Donald R. Buhler

Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression.

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.

Status and opportunities for genomics research with rainbow trout

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus

1. Several unique flavonoid compounds have recently been isolated from hops, Humulus lupulus, and their presence has been detected in beer. Their chemical structures are similar to other plant-derived compounds, many present in the human diet, that have been shown to have cancer chemopreventive properties due, in part, to inhibition of cytochrome P450 enzymes that activate carcinogens. Additionally, preliminary studies have shown these flavonoids (at 100 muM) to be inhibitory of P450-mediated activation reactions in a variety of in vitro systems. Thus, the in vitro effects of these phytochemicals on cDNA-expressed human CYP1A1, CYP1B1, CYP1A2, CYP3A4 and CYP2E1 were currently examined by the use of diagnostic substrates and the carcinogen AFB1. 2. At 10 muM, the prenylated chalcone, xanthohumol (XN), almost completely inhibited the 7-ethoxyresorufin O-deethylase (EROD) activity of CYP1A1. At the same concentration, other hop flavonoids decreased the EROD activity by 90.8-27.0%. 3. At 10 muM, XN completely eliminated CYP1B1 EROD activity, whereas the other hop flavonoids showed varying degrees of inhibitory action ranging from 99.3 to 1.8%. 4. In contrast, the most effective inhibitors of CYP1A2 acetanilide 4-hydroxylase activitywere the two prenylated flavonoids, 8-prenylnaringenin (8PN) and isoxanthohumol (IX), which produced > 90% inhibition when added at concentrations of 10 mu M. 5. CYP1A2 metabolism of the carcinogen AFB1 was also inhibited by IX and 8PN as shown by decreased appearance of dihydrodiols and AFM1 as analysed by hplc. IX and 8PN also decreased covalent binding of radiolabelled AFB1 to microsomal protein in a concomitant manner. 6. XN, IX and 8PN, however, were poor inhibitors of CYP2E1 and CYP3A4 as measured by their effect on chorzoxazone hydroxylase and nifedipine oxidase activities respectively. 7. These results suggest that the hop flavonoids are potent and selective inhibitors of human cytochrome P450 and warrant further in vivo investigations.

Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring.

Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds. This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout (Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities. Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals. Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content. cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary. Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank. Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms. CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout. In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells. Some of the overexpressed P450 isoforms have been partially characterized. Potential future research directions are discussed.

Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells.

The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR.

Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from β-naphthoflavone-fed rainbow trout

We have purified five isozymes of liver microsomal (LM) P-450 from beta-naphthoflavone-fed rainbow trout. Four forms (LM3, LM1, LM4a and LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMx into two components, LM2 and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a lambda max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4b by Mr, lambda max, or activity towards benzo[a]pyrene. Form LM2 (2% yield) had a specific content of 10.8, a Mr of 54,000, a lambda max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, LM1 and LM3. Neither LM1 nor LM3 was active towards benzo[a]pyrene. The properties of LM2, LM4a and LM4b were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4a and LM4b each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4a or LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4a and LM4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to alpha-naphthoflavone, and regioselectivity towards benzo[a]pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.

Mechanisms of the antiangiogenic activity by the hop flavonoid xanthohumol: NF-κB and Akt as targets

Xanthohumol (XN), the principal flavonoid of the hop plant (Humulus lupulus L.) and a constituent of beer, has been suggested to have potential cancer chemopreventive activities. We have observed that most cancer chemopreventive agents show antiangiogenic properties in vitro and in vivo, a concept we termed “angioprevention.” Here we show for the first time that XN can inhibit growth of a vascular tumor in vivo. Histopathology and in vivo angiogenesis assays indicated that tumor angiogenesis inhibition was involved. Further, we show the mechanisms for its inhibition of angiogenesis in vivo and related endothelial cell activities in vitro. XN repressed both the NF‐?B and Akt pathways in endothelial cells, indicating that components of these pathways are major targets in the molecular mechanism of XN. Moreover, using in vitro analyses, we show that XN interferes with several points in the angiogenic process, including inhibition of endothelial cell invasion and migration, growth, and formation of a network of tubular‐like structures. Our results suggest that XN can be added to the expanding list of antiangiogenic chemopreventive drugs whose potential in cancer prevention and therapy should be evaluated.

Immunochemical cross-reactivity ofβ-naphthoflavone-inducible cytochrome P450 (P450IA) in liver microsomes from different fish species and rat.

Antibodies prepared against the major β-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNF-treated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000–59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin O-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr=54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50–200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P450IA gene family).

Purification and characterization of hepatic steroid hydroxylases from untreated rainbow trout

Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.

The role of biotransformation in the toxicity of chemicals

Aquatic and terrestrial animals metabolize foreign chemicals mainly by oxidation, reduction, hydrolysis and conjugation reactions catalyzed by various enzymes, predominantly localized in the liver, but also found in lower concentrations in other tissues. Xenobiotics are thus biotransformed via several different pathways to less toxic, more polar products which are then readily excreted by the liver, kidney or gill. Whereas biotransformation reactions normally result in detoxification, some chemicals also may be enzymatically converted to highly reactive, electrophilic metabolites (epoxides, free radicals, etc.) which may induce cytotoxic, teratogenic, mutagenic or carcinogenic effects through reaction with various cellular constituents. The initial biotransformation of a less toxic chemical to one or more reactive metabolites, a process commonly referred to as ‘metabolic activation’, is now recognized as an essential initial step for many chemically induced toxicities. The metabolic activation of many mutagens and carcinogens, for example, is primarily catalyzed by the cytochrome P-450-dependent mixed-function oxygenase system in the endoplasmic reticulum, with the formation of epoxides or other reactive species capable of covalently binding to nucleic acids or proteins. Whether or not a xenobiotic causes toxicity, is detoxified, or undergoes metabolic activation to more toxic products, depends not only on the chemical and physical properties of the xenobiotic and its metabolites, but also on the species, strain, age, and sex of the animal, the dose and route of exposure, and the effect of various environmental, nutritional and physiological factors.