The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR(2)) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC(50) values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2nM PCB126 approximately 30% of eleutheroembryos(3) failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells.

Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.

An AHH-1 TK+/- cell derivative was developed that stably expresses human cytochrome P4501B1 (CYP1B1) cDNA in an extrachromosomal vector which confers resistance to 1-histidinol and co-expresses NADPH cytochrome P450 oxidoreductase (OR). The CYP1B1-expressing cell line was designated h1B1/OR. Microsomes prepared from CYP1B1 cDNA expressing cells exhibit elevated levels of 7-ethoxy-resorufin deethylase (EROD), 7-ethoxy-4-trifluoromethyl-coumarin deethylase (EFCD), benzo(alpha)pyrene hydroxylase (BPH), bufuralol 1'-hydroxylase, testosterone hydroxylase activities and spectrally quantifiable cytochrome P450. CYP1B1-containing microsomes did not contain detectable coumarin 7-hydroxylase, p-nitrophenol hydroxylase, lauric acid hydroxylase, (S)-mephenytoin 4'-hydroxylase or diclofenac 4'-hydroxylase activities. Kinetic parameters for selected substrates were compared among CYP1B1 and the two additional members of the CYP1 family, CYP1A1 and CYP1A2. For BPH and EFCD, the rank order of rates of substrate metabolism were CYP1A1>> CYP1B1>> CYP1A2. For EROD, the rank order of substrate metabolism was CYP1A1>> CYP1A2>> CYP1B1. For both EROD and EFCD the apparent K(m) values for CYP1B1 were more similar to CYP1A1 than to CYP1A2. In order to begin to characterize the promutagen activating ability of CYP1B1, the mutagenicity of selected chemicals was examined in h1B1/OR cells; there was increased sensitivity (CYP1B1-expressing relative to control cells) to the mutagenicity of benzo(a)pyrene, cyclopenta(c,d)pyrene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and aflatoxin B1 (AFB). CYP1B1, expressed in this system, appears to be particularly efficient at activating AFB.

Endpoints of planar halogenated aromatic hydrocarbon (pHAH) and polycyclic aromatic hydrocarbon (PAH) toxicity are mediated via activation of the aryl hydrocarbon receptor (AhR) followed by activation of the so called "AhR-battery" of genes including the cytochrome P450 1 (CYP1) isoforms. The aim of this study was to develop a method to identify CYP1 activity in early life-stages of zebrafish (Danio rerio) in order to elucidate the spatio-temporal pattern of basal and induced CYP1 activities. Preliminary experiments with the fish embryo toxicity test (FET) were carried out to determine toxic effect thresholds of the AhR agonist β-naphthoflavone. To assess basal and β-naphthoflavone-induced CYP1 activity during early life-stages of zebrafish, the commonly used 7-ethoxyresorufin-O-deethylase (EROD) assay was developed further for use in confocal laser scanning microscopy (CLSM) and spectrometry. Following exposure to selected cytochrome P450 inducers, zebrafish embryos were dechorionated, anaesthetized and inspected in vivo under the CLSM. Alternatively, embryos were homogenized, and EROD activity was measured using classical spectrometry in vitro. CLSM of CYP-induced fluorescence allowed for the in vivo detection of CYP1 enzyme activity down to the cellular level as early as in the gastrulation stage. Basal and induced CYP1 activity was detected at all time points examined from 8h post-fertilization to early adulthood and showed a highly dynamic spatio-temporal pattern throughout zebrafish development. Basal and induced EROD activity was prominent in tissues of the cardiovascular system, the urinary tract, the digestive system, and parts of the brain as well as in the central portion of the eye and the otic vesicle during distinct stages of development. The differentiation between constitutive and induced spatio-temporal patterns of CYP1 activity even as early as the gastrula stage provide further insights into the endogenous role of CYP1 activity.

Hepatic cytochrome P450 (CYP) genes, including members of CYP1 to CYP4 families, comprise the majority of the CYP gene superfamily. Previous study has demonstrated that HepG2-specific transcriptional activation of two CYP2C genes was dependent on a common element that bound a HepG2 nuclear protein designated HPF-1 (Venepally, P., Chen, D., and Kemper, B. (1992) J. Biol. Chem. 267, 17333-17338). This cis-acting element is highly homologous to the hepatocyte nuclear factor 4 (HNF-4) binding motif and is present in the promoters of more than 20 other CYP2 genes. To investigate the relationship between HPF-1 and HNF-4, we have compared their tissue distribution, DNA binding, and immunochemical characteristics, as well as transcriptional activity of their recognition elements. DNase I footprint analyses and gel-shift assays indicated that HPF-1, like HNF-4, was present in liver and kidney, but not brain and spleen. Both factors bound to either the HPF-1 site in the CYP2C2 promoter or an HNF-4 site in the human apolipoprotein CIII promoter. These complexes could be "supershifted" by an antiserum specific for HNF-4. When the sequence of the HPF-1 site in the CYP2C2 promoter was changed to that of the apolipoprotein CIII HNF-4 site, comparable transcriptional activities were obtained with the wild-type promoter and the HNF-4 mutant in transfected HepG2 cells. Cotransfection of HNF-4 with CYP2C2 promoter-luciferase constructs in COS-1 cells indicated that HNF-4 could trans-activate the promoters containing the HPF-1 site. These results indicate that the HPF-1 motif is a functional HNF-4-binding site, and the common immunological properties indicate that HPF-1 and HNF-4 are closely related and possibly identical. HNF-4, therefore, may act as a common regulator for the liver-specific expression of many CYP2 genes.

The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that controls estrogen action through two distinct pathways. In one pathway, AhR acts as a transcription factor that induces the expression of the CYP1 family of estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation of the estrogen receptor and suppresses estrogen signaling. The AhR ligand 3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents breast tumors in rodents and is associated with decreased breast cancer risk in humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to gain insight into how two AhR ligands can exert such fundamentally different health effects. We find that DIM and TCDD have differing abilities to activate the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1 gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD has a notably weaker effect on these processes. Small interfering RNA knockdown of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our findings reveal that DIM and TCDD each elicit a unique pattern of change in pathways that control estrogen action; such patterns may determine if an AhR ligand has beneficial or adverse health effects.

Knowledge of the complement of cytochrome P450 (CYP) genes is essential to understanding detoxification and bioactivation mechanisms for organic contaminants. We cloned three new CYP1 genes, CYP1B1, CYP1C2 and CYP1D1, from the killifish Fundulus heteroclitus, an important model in environmental toxicology. Expression of the new CYP1s along with previously known CYP1A and CYP1C1 was measured by qPCR in eight different organs. Organ distribution was similar for the two CYP1Cs, but otherwise patterns and extent of expression differed among the genes. The AHR agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) (31 pmol/g fish) induced expression of CYP1A and CYP1B1 in all organs examined, while CYP1C1 was induced in all organs except testis. The largest changes in response to PCB126 were induction of CYP1A in testis (approximately 700-fold) and induction of CYP1C1 in liver (approximately 500-fold). CYP1B1 in liver and gut, CYP1A in brain and CYP1C1 in gill also were induced strongly by PCB126 >> 100-fold). CYP1C1 expression levels were higher than CYP1C2 in almost all tissues and CYP1C2 was much less responsive to PCB126. In contrast to the other genes, CYP1D1 was not induced by PCB126 in any of the organs. The organ-specific response of CYP1s to PCB126 implies differential involvement in effects of halogenated aromatic hydrocarbons in different organs. The suite of inducible CYP1s could enhance the use of F. heteroclitus in assessing aquatic contamination by AHR agonists. Determining basal and induced levels of protein and the substrate specificity for all five CYP1s will be necessary to better understand their roles in chemical effects and physiology.

RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38 degrees C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.

Monkeys, especially macaques, including cynomolgus (Macaca fascicularis) and rhesus monkeys (Macaca mulatta), are frequently used in drug metabolism studies due to their evolutionary closeness to humans. Recently, numerous cytochrome P450 (P450 or CYP) cDNAs have been identified and characterized in cynomolgus and rhesus monkeys and were named by the P450 Nomenclature Committee. However, recent advances in genome analysis of cynomolgus and rhesus monkeys revealed that some monkey P450s are apparently orthologous to human P450s and thus need to be renamed corresponding to their human orthologs. In this review, we focus on the P450s identified in cynomolgus and rhesus monkeys and present an overview of the identity and functional characteristics of each P450 cDNA in the CYP1-4 families. Information on the Japanese monkey (Macaca fuscata), African green monkey (Cercopithecus aethiops), and marmoset (Callithrix jacchus), primate species used in some drug metabolism studies, are also included. We compared the genomic structure of the macaque P450 genes to those of human and rat P450 genes in the CYP1-4 families. Based on sequence identity, phylogeny, and genomic organization of monkey P450s, we determined orthologous relationships of monkey P450s and, in this article, propose a revised nomenclature: CYP2B17/CYP2B30 to CYP2B6, CYP2C20/CYP2C74 to CYP2C8, CYP2C43/CYP2C83 to CYP2C9, CYP2C75 to CYP2C19, CYP2F6 to CYP2F1, CYP3A8/CYP3A21/CYP3A64 to CYP3A4, CYP3A66 to CYP3A5, and CYP4F45 to CYP4F2. The information presented in this review is expected to promote a better understanding of monkey P450 genes through comparative genomics and thereby make it more feasible to use monkeys in drug metabolism studies.

Fundulus heteroclitus (Atlantic killifish) found at the Atlantic Wood Industries Superfund site on the Elizabeth River (ER) in Portsmouth, VA (USA), have been shown to be resistant to the teratogenic effects of creosote-contaminated sediments found at this highly contaminated site. Many of the polycyclic aromatic hydrocarbons (PAHs) found at the ER are known to activate the aryl hydrocarbon receptor (AHR), and are thought to mediate their toxic effects through this pathway. Activation of the AHR results in the induction of several Phase I and II metabolic enzymes. It has been previously shown that the AHR of killifish from the ER are refractory to induction by AHR agonists. To more fully characterize this altered AHR response, we exposed embryos from the ER and from a reference site on King's Creek, VA (KC) to two PAHs, benzo[alpha]pyrene (BaP) and benzo[k]fluoranthene (BkF), and to the dioxin-like compound (DLC), 3,3',4,4',5-pentachlorobiphenyl (PCB126). We compared their developmental and molecular responses by screening the embryos for CYP1A enzyme activity, cardiac deformities, and mRNA expression of CYP1A, CYP1B1, CYP1C1, and AHR2. Basal gene expression of both CYP1A and CYP1B1 was 40% higher in the KC control embryos compared to those from the ER, while AHR2 and CYP1C1 were not significantly different between the populations. Exposure of KC embryos to BaP, BkF, and PCB126 induced CYP1A activity and cardiac deformities. In contrast, CYP1A activity was induced in ER embryos only in response to BkF exposure, although this induction in ER embryos was significantly lower than that observed in KC fish at comparable concentrations. ER embryos did not develop cardiac deformities in response to any of the chemicals tested. CYP1A, CYP1B1 and CYP1C1 mRNA were all significantly induced in the KC embryos after exposure to BaP, BkF and PCB126. Exposure to BaP and BkF in ER embryos resulted in a significant induction of CYP1A mRNA, albeit significantly lower than observed in KC fish. Interestingly, BaP exposure resulted in induction of CYP1B1 at comparable levels in embryos from both populations. CYP1s were not induced in ER embryos in response to PCB126, nor was CYP1C1 for any treatment examined. Additionally, AHR2 was not significantly induced for any of the treatment groups. This study further characterizes the AHR response in killifish, and provides greater insight into the adapted ER phenotype. The ER adaptation involves the suppression of normal AHR-inducible gene expression for all three CYP1 genes, and therefore is likely an alteration in AHR signaling or control.

Recent epidemiological studies have disputed whether females are at increased risk of lung cancer compared to males. However, several molecular studies are in support of an increased susceptibility to tobacco smoke carcinogens among females. Our earlier findings suggest that women display higher levels of smoking-induced bulky/hydrophobic DNA adducts which may be related to an increased expression of CYP1A1 in their lungs, compared to men. In this in vitro study, 11 lung adenocarcinoma cell lines, 6 of male and 5 of female origin, were exposed to benzo[a]pyrene, cigarette smoke condensate (CSC), or vehicle control. Subsequent expression analysis of genes in the polycyclic aromatic hydrocarbon bioactivation pathway was conducted with Real-Time RT-PCR. DNA adducts were measured in benzo[a]pyrene-exposed cells by ³²P-postlabelling analysis, and CYP1 activity was measured by EROD assay. Analysis of benzo[a]pyrene-DNA adducts showed higher levels of adducts in cell lines from women compared to cell lines from men (p=0.03). The results also revealed significant sex differences in CYP1A1 gene expression, both in untreated cells (p=0.03), and in cells exposed to benzo[a]pyrene (p=0.017) and cigarette smoke condensate (p=0.0043). In CSC-exposed cells, significantly higher levels of CYP1 activity was found in cell lines of female origin (p=0.049). These results are in support of the previously published in vivo data, providing evidence for a higher susceptibility to PAH of women's lungs.

CYP 1B1 is involved in metabolizing both polycyclic aromatic hydrocarbons and estradiol to potentially carcinogenic intermediates, and it is also over-expressed in human cancer cells. In order to investigate whether flavonoids could specifically inhibit CYP 1B1, seven flavonoids in St. John's wort and apigenin were screened for their inhibition of recombinant human CYP 1B1 and CYP 1A1. While seven flavonoids (myricetin, apigenin, kaempferol, quercetin, amentoflavone, quercitrin and rutin) were slightly more selective for CYP 1B1 EROD inhibition (K(i)s 0.06-5.96 microM) compared to CYP 1A1 (K(i)s 0.20-1.6 microM) the difference in K(i)s for the P450s were not significantly different. Rutin did not inhibit CYP 1A1 at concentrations up to 10 microM. Kinetic analyses determined that apigenin and amentoflavone were competitive inhibitors of CYP 1B1, while quercetin showed mixed type inhibition. To characterize the inhibition potential of these flavonoids, five were studied further for their ability to inhibit TCDD-induced EROD activity in 22Rv1 human prostate cancer cells. 22Rv1 cells express constitutive and TCDD-inducible CYP 1A1 and CYP 1B1 mRNA. In the cells, the IC(50)s were similar to those measured for the recombinant CYP 1A1 except for amentoflavone. Quercetin (IC(50): 4.1 microM), kaempferol (3.8 microM), myricetin (3.0 microM) and apigenin (3.1 microM) caused significant inhibition of EROD activity whereas amentoflavone did not cause inhibition. Depending on their bioavailability, flavonoids that can selectively inhibit CYP1 enzymes may be useful as chemoprotective agents in prostate cancer prevention.

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries Superfund Site (Elizabeth River, Portsmouth, VA, USA) are resistant to the acute toxicity and cardiac teratogenesis caused by high levels of polycyclic aromatic hydrocarbons (PAHs) from creosote. The resistance is linked to down regulation of the aryl hydrocarbon receptor (AHR) pathway. We investigated the association between CYP1 activity, as a marker of potential AHR pathway suppression, and contaminant resistance in killifish subpopulations from sites throughout the estuary that varied significantly in PAH contamination level. Adult killifish and sediments were collected from seven sites across approximately 13.7 km in river length within the estuary and from a nearby reference site. Sediment PAH levels were determined using gas chromatography mass spectrometry. Embryos obtained via manual spawning were exposed to individual AHR agonists and PAH mixtures 24 h post fertilization (hpf); CYP1 activity was determined by in ovo ethoxyresorufin-o-deethylase (EROD) at 96 hpf, and cardiac deformity severity was scored at 144 hpf. The total PAH levels measured among the sites varied from approximately 200 to 125,000 ng/g dry sediment. Overall, the resistance to teratogenesis was strongest in the subpopulations from sites in or closest to the major PAH contamination sites, but even embryos from less-contaminated sites within the Elizabeth River demonstrated at least partial resistance to many challenges. Surprisingly, all of the subpopulations tested were highly resistant to PCB-126 (3,3',4,4',5-pentachlorobiphenyl). However, the degree of CYP1 activity response varied significantly among subpopulations and did not always correlate strongly with resistance to teratogenesis; some subpopulations resisted the cardiac teratogenesis caused by the challenges at doses that still elicited strong EROD induction. Our results suggest that there is variation in the adaptive phenotype exhibited by laboratory-spawned embryos from killifish subpopulations throughout the estuary. Furthermore, the results show that contaminants have affected killifish subpopulations throughout the estuary, even in sites with lower levels of PAHs.

Flavonoids constitute a large class of polyphenolic compounds with cancer preventative properties. We have examined the ability of the natural flavone diosmetin to inhibit proliferation of breast adenocarcinoma MDA-MB 468 and normal breast MCF-10A cells and found that this compound is selective for the cancer cells with slight toxicity in the normal breast cells. Diosmetin was metabolised to the structurally similar flavone luteolin in MDA-MB 468 cells, whereas no metabolism was seen in MCF-10A cells. Diosmetin caused G1 arrest at 10 microM in MDA-MB 468 cells after 48-h treatment whereas this effect was not observed in MCF-10A cells. We suggest that diosmetin exerts cytostatic effects in MDA-MB 468 cells, due to CYP1A1 and CYP1B1 catalyzed conversion to the flavone luteolin.

The dose-effect relationships were analysed for several noncarcinogenic endpoints, such as immunological and biochemical responses at subchronic, low dose exposure of female C57BL/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The animals were treated i.p. with TCDD according to the initial- and maintenance-dose principal for a period of 135 days. The initial doses were 1, 10 and 100 ng TCDD/kg, the weekly maintenance doses were 0.2, 2 and 20 ng TCDD/kg, respectively. At days 23, 79 and 135 of TCDD/kg, treatment 10 animals of each dose group were killed. As immunological parameters the number of thymocytes and the pattern of thymocyte subpopulations were determined. In liver, lung and thymus, mRNA expression of TGF-alpha, TGF-beta(1), TGF-beta(2), TGF-beta(3), TNF-alpha, IL-1 beta and different CYP1 isoforms (CYP1A1, CYP1A2, CYP1B1) was analysed. In the livers, activities of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) were measured. TCDD content in the liver was determined. The main results are summarized as follows: (1) The TCDD doses were not sufficient to elicit dose-dependent changes of pattern of thymocyte subpopulation. (2) TCDD failed to change the mRNA expression of TGF-alpha, TGF-beta and TNF-alpha, but led to an increase of IL-1 beta mRNA expression in liver, lung and thymus. The results show that the TCDD induced IL-1 beta mRNA increase is at least as sensitive a marker as the induction of CYP1A isoforms. (3) The expression of CYP1B1 mRNA remained unchanged at the doses tested, while CYP1A1 and CYP1A2 mRNA expression was dose-dependently enhanced. EROD and MROD activities in the liver paralleled the increases of CYP1A1 and CYP1A2 mRNA expression. (4) Regression analysis of the data showed that most of the parameters tested fit a linear model. (5) From the data, a benchmark dose for EROD/MROD activities in the livers of female C57BL/6 mice of about 0.03 ng TCDD/kg per day was calculated.

Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.

Polycyclic aromatic hydrocarbons (PAHs) induce developmental defects including cardiac deformities in fish. The aryl hydrocarbon receptor (AHR) mediates the toxicity of some PAHs. Exposure to a simple PAH mixture during embryo development consisting of an AHR agonist (benzo(a)pyrene-BaP) with fluoranthene (FL), an inhibitor of cytochrome p450 1(CYP1)--a gene induced by AHR activation--results in cardiac deformities. Exposure to BaP or FL alone at similar concentrations alters heart rates, but does not induce morphological deformities. Furthermore, AHR2 knockdown prevents the toxicity of BaP + FL mixture. Here, we used a zebrafish microarray analysis to identify heart-specific transcriptomic changes during early development that might underlie cardiotoxicity of BaP + FL. We used AHR2 morphant embryos to determine the role of this receptor in mediating toxicity. Control and knockdown embryos at 36 h post-fertilization were exposed to DMSO, 100 μg/l BaP, 500 μg/l FL, or 100 μg/l BaP + 500 μg/l FL, and heart tissues for RNA were extracted at 2, 6, 12, and 18 h-post-exposure (hpe), prior to the appearance of cardiac deformities. Data show AHR2-dependent BaP + FL effects on expression of genes involved in protein biosynthesis and neuronal development in addition to signaling molecules and their associated molecular pathways. Ca(2+)-cycling and muscle contraction genes were the most significantly differentially expressed category of transcripts when comparing BaP + FL-treated AHR2 morphant and control embryos. These differences were most prominent at 2 and 6 hpe. Therefore, we postulate that BaP + FL may affect cellular Ca(2+) levels and subsequently cardiac muscle function, potentially underlying BaP + FL cardiotoxicity.

Interindividual variability in drug disposition is a major cause of lack of efficacy and adverse effects of drug therapies. The majority of hepatically cleared drugs are metabolized by cytochrome P450 (CYP) enzymes, mainly in families CYP1, CYP2, and CYP3. Genes encoding these enzymes are highly variable with allele distribution showing considerable differences between populations. Genetic variability of especially CYP2C9, CYP2C19, CYP2D6, and CYP3A5 is known to have clear clinical impact on drugs that are metabolized by these enzymes. CYP1A2, CYP2A6, CYP2B6, CYP2C8, and CYP3A4 all show variability that affects pharmacokinetics of drugs as well, but so far the evidence regarding their clinical implications is not as conclusive. In this review, we provide an up-to-date summary of the pharmacogenetics of the major human drug-metabolizing CYP enzymes, focusing on clinically significant examples.

Cytochrome P450 (CYP) 1A1 (CYP1A1) and CYP1B1, dioxin-inducible CYP1s, are associated with carcinogenesis in extrahepatic tissues. CYP1B1 is featured in carcinogenesis of hormone-responsive tissues, where the CYP1B1 level is considerably high. Although aberrant expression of these enzymes is also observed in cancers that are not related to hormone response, their roles in carcinogenesis are not yet fully understood. We examined DNA methylation status of the CpG islands within the 5'-flanking region of the CYP1B1 and CYP1A1 genes in 7 colorectal cancer cell lines and 40 primary colorectal cancers. By bisulfite-modified direct sequencing, CYP1B1 gene methylation was detected in 2 cell lines (SW48 and Caco-2) and 2 (5%) cancers, but not in corresponding normal tissues. Treatment of the cells with 5-aza-2'-deoxycytidine revealed a clear increase in the CYP1B1 mRNA levels in SW48 and Caco-2 cells, while the amount of methylated alleles decreased. Only HT29 cells showed a clear increase in CYP1A1 mRNA, although there were no apparent differences in methylation status among these cell lines. None of these cell lines showed significant change in mRNA levels of aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT), which are known to directly activate CYP1 transcription. This observation suggested that expression of CYP1B1, but not CYP1A1, was downregulated by promoter methylation rather than decreased expression of AhR/ARNT. In conclusion, CpG methylation of the CYP1B1 promoter region epigenetically regulates CYP1B1 expression during development of some colorectal cancers. Moreover, cancers with aberrant CYP1B1 expression might show altered response to procarcinogen metabolism and chemotherapy.

OBJECTIVE

Pharmacokinetics of theophylline was investigated in Cyp1(+/+) wild-type mice, Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and humanized hCYP1A1_1A2 mice lacking either the mouse Cyp1a1 or Cyp1a2 gene.

RESULTS

Animals received a single dose of theophylline (8 mg/kg i.p.), either alone or pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 microg/kg i.p.) 24 h prior to theophylline. We found that mouse or human CYP1A2 is the predominant enzyme for theophylline metabolism, the contribution of mouse or human CYP1A1 to theophylline metabolism is negligible, and another TCDD-inducible enzyme plays a minor role in 1-methyluric acid and 1,3-dimethyluric acid formation as well as enhanced theophylline clearance from the body. The half-life of elimination from plasma was more than four times longer in Cyp1a2(-/-) than Cyp1(+/+) mice and more than 10 times different after TCDD pretreatment. In humanized hCYP1A1_1A2 mice lacking the mouse Cyp1a2 gene, the half-life of elimination from plasma was two to three times longer than that in Cyp1(+/+) mice and four to five times different after TCDD pretreatment.

CONCLUSIONS

Replacement of mouse Cyp1a2 with a functional human CYP1A2 gene restored the ability to metabolize theophylline, and the metabolism changed to a humanized profile (i.e. 3-methylxanthine formation, not seen in the wild-type mouse). TCDD-pretreated hCYP1A1_1A2 Cyp1a2(-/-) mice exhibited enhanced theophylline metabolism and clearance, due to induction of the human CYP1A2 enzyme. Comparing the hCYP1A1_1A2 Cyp1a2(-/-) and wild-type mice with published clinical studies, we found theophylline clearance to be about 5 times and 12 times, respectively, greater than that reported in humans.

In the accompanying paper, we present and analyse the sequence of a "superactivator" mutant allele of the CYP1 (HAP1) gene. This locus encodes a trans-acting pleiotropic positive regulator of the transcription of both isocytochrome c structural genes. In this paper, we present the genetic localization of the mutation and the sequence of the wild-type fragment that includes the mutation. The mutated phenotype that commutes the expression of the two isocytochrome structural genes (superactivation of CYP3 and inhibition of CYC1) results from a transversion in an AGT codon (serine) in the wild-type to an AGG codon (arginine) in the mutant. Moreover, we show that the missense mutation that affects the amino acid preceding the first cysteine of the "Zn finger" is responsible on its own account for the entire mutated phenotype. In all seven yeast regulatory proteins analysed so far, this position is occupied by a neutral amino acid (serine, alanine or glycine), thus the serine-arginine replacement is a radical one. This result is consistent with the hypothesis of alternative and mutually exclusive Zn fingers, formed either at low or high redox potential, recognizing the target sequences identified in the upstream regions of the CYC1 and CYP3 isocytochrome c structural genes.

Cells of Saccharomyces cerevisiae contain a major cytosolic cyclophilin (Cyp)-related peptidyl-prolyl cis-trans isomerase (PPIase) which is the target for cyclosporin A (CsA) cytotoxicity and which is encoded by the CYP1 gene [Haendler et al., Gene 83 (1989) 39-46]. We recently identified a second Cyp-related gene in yeast, CYP2 [Koser et al., Nucleic Acids Res. 18 (1990) 1643] which predicts a protein with a hydrophobic leader sequence. A sequence lacking 33 codons from the 5'-end of the CYP2 open reading frame was generated by the polymerase chain reaction and engineered for expression in Escherichia coli. The corresponding recombinant truncated protein was purified and found to exhibit PPIase activity which was inhibited by CsA. The CYP2 gene is genetically unlinked to CYP1. As with CYP1, genomic disruption of CYP2 had no effect on haploid cell viability. Disruption of all three of the known yeast PPIase-encoding genes [CYP1, CYP2, and RBP1 for rapamycin-binding protein; Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] in the same haploid cell also resulted in no apparent cellular phenotype, suggesting either that none of these enzymes have an essential function or that additional PPIases can compensate for their specific absence. Whereas cells containing a genomic disruption of CYP1 exhibited a CsA-resistant phenotype, genomic disruption of CYP2 had no effect on CsA sensitivity. This suggests that the CYP1 gene product is the primary cellular target for CsA toxicity in yeast. Since both purified Cyps display CsA sensitivity in vitro, our data suggest that Cyp1 and Cyp2 differ in terms of their cellular function and/or localization.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed when meat containing food is cooked, induces cancer of the colon, prostate and mammary gland of rats, tumours that are strongly associated with a Western diet. After consumption of a meat meal, PhIP is rapidly absorbed, metabolised and bioactivated to DNA damaging species. Thus, PhIP should be considered as a candidate etiological agent for human cancer. Studies in vitro in model mammalian cell culture systems, and in vivo in transgenic animals, have shown that mutation induced by PhIP is dose dependent and describes a mutational "fingerprint" that is characteristic of the chemical. This genetic toxicity is dependent upon CYP1 family metabolic activation and is detectable in these model systems at micro M concentrations. At early time points, PhIP treated cells show subtle signs of toxicity that lead to altered growth and cycling. Using co-culture systems where one cell line bioactivates PhIP with a second cell line as target, we showed in human lymphoblastoid target cells that PhIP induced a dose- and time-dependent S-phase delay of the cell cycle. With time, the cell population became increasingly apoptotic with remaining survivors carrying a mutated gene set. Transcript profiling of treated cells indicated differential expression of genes involved in cell cycle regulation, stress response, receptors and tumour related genes. Prominent was elevation of p21(cip1/waf1) transcript and Western blot analysis confirmed induction of p21(cip1/waf1) and p53 proteins. The dose dependency and temporal aspects of these changes indicate that manipulation of the cell cycle and growth in response to PhIP is a precursor to mutant selection. Reduction of the PhIP dose allows dissection of a different battery of cellular responses that favour cell growth rather than inhibition. This pro-growth stimulus is oestrogen-like and encompasses altered gene expression, proliferation and cell behaviour. In human breast cell lines, these PhIP-mediated pro-oestrogenic responses are inhibited by the anti-oestrogen ICI 182780. This range of molecular and genetic responses induced in cells by PhIP is quite remarkable. Its ability to activate S-phase cell cycle checkpoint, alter gene expression leading to apoptosis and an increased frequency of mutation are probably direct consequences of its genetic toxicity. In contrast, its pro-oestrogenic activity is likely to be a driver of clonal expansion. We suggest that these PhIP-induced genomic and cellular events contrive to manipulate cell cycle and survival. Understanding these molecular processes as well as the genetic toxicology of the chemical will help to define the involvement of PhIP in carcinogenesis and shed light upon its tissue specificity.

Mammalian cytochrome P450 1 (CYP1) genes are well characterized, but in other vertebrates only the functions of CYP1A genes have been well studied. We determined the catalytic activity of zebrafish CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1 proteins using 11 fluorometric substrates and benzo[a]pyrene (BaP). The resorufin-based substrates, 7-ethoxyresorufin, 7-methoxyresorufin, and 7-benzyloxyresorufin, were well metabolized by all CYP1s except CYP1D1. CYP1A metabolized nearly all substrates tested, although rates for non-resorufin substrates were typically lower than resorufin-based substrates. Zebrafish CYP1s did not metabolize 7-benzyloxyquinoline, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin or 7-methoxy-4-(aminomethyl)-coumarin. CYP1B1 and CYP1C2 had the highest rates of BaP metabolism. 3-Hydroxy-BaP was a prominent metabolite for all but CYP1D1. CYP1A showed broad specificity and had the highest metabolic rates for nearly all substrates. CYP1C1 and CYP1C2 had similar substrate specificity. CYP1D1 had very low activities for all substrates except BaP, and a different regioselectivity for BaP, suggesting that CYP1D1 function may be different from other CYP1s.