Genomic profiling of late-onset basal cell carcinomas from two brothers with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant genetic disorder. It is commonly caused by mutations in PTCH1 and chiefly characterized by multiple basal cell carcinomas (BCCs) developing prior to the age of 30 years. In rare cases, NBCCS presents with a late onset of BCC development. OBJECTIVE: To investigate BCC tumorigenesis in two brothers, who showed characteristic features of NBCCS but developed their first BCCs only after the age of 40 years. Two other siblings did not show signs of NBCCS. RESULTS: We obtained blood samples from four siblings and nine BCCs from the two brothers with NBCCS. Whole exome sequencing and RNA sequencing revealed loss of heterozygosity (LOH) of PTCH1 in eight out of nine tumours that consistently involved the same haplotype on chromosome 9. This haplotype contained a germinal splice site mutation in PTCH1 (NM_001083605:exon9:c.763-6C>A). Analysis of germline DNA confirmed segregation of this mutation with the disease. All BCCs harboured additional somatic loss-of-function (LoF) mutations in the remaining PTCH1 allele which are not typically seen in other cases of NBCCS. This suggests a hypomorphic nature of the germinal PTCH1 mutation in this family. Furthermore, all BCCs had a similar tumour mutational burden compared to BCCs of unrelated NBCCS patients while harbouring a higher number of damaging PTCH1 mutations. CONCLUSIONS: Our data suggest that a sequence of three genetic hits leads to the late development of BCCs in two brothers with NBCCS: a hypomorphic germline mutation, followed by somatic LOH and additional mutations that complete PTCH1 inactivation. These genetic events are in line with the late occurrence of the first BCC and with the higher number of damaging PTCH1 mutations compared to usual cases of NBCCS.

Compact acousto-optic multimode interference device in (Al,Ga)As.

Multimode interference (MMI) devices are key components in modern integrated photonic circuits. Here, we present acoustically tuned optical switches on an (Al,Ga)As platform that enable robust, compact and fast response systems improving on recently demonstrated technology. The device consists of a 2 × 2 MMI device fine-tuned in its center region by a focused surface acoustic wave (SAW) beam working in the low GHz range. In this way, we can tune the refractive index profile over a narrow modulation region and thus control the optical switching behaviour via the applied SAW intensity. Direct tuning of the MMI device avoids losses and phase errors inherent to arrayed waveguide based switches, while also reducing the dimensions of the photonic circuit.

Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Plant polyphenols and multidrug resistance: effects of dietary flavonoids on drug transporters in Caco-2 and MDCKII-MDR1 cell transport models.

The hypothesis tested was that specific flavonoids such as epicatechin gallate, epigallocatechin gallate, genistein, genistin, naringenin, naringin, quercetin and xanthohumol will modulate cellular uptake and permeability (P(e)) of multidrug-resistant substrates, cyclosporin A (CSA) and digoxin, across Caco-2 and MDCKII-MDR1 cell transport models. (3)H-CSA/(3)H-digoxin transport and uptake experiments were performed with and without co-exposure of the flavonoids. Aglycone flavonoids reduced the P(e) of CSA to a greater extent than glycosylated flavonoids with 30 microM xanthohumol producing the greatest effect (7.2 x 10(-6) to 6.6 x 10(-7) and 17.9 x 10(-6) to 4.02 x 10(-6) cm s(-1) in Caco-2 and MDCKII-MDR1 cells, respectively); while no measurable effects were seen with digoxin. Xanthohumol significantly demonstrated (1) saturable efflux, (2) increased uptake of (3)H-digoxin and (3) decreased uptake of (3)H-CSA in the Caco-2 cells. The transport data suggests that xanthohumol effects transport of CSA in a manner that is distinct from the digoxin efflux pathway and suggests that intestinal transport of these MDR1 substrates is more complex than previously reported.

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Phytochemical-induced changes in gene expression of carcinogen-metabolizing enzymes in cultured human primary hepatocytes.

1. The naturally occurring compounds curcumin (CUR), 3,3'-diindolylmethane (DIM), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) protect animals against chemically induced tumours. Putative chemoprotective mechanisms include modulated expression of hepatic biotransformation enzymes. However, few, if any, studies have used human primary cells as test models. 2. The present study investigated the effects of these phytochemicals on the expression of four carcinogenesis-relevant enzymes--cytochrome P450 (CYP)1A1 and 1A2, NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S-transferase A1 (GSTA1)--in primary cultures of freshly isolated human hepatocytes. 3. Quantitative RT-PCR analyses demonstrated that CYP1A1 was up-regulated by PEITC and DIM in a dose-dependent manner. CYP1A2 transcription was significantly activated following DIM, IXN, 8PN and PEITC treatments. DIM exhibited a remarkably effective induction response of CYP1A1 (474-, 239- and 87-fold at 50, 25 and 10 microM, respectively) and CYP1A2 (113-, 70- and 31-fold at 50, 25 and 10 microM, respectively), that was semiquantitatively reflected in protein levels. NQO1 expression responded to PEITC (11 x at 25 microM), DIM (4.5 x at 50 microM) and SFN (5 x at 10 microM) treatments. No significant effects on GSTA1 transcription were seen. 4. The findings show novel and unexpected effects of these phytochemicals on the expression of human hepatic biotransformation enzymes that play key roles in chemical-induced carcinogenesis.

Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln.

Seven Sm proteins, termed B/B', D1, D2, D3, E, F, and G, assemble in an ordered manner onto U snRNAs to form the Sm core of the spliceosomal snRNPs U1, U2, U4/U6, and U5. The survival of motor neuron (SMN) protein binds to Sm proteins and mediates in the context of a macromolecular (SMN-) complex the assembly of the Sm core. Binding of SMN to Sm proteins is enhanced by modification of specific arginine residues in the Sm proteins D1 and D3 to symmetrical dimethylarginines (sDMAs), suggesting that assembly might be regulated at the posttranslational level. Here we provide evidence that the previously described pICln-complex, consisting of Sm proteins, the methyltransferase PRMT5, pICln, and two novel factors, catalyzes the sDMA modification of Sm proteins. In vitro studies further revealed that the pICln complex inhibits the spontaneous assembly of Sm proteins onto a U snRNA. This effect is mediated by pICln via its binding to the Sm fold of Sm proteins, thereby preventing specific interactions between Sm proteins required for the formation of the Sm core. Our data suggest that the pICln complex regulates an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN-complex.

Immunohistochemical localization and differential expression of cytochrome P450 3A27 in the gastrointestinal tract of rainbow trout.

In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), a fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed by the proximal descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism.

A multiprotein complex mediates the ATP-dependent assembly of spliceosomal U snRNPs.

The spliceosomal snRNPs U1, U2, U4 and U5 contain a common RNP structure termed the Sm core that is formed by the binding of Sm proteins onto the U snRNA. Although isolated Sm proteins assemble spontaneously onto U snRNAs in vitro, there is increasing evidence that SMN and its interactor Gemin2 are involved in this process in vivo. Here, we describe a cell-free assay system for the assembly of U snRNPs that closely reproduces in vivo conditions. Using this system, we show that assembly of U1 snRNP depends on ATP. Immunodepletion of SMN-Gemin2 from the extract abolished assembly even though the extract contained high levels of Sm proteins. An affinity-purified macromolecular SMN complex consisting of 16 components including all Sm proteins restored assembly in the immunodepleted extract. These data provide the first direct evidence that a complex containing SMN and Gemin2 mediates the active assembly of spliceosomal U snRNPs.

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.

In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes.

Xanthohumol (XN) is the major prenylated flavonoid of hop plants and has been detected in beer. Previous studies suggest a variety of potential cancer chemopreventive effects for XN, but there is no information on its metabolism. The aim of this study was to investigate in vitro glucuronidation of XN by rat and human liver microsomes. Using high-performance liquid chromatography, two major glucuronides of XN were found with either rat or human liver microsomes. Release of the aglycone by enzymatic hydrolysis with beta-glucuronidase followed by liquid chromatography/mass spectrometry and nuclear magnetic resonance analysis revealed that these were C-4' and C-4 monoglucuronides of XN.

In vitro biotransformation of xanthohumol, a flavonoid from hops (Humulus lupulus), by rat liver microsomes.

Xanthohumol (XN) is the major prenylated flavonoid of the female inflorescences (cones) of the hop plant (Humulus lupulus). It is also a constituent of beer, the major dietary source of prenylated flavonoids. Recent studies have suggested that XN may have potential cancer-chemopreventive activity, but little is known about its metabolism. We investigated the biotransformation of XN by rat liver microsomes. Three major polar metabolites were produced by liver microsomes from either untreated rats or phenobarbital-pretreated rats as detected by reverse-phase high-performance liquid chromatography analysis. Liver microsomes from isosafrole- and beta-naphthoflavone-pretreated rats formed another major nonpolar metabolite in addition to the three polar metabolites. As determined by liquid chromatography/mass spectrometry and (1)H NMR analyses, the three major polar microsomal metabolites of XN were tentatively identified as 1) 5"-isopropyl-5"-hydroxydihydrofurano[2",3":3',4']-2',4-dihydroxy-6'-methoxychalcone; 2) 5"-(2"'-hydroxyisopropyl)-dihydrofurano[2",3":3',4']-2',4-dihydroxy-6'-methoxychalcone; and 3) a derivative of XN with an additional hydroxyl function at the B ring. The nonpolar XN metabolite was identified as dehydrocycloxanthohumol.

Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by the degeneration of motor neurons in the spinal cord. The disease is caused by mutations of the survival of motor neuron 1 gene (SMN1), resulting in a reduced production of functional SMN protein. A major question unanswered thus far is why reduced amounts of ubiquitously expressed SMN protein specifically cause the degeneration of motor neurons without affecting other somatic cell types. In a first attempt to address this issue we have investigated the Smn interacting protein 1 (Sip1), with an emphasis on its developmental expression and subcellular distribution in spinal motor neurons in relation to Smn. By confocal immunofluorescence studies we provide evidence that a significant amount of Smn does not co-localize with Sip1 in neurites of motor neurons, indicating that Smn may exert motor neuron-specific functions that are not dependent on Sip1. Sip1 is highly expressed in the spinal cord during early development and expression decreases in parallel with Smn during postnatal development. Strikingly, reduced production of Smn as observed in cell lines derived from SMA patients or in a mouse model for SMA coincides with a simultaneous reduction of Sip1. The finding that expression of Sip1 and Smn is tightly co-regulated, together with the unique localization of Smn in neurites, may help in understanding the motor neuron-specific defects observed in SMA patients.

SMN tudor domain structure and its interaction with the Sm proteins.

Spinal muscular atrophy (SMA) is a common motor neuron disease that results from mutations in the Survival of Motor Neuron (SMN) gene. The SMN protein plays a crucial role in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes via binding to the spliceosomal Sm core proteins. SMN contains a central Tudor domain that facilitates the SMN-Sm protein interaction. A SMA-causing point mutation (E134K) within the SMN Tudor domain prevents Sm binding. Here, we have determined the three-dimensional structure of the Tudor domain of human SMN. The structure exhibits a conserved negatively charged surface that is shown to interact with the C-terminal Arg and Gly-rich tails of Sm proteins. The E134K mutation does not disrupt the Tudor structure but affects the charge distribution within this binding site. An intriguing structural similarity between the Tudor domain and the Sm proteins suggests the presence of an additional binding interface that resembles that in hetero-oligomeric complexes of Sm proteins. Our data provide a structural basis for a molecular defect underlying SMA.

Cloning, sequencing, and characterization of CYP1A1 cDNA from leaping mullet (Liza Saliens) liver and implications for the potential functions of its conserved amino acids.

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambdaZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method.

Oregon's Toxic Household Products Law.

In 1991, Oregon became the first state in the U.S. to require the addition of an aversive agent to ethylene glycol-containing antifreeze and methanol-containing windshield wiper fluid. This new law, entitled "Toxic Household Products (THP) Act," was designed to reduce pediatric and animal poisonings from accidental ingestion of these two potentially lethal consumer automotive products. While not the stated intention of the law, addition of aversive agents to consumer automotive products could also reduce adult poisonings associated with intentional (suicides or alcoholics ingesting methanol-containing windshield wiper fluid) or accidental exposures. This law went into effect April 30, 1995, following settlement of a lawsuit brought by the Chemical Manufacturing Specialties Association (CSMA), a trade group representing the five largest manufacturers of ethylene glycol-based antifreeze in the U.S. This paper discusses the major policy issues that arose following the passage of Oregon's THP Act. Major provisions of the law are provided along with a discussion of CSMA's opposition to the Act's implementation. A description of the eventual settlement that was reached with CSMA as well as the major components of Oregon Health Division's (OHD) enforcement program are also highlighted. Data are presented for 1987 through 1998 on the number of exposures and severity of effects for pediatric cases (children < 6 years old) following exposure to both of these potentially lethal automotive products. However, because of the low incidence of exposures each year, these data are insufficient to draw any conclusions on the impact of the THP Act.

Effects of 17beta-estradiol and testosterone on hepatic mRNA/protein levels and catalytic activities of CYP2M1, CYP2K1, and CYP3A27 in rainbow trout (Oncorhynchus mykiss).

There is growing concern that exposure to chemicals in the environment can disrupt the endocrine systems of wildlife and humans, causing reproductive problems or other adverse effects. The expression of many cytochrome P450s (CYPs) is under hormonal control, hence, levels of these enzymes can be affected by exposure to endocrine-disrupting chemicals. Previous research has reported that treatment of fish and other animals with the estrogenic and androgenic hormones 17beta-estradiol (E2) and testosterone (T) alters the P450 content or enzyme activities in the treated animals. However, the results of many of these studies are either incomplete or in disagreement and in most cases the effect on specific P450 forms has not been determined. Therefore, to better understand the effects of gonadal hormones on the expression of P450s and their associated enzyme activities, it was of interest to undertake a comprehensive investigation of the transcriptional and translational expression of three constitutive hepatic P450s in the rainbow trout (Oncorhynchus mykiss) following hormone exposure. Accordingly, juvenile trout were injected intraperitoneally with propylene glycol vehicle and the most active estrogenic and androgenic hormones E2 (3 mg/kg) or T (3 mg/kg) on days 1, 4, 7, 13, and 15 and euthanized on day 19. After treatment with E2, hepatic microsomes showed significantly lower levels (percentage of control) in total P450 contents (52%), lauric acid hydroxylase (32%), and 6beta-progesterone hydroxylase activities (27%), [(3)H]aflatoxin-DNA binding (31%), and the protein levels of individual cytochrome P450s (CYPs) LMC1 (CYP2M1), LMC2, (CYP2K1), and LMC5 (CYP3A27) (average for three isoforms a reduction to 29% of control values) with only minor differences between sexes. Treatment with T had either no effect or resulted in small increases in total P450 in males (42%), in lauric acid hydroxylase in females (24%), and in 6beta-progesterone hydroxylase activity in males (21%). Biological variabilities among fish were high and a polymorphic or new LMC2-like form was detected at about 52 kDa in some liver microsomal samples after exposure of fish to either hormone. Female liver RNAs were analyzed through Northern blots and an average decrease of 94% in CYP2 M1, CYP2K1, and CYP3A27 mRNA levels occurred in the E2-treated trout. In livers from T-treated trout, the changes of mRNA levels of CYP2M1 and CYP3A27 were negligible, but CYP2K1 mRNA level decreased by about 60%. Additional CYP2K1 cDNA hybridizable mRNAs were seen in some fish as faint bands at about 2.8 kb for both hormone treatments. Results of this study, therefore, indicated that E2 down-regulated while T produced small but variable effects on the hepatic mRNA/protein levels of CYP2K1, CYP2M1, and CYP3A27 in juvenile rainbow trout. This study, therefore, suggests that exposure of fish and other wildlife to environmental endocrine disruptors, especially estrogen mimics, can adversely affect a number of physiological processes through mechanisms involving altered levels of expression of specific P450 isozymes.

Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.

LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.

Prenylflavonoids from hops inhibit the metabolic activation of the carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4, 5-f]quinoline, mediated by cDNA-expressed human CYP1A2.

The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potential human carcinogen found in cooked food that requires initial metabolic activation by cytochrome P450s, primarily CYP1A2. The present study was conducted to examine whether recombinant human CYP1A2 expressed in insect cells mediates the metabolic activation of IQ and whether prenylflavonoids found in hops and beer would modulate the CYP1A2-mediated activation of IQ. The cDNA-expressed human CYP1A2 was found to strongly activate IQ as measured by the Ames Salmonella assay and by the covalent binding of IQ metabolites to calf thymus DNA and protein. Inhibition studies showed that the prenylchalcone xanthohumol and the prenylflavanones 8-prenylnaringenin and isoxanthohumol strongly inhibited the mutagenic activation of IQ mediated by cDNA-expressed human CYP1A2 in the Ames Salmonella assay. The three prenylflavonoids also markedly inhibited the human CYP1A2-mediated binding of IQ to metabolites that bind to DNA. The inhibition of the metabolic activation of IQ was paralleled by the inhibition of acetanilide 4-hydroxylase activity of human CYP1A2. Thus, xanthohumol, isoxanthohumol, and prenylflavanones 8-prenylnaringenin are potent inhibitors of the metabolic activation of IQ and may have the potential to act as chemopreventive agents against cancer induced by heterocyclic amines activated by CYP1A2.