Metabolizing enzyme localization and activities in the first trimester human placenta: the effect of maternal and gestational age,smoking and alcohol consumption

BACKGROUND: The rationale for this study was to assess the expression, activity and localization of the enzymes uridine diphosphate glucuronosyltransferase (UGT), beta-glucuronidase, cytochrome P450 1A (CYP1A) and cytochrome P450 2E1 (CYP2E1) in first trimester human placenta and to gauge the effects of maternal variables on placental metabolism. METHODS: CYP1A, CYP2E1, UGT and beta-glucuronidase activities were assessed in 25 placentas using ethoxyresorufin, chlorzoxazone, 4-methylumbelliferone and 4-methylumbelliferone glucuronide respectively. Protein expression and localization were detected by immunoblot and immunohistochemistry. All statistics were non-parametric. RESULTS: UGT, beta-glucuronidase and CYP1A activities were detected in all placentas sampled; CYP2E1 was undetectable. CYP1A, UGT1A UGT2B proteins were detected in all placentas (n = 6) tested and CYP2E1 in 4/6 placentas sampled and were localized to the syncytium. UGT and CYP1A activities were significantly elevated in the placentas of mothers who smoked (P < 0.05 and P < 0.001 respectively) and were greatest in women who both smoked and drank alcohol (P < 0.05 and P < 0.01 respectively). Enzyme activities were significantly negatively correlated with gestational age (P < 0.05, r = 0.54, UGT) and maternal age respectively (P < 0.001, r = 0.63, CYP1A). beta-Glucuronidase activity did not differ with patient variables. CONCLUSIONS: Metabolism of compounds by the human placenta in the first trimester may be affected by maternal and environmental factors altering the activity of constitutive metabolizing enzymes.     

Cytochrome P450 1A1 (<Emphasis Type="Italic">CYP1A1</Emphasis>) T3801C and A2455G polymorphisms in breast cancer risk: a meta-analysis

The cytochrome P450 1A1 gene (CYP1A1), encoding Phase I metabolic enzymes, appeared to be a candidate gene for breast cancer risk. However, studies on the association between polymorphisms in this gene and breast cancer have yielded conflicting results. We performed a meta-analysis to investigate the association with breast cancer of the CYP1A1 polymorphisms T3801C (9,316 cases and 12,714 controls) and A2455G (9,552 cases and 9,320 controls). In the genotype contrast of A2455G, both additive [GG vs AA, P = 0.04, fixed-effects OR 0.72; 95% CI (0.53–0.99), P = 0.95 for heterogeneity] and recessive [GG vs (GA + AA), P = 0.04, fixed-effects OR 0.73; 95% CI (0.53–0.99), P = 0.97 for heterogeneity] models produced significant results in east-Asians. In pre-menopausal women in a worldwide population, significant association between A2455G and breast cancer was also found using both models [additive model: P = 0.02, fixed-effects OR 0.52; 95% CI (0.29–0.92), P = 0.39 for heterogeneity; recessive model: P = 0.02, fixed-effects OR 0.51; 95% CI (0.29–0.90), P = 0.38 for heterogeneity]. Our meta-analysis suggests that an A2455G G/G genotype is associated with a trend of reduced breast cancer risk, both in east-Asian women and in pre-menopausal women worldwide, while the T3801C C allele might not be a risk factor for breast cancer. Larger scale primary studies are required to further evaluate the interaction of CYP1A1 polymorphisms and breast cancer risk in specific populations.     

<Emphasis Type="Italic">CYP3A4</Emphasis> and <Emphasis Type="Italic">CYP3A5</Emphasis>genotyping by Pyrosequencing

Background  

Human cytochrome P450 3A enzymes, particularly CYP3A4 and CYP3A5, play an important role in drug metabolism. CYP3A expression exhibits substantial interindividual variation, much of which may result from genetic variation. This study describes Pyrosequencing assays for key SNPs in CYP3A4 (CYP3A4*1B, CYP3A4*2, and CYP3A4*3) and CYP3A5 (CYP3A5*3C and CYP3A5*6).     

Gadolinium chloride suppresses styrene-induced cytochrome P450s expression in rat liver

To assess the effect of gadolinium (Gd) on the expression of several forms of cytochrome P450 (P450s) and antioxidant enzymes, we treated rats with gadolinium chloride (25 mg as Gd/kg body weight) 4 h after styrene (a multiple P450 inducer) treatment (600 mg/kg). Gd treatment significantly suppressed styrene-inducible cytochrome P4502B1 (CYP2B1), CYP2B2, CYP2E1, and CYP3A2 mRNA expressions to 48.6%, 69.8%, 61.1%, and 38.5%, accompanying with the reduction of proteins expression to 1.42%, 31.2%, 21.1% and 21.1%, respectively, compared with styrene alone treatment. Gd suppressed styrene-inducible CYP1A2 expression, but only at the protein level. On the other hand, styrene treatment caused a decrease in reduced form of glutathione (GSH), as well as increases in lipid peroxide and serum ALT and AST activities, suggesting the occurrence of hepatic damage probably due to styrene-induced oxidative stress in rat liver. Post-treatment of Gd attenuated this styrene-caused hepatic damage. Moreover, mRNA expressions of cellular antioxidant enzymes such as catalase, CuZn-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX) were hardly changed by styrene and/or Gd treatment. In summary, Gd suppressed styrene-inducible expression of not only CYP2B1 but also several forms of P450 at both the mRNA and protein levels, along with attenuation of styrene-caused liver damage. These findings suggested that Gd is a chemo-preventive agent against hepatic damage caused by xenobiotics requiring biotransformation.     

CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation

Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti‐cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P < 0.05). CYP3A5 and CYP3A7 were expressed in 5 and 13 cases respectively (15.6%, 40.6%). There was no association between the expression of CYP3A isoforms and age, sex, tumour size, or location (pelvic or extra‐pelvic). None of the biomarkers showed any correlation with overall or disease‐free survival. In conclusion, expression of CYP3A isoforms is noted in Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours.     

Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae

The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr³) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strain at higher levels than in the insecticide-sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH-cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B₁ to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7, 12-dimethylbenz-[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), were metabolized in the transformed strain to cytotoxic products. © 1996 Wiley-Liss, Inc.     

Genetic variation in UGT genes modify the associations of NSAIDs with risk of colorectal cancer: Colon cancer family registry

The use of non‐steroidal anti‐inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal neoplasia. Previous studies have reported that polymorphisms in NSAID‐metabolizing enzymes central to NSAID metabolism including UDP‐glucuronosyltransferases (UGT) and cytochrome P450 (CYP) 2C9 may modify this protective effect. We investigated whether 35 functionally relevant polymorphisms within CYP2C9 and UGT genes were associated with colorectal cancer risk or modified the protective effect of NSAIDs on colorectal cancer susceptibility, using 1,584 colorectal cancer cases and 2,516 unaffected sibling controls from the Colon Cancer Family Registry. A three‐SNP genotype in UGT1A6 (G–A–A; Ala7–Thr181–Arg184) and the Asp85 variant in UGT2B15 increased the risk of colorectal cancer (OR 3.87; 95% CI 1.04–14.45 and OR 1.34; 95% CI 1.10–1.63, respectively). We observed interactions between UGT1A3 Thr78Thr (A>G) and NSAID use (P‐interaction = 0.02), a three‐SNP genotype within UGT2B4 and ibuprofen use (P‐interaction = 0.0018), as well as UGT2B15 Tyr85Asp (T>G) and aspirin use (P‐interaction = 0.01). The interaction with the UGT2B4 and the UGT2B15 polymorphisms were noteworthy at the 25% FDR level. This study highlights the need for further pharmacogenetic studies to identify individuals who might benefit from NSAID use as part of developing effective strategies for prevention of colorectal neoplasia. © 2014 Wiley Periodicals, Inc.     

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M
(2011) Histopathology  59, 1249–1260
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma Aims: It has recently been shown that podoplanin, a mucin‐type glycoprotein, is expressed by cancer cells and cancer‐associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. Methods and results: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real‐time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin‐positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High‐level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). Conclusions: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.     

Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness

Dhakal H P, Naume B, Synnestvedt M, Borgen E, Kaaresen R, Schlichting E, Wiedswang G, Bassarova A, Holm R, Giercksky K‐E & Nesland J M
(2012) Histopathology  61, 350–364 Expression of vascular endothelial growth factor and vascular endothelial growth factor receptors 1 and 2 in invasive breast carcinoma: prognostic significance and relationship with markers for aggressiveness Aims: Vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR‐1) and VEGF receptor 2 (VEGFR‐2) play a role in breast cancer growth and angiogenesis. We examined the expression and relationship with clinical outcome and other prognostic factors. Methods and results: Tumour sections from 468 breast cancer patients were immunostained for VEGF, VEGFR‐1, and VEGFR‐2, and their relationships with tumour vascularity, disseminated tumour cells (DTCs) in bone marrow and other clinicopathological parameters were evaluated. VEGF, VEGFR‐1 and VEGFR‐2 immunoreactivities were observed in invasive breast carcinoma cells. VEGF expression was significantly associated with VEGFR‐1 and VEGFR‐2 expression (P < 0.001). High‐level cytoplasmic expression of VEGFR‐1 was associated with significantly reduced distant disease‐free survival (DDFS) (P = 0.017, log‐rank) and breast cancer‐specific survival (BCSS) (P = 0.005, log‐rank) for all patients, and for node‐negative patients without systemic treatment (DDFS, P = 0.03, log‐rank; BCSS, P = 0.009, log‐rank). VEGFR‐1 expression was significantly associated with histopathological markers of aggressiveness (P < 0.05). Significantly reduced survival was observed in DTC‐positive patients as compared with DTC‐negative patients in the combined moderate/high VEGFR‐1 group (P < 0.001 for DDFS and BCSS), and the same was true for DDFS in the moderate VEGFR‐2 group (P = 0.006). Conclusions: High‐level expression of VEGFR‐1 indicates reduced survival. Higher‐level expression of VEGFR‐1 or VEGFR‐2 in primary breast carcinomas combined with the presence of DTC selects a prognostically unfavourable patient group.     

CYP17 gene polymorphism and its association with high-risk north Indian breast cancer patients

A single T > C change at the 5′ promoter region of the CYP17 gene is reported to be associated with increased risk of breast cancer. This study evaluates the influence of genetic polymorphism of CYP17 on breast cancer susceptibility. Two hundred and forty-two patients with histopathologically confirmed breast cancer and 212 age-matched controls were included in the present study. Information relating to age at onset of the disease, family history and estrogen receptor status was elicited. Investigation for CYP17 polymorphism was carried out in 106 early onset, 80 late onset and 56 familial cases. The frequencies of two CYP17 alleles were also analyzed in 116 (47.9%) cases with known estrogen receptor (ER) status confirmed immunohistochemically. A polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method was used to detect the polymorphism, and the genotypes identified were assigned as homozygous wild type (A1A1), heterozygous variant (A1A2), and homozygous variant (A2A2). Associations between the various genotypes in patients and controls were investigated with Fisher’s exact test. All the tests were two tailed. The results showed that the frequency of heterozygous and homozygous CYP17 genotype was higher in early onset breast cancer patients (94.3%) than in controls (80.3%), and the difference was significant (P = 0.001). A highly statistically significant increased risk in carriers of homozygous A2 allele was found in young patients (P ≤ 0.001) in comparison with patients having late onset condition (P = 0.260). However, no significant association between the genotype and breast cancer risk was observed among women with strong family history. Further, data had showed that patients (80.6%) with at least one A2 allele tended to exhibit ER-independent cell proliferation, although statistical significance could not be established (P = 0.160). The present findings suggest that CYP17 A2 allele gene polymorphism might play a significant role in breast cancer development in young Indian women.     

Hepatotoxicity and gene expression down-regulation of CYP isozymes caused by renal ischemia/reperfusion in the rat

Renal ischemia/reperfusion (I/R) occurs in many clinical scenarios, including trauma, elective surgery, and transplantation. Events initiated by this process can lead to inflammation in the kidneys, culminating in local injury as well as distant organ dysfunction. The objectives of this study were to investigate the changes in the functions of the liver and the regulation of gene expression of cytochrome P450 (CYP) isozymes after renal I/R. Hepatoxocity was assessed by serum alanine aminotransferase (sALT), serum aspartate aminotransferase (sAST) and liver glutathione-S-transferase (GST) activities, liver glutathione (GSH) level, and histopathological examination. Hepatic cytochrome P4503A1 (CYP3A1) and cytochrome P4502E1 (CYP2E1) activities were measured by erythromycin N-demethylase (ERD) and aniline hydroxylase (ANH) activities, respectively. CYP3A1 and CYP2E1 mRNA expression was determined by RT-PCR. Results showed that activities of sALT and sAST were significantly increased, while hepatic CYP3A1and CYP2E1 activities as well as their respective mRNA levels were significantly decreased after renal I/R. Moreover, hepatic tissue congestion, degeneration, and local necrosis were observed in rats after 1, 4, and 8 h renal reperfusion following 2 h renal ischemia. In conclusion, the present study suggests that renal I/R can cause hepatotoxicity and gene expression down-regulation of CYP isozymes in rats.     

Possible risk modification by CYP1A1, GSTM1 and GSTT1 gene polymorphisms in lung cancer susceptibility in a South Indian population

Susceptibility to lung cancer has been shown to be modulated by inheritance of polymorphic genes encoding cytochrome P450 1A1 (CYP1A1) and glutathione S transferases (GSTM1 and GSTT1), which are involved in the bioactivation and detoxification of environmental toxins. As the incidence of lung cancer is known to differ according to ethnicity, we have conducted a case-control study of 146 South Indian lung cancer patients along with 146 healthy controls, to assess any association between CYP1A1, GSTM1 and GSTT1 polymorphisms, either separately or in combination, with the likelihood of development of lung cancer in our population. The current weight of evidence from our study indicated that the frequency of CYP1A1 MspI homozygous variant alleles was significantly higher in cases (OR=3.178). We observed a considerable difference in the GSTT1 null deletion frequency in this population when compared with other populations (OR=2.472, 95% CI: 1.191–5.094, P=0.014). There was no relative risk in GSTM1 null genotype when analysed singly (P=0.453). Considering genotype combinations, risk of lung cancer increased remarkably significantly in individuals having one variant allele of CYP1A1, GSTM1, or GSTT1, suggesting gene–gene interactions. Rare genotypic combinations (such as CYP1A1 wild GSTM1 or GSTT1 either null; CYP1A1 variant both GSTM1 and GSTT1 present; CYP1A1 variant GSTM1 or GSTT1 either null), were at higher risk compared to the reference group. Moreover, patients who had smoked <20 pack years and harboured the CYP1A1 variant allele or the GSTT1 null genotype also had a significant risk of lung cancer. Hence our study—the first to analyse a South Indian population—suggests the importance of combined CYP1A1, GSTM1 and GSTT1 polymorphisms in the development of smoking-induced lung cancer.     

Amplified in breast cancer 1 expression in breast cancer

Aims: The amplified in breast cancer 1 (AIB1), steroid receptor co‐activator family member, acts as an oestrogen receptor (ER) co‐activator. Acting with HER‐2, it is thought to play a role in endocrine resistance by facilitating ER–growth factor crosstalk. The aim was to analyse AIB1 expression by immunohistochemistry and study its correlations with other prognostic variables in breast cancer and its effect on survival. Methods: A tissue microarray comprising tumours from 438 patients with 15.4 years’ median follow‐up was used. Interpretable AIB1 expression obtained in 395 patients was analysed along with other prognostic factors in breast cancer. Results: AIB1 expression scores ranged from 0 to 30; positive AIB1 expression (score > 14) was seen in 146/395 breast cancers; it correlated negatively with ER (P = 0.003) and progesterone receptor (PR) (P = 0.007), and positively with HER‐2 (P = 0.005) and tumour grade (P = 0.014). It did not correlate with nodal status (P = 0.437). Among ER+ patients, AIB1 expression showed a trend towards loss of PR expression (29% versus 20%; P = 0.14). AIB1 did not predict survival on univariate or multivariate analysis. Conclusions: AIB1 expression correlates with HER‐2 expression in breast cancer and shows a trend of association with loss of PR expression in ER+ tumours. Our study supports the postulated role of AIB1 in ER–growth factor interactions.     

Expression of autophagy‐related markers beclin‐1, light chain 3A,light chain 3B and p62 according to the molecular subtype of breast cancer

Aims: To investigate the relationship between the expression of autophagy‐related proteins, including beclin‐1, light chain (LC) 3A, LC3B, and p62, and prognosis in invasive breast cancer. Methods and results: We constructed tissue microarrays from the breast cancer cells of 489 patients, and classified molecular subtypes using surrogate immunohistochemical stains. The tumoral expression levels of LC3A and LC3B were highest in triple‐negative breast cancer (TNBC) (P < 0.001), whereas these types of tumour had the lowest expression levels of these markers in the stroma (P = 0.005 and P < 0.001, respectively). Cytoplasmic beclin‐1 expression was highest in TNBC, but nuclear expression was lowest (P < 0.001). p62 cytoplasmic and nuclear expression were highest in HER2‐type tumours (P = 0.001 and P < 0.001, respectively). Tumoral LC3A and LC3B expression were associated with high histological grade (P < 0.001, and P < 0.028, respectively), but nuclear p62 expression was associated with lower histological grade (P = 0.004). Conclusions: Autophagy‐related markers are differentially expressed according to the molecular subtype of breast cancer. In particular, expression of LC3A, LC3B and beclin‐1 was highest in TNBC tumour cells, whereas that of LC3A and LC3B in the tumour stroma was lowest in TNBC.     

Identification of the metabolites of polybrominated diphenyl ether 99 and its related cytochrome P450s

Objective

To investigate the metabolites of polybrominated diphenyl ether 99 (BDE-99) and its related cytochrome P450s in an in vitro system.

Methods

Rat primary hepatocytes were isolated and treated with BDE-99 for 24-72 h. Metabolites were then extracted from the hepatocytes and media, and detected by GC/MS. Several mRNAs of metabolic enzymes were also extracted from the same cells and the gene expression levels were determined using quantitative real-time PCR. In addition, selected recombinant cytochrome P450s (CYPs) were expressed in a bacurovirus/sf9 system, and these were further used to explore the metabolism of BDE-99 in vitro. The parent depletion approach was used for screening the ability of CYPs to eliminate BDE-99.

Results

A reductively debrominated metabolite, BDE-47, and three oxidative metabolites, 2, 4, 5-tribromophenol, 5-OH-BDE-47, and 5′-OH-BDE-99, were identified from the BDE-99-treated rat hepatocytes, whereas no MeO metabolite was detected in the system. RT-PCR analysis showed that CYP 3A23/3A1, 1A2, and 2B1/2 were induced by BDE-99. Furthermore, using the heterological expressed CYP proteins in in vitro BDE-99 metabolism experiments we found that CYP1A2 and CYP3A4 showed the highest metabolic efficiency for BDE-99, with the metabolic clearance rates of CYP1A2 and CYP3A4 being 30.3% and 27.7%, respectively. CYP1A1 and CYP2A6 displayed relatively low clearance rates, while CYP2E1 seemed not to be associated with the BDE-99 metabolism.

Conclusions

In our in vitro rat primary hepatocyte metabolism system, four metabolites of BDE-99 were identified, and CYP3A4 and CYP1A2 were demonstrated to be involved in the BDE-99 metabolism.     

Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and normal bronchial tissue

Cytochrome P450 2E1 (CYP2E1) is a major xenobiotic-metabolizing enzyme but data concerning its extrahepatic expression are few. CYP2E1 can metabolically activate many procarcinogens and therefore its presence in the lung might play a role in bioactivation of procarcinogens, so we studied the expression and localization of CYP2E1 in primary pulmonary carcinomas and surrounding normal bronchial tissue from 28 patients. Seromucous glands showed expression of CYP2E1 in 19 and bronchial epithelium in 18 of the 28 samples of normal bronchial tissue. Thirteen of the corresponding cases of primary pulmonary carcinoma showed staining for CYP2E1. In 11 of these 13 cases, CYP2E1 was also present in normal bronchial tissue. There was no statistically significant difference in the expression of CYP2E1 between adenocarcinomas and squamous cell carcinomas. No association was observed between the expression of CYP2E1 in tumour tissue and normal bronchial tissue. However, there was a significant correlation between the expression of CYP2E1 in seromucous glands and bronchial epithelium (r=0.61, P<0.01) of normal tissue. We conclude that CYP2E1 can be present in both normal and neoplastic bronchial tissue.     

Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase.

A series of Salmonella typhimurium (S. typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo-protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved.     

Interaction of nitric oxide and the cytochrome P-450 system on blood pressure and renal function in the rat: dependence on sodium intake

Aim: Interaction was examined of nitric oxide (NO) and cytochrome P‐450 (CYP‐450)‐dependent arachidonic acid derivatives, 20‐HETE and EETs, in control of arterial pressure (MABP) and renal function. Modification of this interaction by changing sodium intake was also studied. Methods: On low, standard or high Na diet (LS, STD and HS rats respectively) effects of sequential blockade of NO synthases (NOS) and CYP‐450 enzyme activity on MABP, renal blood flow (RBF, Transonic probe), renal medullary perfusion (MBF, laser‐Doppler technique), medullary tissue NO (selective electrode) and renal excretion were examined in anaesthetized rats. All NOS were blocked with N^?‐nitro‐l ‐arginine methyl ester (l ‐NAME), the neuronal NOS with S‐methyl‐l ‐thiocitrulline (SMTC), and CYP‐450 with 1‐aminobenzotriazole (ABT). Results: In each diet group the baseline MABP was highest in rats pre‐treated with l ‐NAME. CYP‐450 inhibition significantly decreased MABP only in LS (?9%) and HS rats (?22%) pre‐treated with l ‐NAME. This MABP decrease correlated directly with the dietary sodium content (r = 0.644, P < 0.001). CYP‐450 inhibition decreased RBF in LS and HS rats (not in HS pre‐treated with l ‐NAME). Acute exclusion of CYP‐450 significantly increased MBF only in STD, SMTC pre‐treated rats; in HS group it significantly increased medullary tissue NO by about 1.0 nA. The post‐ABT changes in renal excretion occurred in LS and HS rats, irrespective of the status of NO synthesis. Conclusions: Both NO‐ and CYP‐450‐dependent agents contribute to blood pressure and kidney function control, however, the role of 20‐HETE and EETs becomes crucial only under conditions of high sodium intake or after NOS inhibition.     

Genetic polymorphisms in oestrogen metabolic pathway and breast cancer: a positive association with combined CYP/GST genotypes

The cytochrome P450 family (CYPs) and the glutathione S-transferase (GSTs) enzymes play an important role in the metabolism of environmental carcinogens and of oestrogen and can affect breast cancer risk. In this study we examine the role of the genes CYP1A1, CYP17, CYP2D6, GSTM1, GSTP1 and GSTT1 in breast cancer risk in Brazilian women. The study population consisted of 102 incident breast cancer cases and 102 healthy controls. Genotyping analyses were performed by PCR-based methods. A significant finding was observed between GSTP1 Ile-Val polymorphism and breast cancer risk (OR = 1.81; CI 95% = 1.04-3.16). A significant association was observed between women with 0-2 risk genotypes and those with 4 or more risk genotypes (OR = 2.42; CI 95% = 1.13-5.18) when the potential combined effects of the risk genotypes were examined. No significant differences between cases and controls were found correlating the genotypes and the clinical-histopathological parameters. In conclusion, in our population only GSTP1 was associated with breast cancer risk. However, when the genes were tested in combination, a significant association in the breast cancer risk was observed.