In vitro biotransformation of imatinib by the tumor expressed CYP1A1 and CYP1B1.

The main objective of the study was to examine the biotransformation of the anticancer drug imatinib in target cells by incubating it with oxidoreductases expressed in tumor cells. The second objective was to obtain an in silico prediction of the potential activity of imatinib metabolites. An in vitro enzyme kinetic study was performed with cDNA expressed human oxidoreductases and LC-MS/MS analysis. The kinetic parameters (Km and Vmax) were determined for six metabolites. A molecular modeling approach was used to dock these metabolites to the target Abl or Bcr-Abl kinases. CYP3A4 isozyme showed the broadest metabolic capacity, whereas CYP1A1, CYP1B1 and FMO3 isozymes biotransformed imatinib with a high intrinsic clearance. The predicted binding modes for the metabolites to Abl were comparable to that of the parent drug, suggesting potential activity. These findings indicate that CYP1A1 and CYP1B1, which are known to be overexpressed in a wide range of tumors, are involved in the biotransformation of imatinib. They could play a role in imatinib disposition in the targeted stem, progenitor and differentiated cancer cells, with a possible contribution of the metabolites toward the activity of the drug.

Polimorfismos de los genes P53, CYP1A1, CYP1B1, GSTM1, GSTT1 y GSTP1 en cáncer de seno familiar en una población colombiana

Se considera que la susceptibilidad al cáncer de seno es poligenética1; es necesario estudiar otros factores genéticos. Se realizó un estudio analítico de casos y controles (1:2) 120:240. Se tomó una muestra de sangre periférica, y posteriormente se realizó la extracción de ADN (PROBE) y se determinó la presencia de los polimorfismos. Se encontraron asociaciones estadísticamente significativas con el cáncer de seno para: del grupo p53 exón 4, la Arginina con un OR 1,923 IC 95% (1,117 – 3.309); del grupo p53 intrón 3 el I3wm con OR 30,887 IC 95% (3,709 – 257,209); del grupo p53 intrón 6 el I6wm con OR 2.061 IC 95% (1.059 - 4,013); del grupo CYP1B1 Val432leu, la Valina con un OR 2.273 con un IC del 95% (1.084 – 4.855); del grupo CYP1B1 Asn453se la Asparagina/Serina con un OR 1,987 con IC 95% (1.076 – 3.670). El polimorfismo gstm 1r reporta un OR 0,366 con IC 95% (0,219 – 0,613) el cual se considera como protector para cáncer de seno.

Avaliação da expressão dos genes cFOS, IL-1b, CYP1a1 e CYP1b1 em Danio rerio expostos a Benzo[a]pireno e tratados com ligantes do receptor P2X7

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cytochrome P450 CYP1B1 determines susceptibility to 7,12-dimethylbenz[a]anthracene-induced lymphomas

CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.

Molecular modelling of human CYP1B1 substrate interactions and investigation of allelic variant effects on metabolism

Molecular modelling of human CYP1B1 based on homology with the mammalian P450, CYP2C5, of known three-dimensional structure is reported. The enzyme model has been used to investigate the likely mode of binding for selected CYP1B1 substrates, particularly with regard to the possible effects of allelic variants of CYP1B1 on metabolism. In general, it appears that the CYP1B1 model is consistent with known substrate selectivity for the enzyme, and the sites of metabolism can be rationalized in terms of specific contacts with key amino acid residues within the CYP1B1 heme locus. Further-more, a mode of binding interaction for the inhibitor, a-naphthoflavone, is presented which accords with currently available information. The current paper shows that a combination of molecular modelling and experimental determinations on the substrate metabolism for CYP1B1 allelic variants can aid in the understanding of structure-function relationships within P450 enzymes. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Variability in Estrogen-Metabolizing Genes and Their Association with Genomic Instability in Untreated Breast Cancer Patients and Healthy Women

In the present study, we investigated the relationship between polymorphisms in the estrogen-metabolizing genes CYP17, CYP1B1, CYP1A1, and COMT and genomic instability in the peripheral blood lymphocytes of 62 BC patients and 62 controls considering that increased or prolonged exposure to estrogen can damage the DNA molecule and increase the genomic instability process in breast tissue. Our data demonstrated increased genomic instability in BC patients and that individuals with higher frequencies of MN exhibited higher risk to BC when belonging Val/Met genotype of the COMT gene. We also observed that CYP17 and CYP1A1 polymorphisms can modify the risk to BC depending on the menopause status. We can conclude that the genetic background in estrogen metabolism pathway can modulate chromosome damage in healthy controls and patients and thereby influence the risk to BC. These findings suggest the importance to ally biomarkers of susceptibility and effects to estimate risk groups.

Relationships between non-occupational cadmium exposure and expression of nine cytochrome P450 forms in human liver and kidney cortex samples

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 kg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 mug/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYPIA2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYPIA2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, WIC, and CIC were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations. (C) 2001 Elsevier Science Inc. All rights reserved.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Specificity of 17 beta-oestradiol and benzo[a] pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg(48)Ala(119)Leu(432), Arg(48)Ala(119)Val(432), Gly(48)Ala(119)Leu(432), Gly(48)Ala(119)Val(432), Arg(48)Ser(119)Leu(432), Arg(48)Ser(119)Val(432), Gly(48)Ser(119)Leu(432) and Gly(48)Ser(119)Val(432) (all with Asn(453)), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17 beta -oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+. CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg(48) variants tended to have higher activities for 17 beta -oestradiol 4-hydroxylation than Gly(48) variants, although there were no significant variations in 17 beta -oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17 beta -oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val(432) forms than the corresponding Leu(432) forms. 4. In contrast, Leu(432) forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7, 8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val(432) forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17 beta -oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K-l of 0.9 muM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Molecular characterization of a de novo t (11;18) translocation associated with Peter´s anomaly

Dissertação para obtenção do grau de Mestre em Genética Molecular e Biomedicina

Selected polymorphisms in sex hormone-related genes, circulating sex hormones and risk of endometrial cancer.

BACKGROUND: The role of estrogen and progesterone in the development of endometrial cancer is well documented. Few studies have examined the association of genetic variants in sex hormone-related genes with endometrial cancer risk. METHODS: We conducted a case-control study nested within three cohorts to examine the association of endometrial cancer risk with polymorphisms in hormone-related genes among 391 cases (92% postmenopausal at diagnosis) and 712 individually-matched controls. We also examined the association of these polymorphisms with circulating levels of sex hormones and SHBG in a cross-sectional analysis including 596 healthy postmenopausal women at blood donation (controls from this nested case-control study and from a nested case-control study of breast cancer in one of the three cohorts). RESULTS: Adjusting for endometrial cancer risk factors, the A allele of rs4775936 in CYP19 was significantly associated (OR(per allele)=1.22, 95% CI=1.01-1.47, p(trend)=0.04), while the T allele of rs10046 was marginally associated with increased risk of endometrial cancer (OR(per allele)=1.20, 95% CI=0.99-1.45, p(trend)=0.06). PGR rs1042838 was also marginally associated with risk (OR(per allele)=1.25, 95% CI=0.96-1.61, p(trend)=0.09). No significant association was found for the other polymorphisms, i.e. CYP1B1 rs1800440 and rs1056836, UGT1A1 rs8175347, SHBG rs6259 and ESR1 rs2234693. Rs8175347 was significantly associated with postmenopausal levels of estradiol, free estradiol and estrone and rs6259 with SHBG and estradiol. CONCLUSION: Our findings support an association between genetic variants in CYP19, and possibly PGR, and risk of endometrial cancer.

Characterizing the Expression of Cytochrome P450s in Breast Cancer Cells

Une résistance aux agents anticancéreux utilisés dans le traitement du cancer du sein est souvent associée à un échec de traitement. Des variations dans le devenir des agents anticancéreux dans l’organisme, sont des facteurs pouvant expliquer des phénomènes de résistance. Notre but était d’évaluer l’impact des isoenzymes du CYP450s, dans le métabolisme local des agents anticancéreux. Notre premier objectif était de valider un gène rapporteur pour nos analyses de PCR en temps réel. Pour ce faire, nous avons criblé l’expression de 6 gènes rapporteurs dans 23 lignées cellulaires. NUP-214 a été démontré comme étant le gène rapporteur le plus stable avec un écart-type de seulement 0.55 Ct. Notre deuxième objectif était de déterminer le niveau d’expression des ARNm de 19 isoformes du CYP450 dans plusieurs lignées cellulaires du cancer du sein. Les ARNm des CYP450s ont démontré une très grande variabilité entre les lignées cellulaires. Les isoformes CYP1B1 et CYP2J2 démontrent l’expression la plus importante pour la majorité des lignées. Notre troisième objectif était d’évaluer la corrélation entre l’expression des isoformes des CYP450s et leur activité métabolique en utilisant les substrats spécifiques du CYP1B1 et 2J2, 7-éthoxyrésorufine et ébastine, respectivement. Une forte corrélation (r2=0.99) fut observée entre l’activité métabolique vis-à-vis l’ébastine et l’expression du CYP2J2. De même, le métabolisme du 7-éthoxyrésorufine était fortement corrélé (r2=0.98) avec l’expression du CYP1B1. En résumé, ces résultats suggèrent que le métabolisme local des agents anticancéreux pourrait significativement moduler le devenir des agents anticancéreux dans l’organisme, et pourrait être ainsi, une source de résistance.

Soy products in the management of breast cancer

Purpose of review: Meta-analyses of epidemiological studies of soy consumption and breast cancer risk have demonstrated modest protective effects, usually attributed to isoflavones. Concern has been expressed, however, that the estrogenic activity of isoflavones may have adverse effects on breast cancer recurrence. Recent findings: The review covers epidemiological studies that have investigated the impact of soy consumption in breast cancer patients on recurrence and mortality. There are preliminary data to suggest that soy has differential effects on recurrence in human epidermal growth factor receptor-2 positive and human epidermal growth factor receptor-2 negative tumours. Recent studies on mechanisms of action of soy in breast cancer provide insights into epigenetic effects and the interaction of isoflavones with IGF-1 and with a number of polymorphisms of genes associated with breast cancer risk such as MDM2 and CYP1B1. Summary: Overall, these studies indicate that soy foods consumed at levels comparable to those in Asian populations have no detrimental effects on risk of breast cancer recurrence and in some cases significantly reduce the risk. Importantly, soy does not appear to interfere with tamoxifen or anastrozole therapy. Recent research suggests that women who are at increased risk of breast cancer due to polymorphisms in genes associated with the disease may especially benefit from high soy isoflavone intake.