodents is well documented. Its mechanism of tumor initiation, however, is not well understood and in vitro mutagenicity studies have yielded inconsistent results. In this study, Big Blue lacI transgenic rats were used to assess the mutagenicity of TCDD in both male and female animals. After 6 weeks of exposure to 2 microg/kg TCDD neither an increase in mutation frequency nor any change in mutation spectrum was observed in either male or female animals.     

Dietary fat modifies the in vivo mutagenicity of some food-borne carcinogens

Female BALB/c mice were fed a low fat diet (1% safflower oil, by weight) or one supplemented with 25% (by weight) of beef fat or olive oil. The abilities of these diets to modify the in vitro and in vivo hepatic conversion of the dietary carcinogens aflatoxin B1, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to bacterial mutagens was evaluated. Dietary olive oil appeared to increase the metabolism of both MeIQ and Trp-P-2 to bacterial mutagens in vivo using the intrasanguineous host-mediated assay. Feeding mice either of the high-fat diets increased hepatic conversion of these two compounds to bacterial mutagens in vitro. Dietary fat had no effect on the metabolism of aflatoxin B1. Subsequent experiments suggested that the in vivo effects of dietary olive oil on MeIQ and Trp-P-2 mutagenesis were due to the induction of hepatic enzyme activities rather than to increased rates of uptake of the carcinogen from the gut-lumen.     

Chemoprevention of photocarcinogenesis by selected dietary botanicals.

Epidemiological, clinical and laboratory studies have implicated solar ultraviolet (UV) radiation as a tumor initiator, tumor promoter and complete carcinogen, and their excessive exposure can lead to the development of various skin disorders including melanoma and nonmelanoma skin cancers. Sunscreens are useful, but their protection is not adequate to prevent the risk of UV-induced skin cancer. It may be because of inadequate use, incomplete spectral protection and toxicity. Therefore new chemopreventive methods are necessary to protect the skin from photodamaging effects of solar UV radiation. Chemoprevention refers to the use of agents that can inhibit, reverse or retard the process of skin carcinogenesis. In recent years, considerable interest has been focused on identifying naturally occurring botanicals, specifically dietary, for the prevention of photocarcinogenesis. A wide variety of botanicals, mostly dietary flavonoids or phenolic substances, have been reported to possess substantial anticarcinogenic and antimutagenic activities because of their antioxidant and antiinflammatory properties. This review summarizes chemopreventive effects of some selected botanicals, such as apigenin, curcumin, grape seed proanthocyanidins, resveratrol, silymarin, and green tea polyphenols, against photocarcinogenesis in in vitro and in vivo systems. Attention has also been focused on highlighting the mechanism of chemopreventive action of these dietary botanicals. We suggest that in addition to the use of these botanicals as dietary supplements for the protection of photocarcinogenesis, these botanicals may favorably supplement sunscreens protection and may provide additional antiphotocarcinogenic protection including the protection against other skin disorders caused by solar UV radiation.     

Pitfalls of enzyme-based molecular anticancer dietary manipulations: food for thought

Dietary approaches to cancer chemoprevention increasingly have focused on single nutrients or phytochemicals to stimulate one or another enzymatic metabolizing system. These procedures, which aim to boost carcinogen detoxification or inhibit carcinogen bioactivation, fail to take into account the multiple and paradoxical biological outcomes of enzyme modulators that make their effects unpredictable. Here, we critically examine the scientific and medical evidence for the idea that the physiological roles of specific enzymes may be manipulated by regular, long-term administration of isolated nutrients and other chemicals derived from food plants. Instead, we argue that consumption of healthful diets is most likely to reduce mutagenesis and cancer risk, and that research efforts and dietary recommendations should be redirected away from single nutrients to emphasize the improvement of dietary patterns as a principal strategy for public health policy.