Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues

Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, P greater than 0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, P less than 0.02). On Western blotting of human liver samples with an antibody specific to cytochrome P450IA2 in the rat, only the orthologue of P450IA2 could be detected. This antibody inhibited greater than 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P450IA2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissue-dependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.     

The stability of dioxin-receptor ligands influences cytochrome P450IA1 expression in human keratinocytes.

Three dioxin-receptor ligands were analyzed for their effect on cytochrome P450IA1 mRNA expression in normal human keratinocytes. Although a 2 h pulsed treatment with the receptor agonists 2,3,7,8-tetrachlorodibenzofuran (TCDF) and beta-naphthoflavone (BNF) gave the same maximal induction response, the effect of BNF was transient compared to effect of TCDF. This was most likely due to metabolism of BNF as exemplified by the fact that a P450IA1 enzyme suicide-inhibitor, 1-ethynylpyrene, could prolong the induction response following a short BNF treatment. The TCDF induction of a reporter gene construct under the control of the -1140 to +2435 part of the CYPIA1 gene transiently transfected into HK was effectively inhibited by the dioxin-receptor antagonist alpha-naphthoflavone (ANF). In addition, ANF inhibited the accumulation of TCDF-activated nuclear receptors with capacity to bind to a xenobiotic response element. Interestingly, ANF could also suppress already maximally induced P450IA1 mRNA levels. The data demonstrate that the stability of the ligand influences the long-term effects on gene expression and that the effect of stable ligands may be masked due to receptor antagonist presence. In addition, the results support the hypothesis that a constant low level of activated nuclear receptors is required to maintain induced P450IA1 expression.     

Induction of cytochrome P450IA1 in rat colon and liver by indole-3-carbinol and 5,6-benzoflavone

It is known that consumption of cruciferous vegetables protects against the chemical induction of cancer in many organs. It has been suggested that this protection is mediated through an effect on the cytochrome P450 monooxygenase system. This system is responsible for the activation of a number of chemical carcinogens to their ultimate forms. In the present study, the effect of indole-3-carbinol (I3C) and 5,6-benzoflavone (5,6BF) on the expression of cytochrome P450IA1 in rat colon and liver has been investigated. Cytochrome P450IA1 mRNA was induced in colon following a single oral administration of I3C or 5,6BF. A biphasic induction profile was obtained with maxima at 4 and 16 h post-administration. Both inducers caused an approximately 2-fold increase in P450IA1 mRNA at 4 h and a 10-fold increase at 16 h. In contrast, both cytochrome P450IA1 and IA2 mRNAs was increased over the control between 4 and 24 h. The total amount of P450IA mRNAs in liver at 4 and 16 h was increased about 2- and 4-fold respectively by I3C; 5,6BF induced the P450IA mRNAs 4- and 5-fold respectively. The expression of cytochrome P450IA1 and IA2 is induced by I3C and several flavones present in cruciferous vegetables. This suggests that one of the protective effects of cruciferous vegetables in the reduction of chemically induced cancer may be regulation of cytochrome P450s involved in the metabolism of the chemical carcinogens.     

Quantitative analysis of estrogen receptor-alpha and -beta messenger RNA expression in normal and malignant thyroid tissues by real-time polymerase chain reaction.

OBJECTIVES: We have conducted a quantitative analysis of estrogen receptor-alpha (ER-alpha) and -beta (ER-beta) mRNA expression in normal thyroid and tumor tissues. METHODS: Normal thyroid tissues (n = 10) and tumor tissues [(follicular adenoma (n = 14), follicular carcinoma (n = 8), papillary carcinoma (n = 14), anaplastic carcinoma (n = 3) and medullary carcinoma (n = 6)] were obtained at surgery from 45 female patients. ER-alpha and ER-beta mRNA expression has been studied by a quantitative polymerase chain reaction. RESULTS: ER-alpha mRNA levels in the normal thyroid were not significantly different from those in follicular adenomas, papillary carcinomas and medullary carcinomas but were marginally (p = 0.08) higher than those in follicular and anaplastic carcinomas. ER-beta mRNA levels in the normal thyroid tissues were not significantly different from those in any other tumor tissues. ER-beta to ER-alpha mRNA ratios were significantly (p < 0.05) higher in the normal thyroid tissues than in follicular adenomas. Proportions of ER-beta mRNA variants (ER-beta 1, 2, 5, and 5') did not significantly differ among the normal and tumor tissues. CONCLUSIONS: A downregulation of ER-alpha mRNA in follicular and anaplastic carcinomas seems to suggest that estrogens are unlikely to play an important role in the carcinogenesis and progression of these carcinomas. On the other hand, a significant decrease in ER-beta to ER-alpha mRNA ratios in follicular adenomas suggests a possible involvement of estrogens in the pathogenesis of this disease since the same phenomenon has been reported on estrogen-dependent breast cancers.     

Effect of dietary fiber on cytochrome P450IA1 induction in rat colonic mucosa.

The effect of dietary fiber on the induction of cytochrome P450IA1 in rat colonic mucosa after a single intragastric injection of 3-methylcholanthrene (3MC, 20 mg/kg) was investigated by examining the drug-metabolizing enzyme activity, immunoblotting for cytochrome P450IA1 and immunohistochemistry. 7-Ethoxycoumarin-O-deethylase activities were approximately 20-fold higher in microsomes from both proximal and distal portions of the colonic mucosa of control diet-fed 3MC-treated rats compared with those of control diet-fed untreated rats. Strong immunofluorescence for cytochrome P450IA1 was localized in the cytoplasm of the colonic mucosa surface epithelium from the control diet-fed 3MC-treated rats. 7-Ethoxycoumarin-O-deethylase activity and cytochrome P450IA1 content determined by immunoblotting were significantly lower in wheat bran-fed 3MC-treated rats than in control diet-fed 3MC-treated rats. Immunohistochemical analysis showed much weaker immunofluorescence for cytochrome P450IA1 in the surface epithelium of the colonic mucosa of the wheat bran-fed 3MC-treated rats. These observations suggested that dietary fiber can affect the induction of cytochrome P450IA1 in colonic mucosa by dietary inducers or carcinogens.     

Quantitative analysis of estrogen receptor-alpha and -beta messenger RNA expression in breast carcinoma by real-time polymerase chain reaction

BACKGROUND: Estrogen action is mediated not only through a classic estrogen receptor (ER) (ER-alpha) but also through a second ER (ER-beta) that has a structure and function similar to ER-alpha. A correlation between ER-beta mRNA expression with ER and progesterone receptor (PR) protein levels as well as prognostic factors remains to be established in breast carcinoma. METHODS: The authors conducted a quantitative analysis of ER-alpha and ER-beta mRNA expression in 116 breast tumors using real-time polymerase chain reaction (PCR), and investigated a possible correlation between ER-alpha and ER-beta mRNA expression and ER and PR status as determined by enzyme immunoassay as well as with various prognostic factors. RESULTS: ER-alpha mRNA levels were significantly (P < 0.01) higher in ER positive compared with ER negative tumors. Conversely, ER-beta mRNA levels were significantly (P < 0.01) lower in ER positive compared with ER negative tumors. Accordingly, the ratio of ER-beta to ER-alpha was significantly (P < 0.01) higher in ER negative compared with ER positive tumors. A subset analysis based on ER and PR status showed that ER-beta mRNA levels as well as the ratios of ER-beta to ER-alpha mRNA level were highest in ER negative and PR negative tumors (P < 0.05). ER-alpha mRNA levels were significantly (P < 0.05) higher in postmenopausal compared with premenopausal tumors. Histologic Grade 3 tumors showed a significant decrease in ER-alpha mRNA levels compared with Grade 1 and 2 tumors (P < 0.01 and P < 0.05, respectively). No significant correlation between ER-alpha and ER-beta mRNA levels and histologic type, tumor size, or lymph node status was observed. CONCLUSIONS: An absolute and relative increase in ER-beta mRNA levels in ER negative and PR negative breast tumors, which rarely respond to endocrine therapy, suggests the possible involvement of up-regulation of ER-beta mRNA in the development of estrogen-independent tumors.     

Cytochrome P450IA expression in adult and fetal human liver.

A monoclonal antibody has been produced that recognizes the cytochrome P450 form, cytochrome P450IA1, but not cytochrome P450IA2 in rats and recognizes a single protein band of similar mol. wt on immunoblots of human liver microsomes. Immunohistochemical studies have been carried out with this antibody to investigate the localization and distribution of cytochrome(s) P450 of the P450IA family in human liver. Cytochrome P450IA was identified in both adult and fetal liver and in each case it was localized predominantly to hepatocytes. In adult liver there was a heterogeneous distribution of cytochrome P450IA immunoreactivity with cytochrome P450IA mainly present in zone 3 hepatocytes of the liver acinus. Within fetal liver there was a uniform distribution of cytochrome P450IA immunoreactivity with no apparent zonal distribution. Bile duct epithelium did not show definite immunostaining for cytochrome P450IA in either adult or fetal liver.     

Expression of mouse cytochrome P450IA1 cDNA in repair-deficient and repair-proficient CHO cells.

Recombinant DNA techniques have been used to develop Chinese hamster ovary cell lines for studying chemically induced genotoxicity. These cell lines express a specific cytochrome P450 isozyme responsible for the metabolism of polycyclic aromatic hydrocarbons and exhibit defined differences in DNA repair capacity. A bacterial gene (neo) conferring resistance to gentamicin was inserted into the pcD expression vector containing the mouse cytochrome P1450 (P450IA1) cDNA to facilitate the selection of transformed cells. This plasmid was introduced into the nucleotide-excision-repair-deficient UV5 cell line by electroporation. Transformed clonal isolates expressing the P1450 cDNA were identified by differential cytotoxicity assays using benzo[a]pyrene (B[a]P). One such clone, termed UV5P1, was mutagenized with ethyl methanesulfonate and selected for resistance to killing by UV radiation to derive a repair-competent clone that expresses similar P1450 activity to that of the parental cell line. Two repair-competent clones were selected and called 5P1R1 and 5P1R3. The resulting cell lines (UV5P1, 5P1R1, and 5P1R3) expressed arylhydrocarbon hydroxylase activity. UV5P1 and 5P1R3 were compared in terms of cytotoxicity and mutagenicity after exposure to B[a]P. Induced mutations were measured at the adenine phosphoribosyltransferase (aprt) and hypoxanthine guanine phosphoribosyltransferase (hprt) loci. Repair-deficient UV5P1 cells were killed by B[a]P at concentrations below 0.1 microM, while the repair-proficient 5P1R3 cells showed no toxicity up to 60 microM. Mutation induction at both loci was also much more efficient in UV5P1 cells. These new cell lines provide a more sensitive system that can be used in a battery of assays to evaluate the genotoxicity of chemicals requiring P450IA1 metabolic activation and to assess the role of DNA repair in modulating the biological effects of DNA damage.     

Effects of the food mutagens MeIQx and PhIP on the expression of cytochrome P450IA proteins in various tissues of male and female rats.

The promutagenic 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) found in cooked food are converted to their active forms mainly by cytochrome P450 forms IA1 and IA2. By induction of these isoenzymes the food mutagens could thus influence their own rate of activation. Male and female Wistar rats were given MeIQx, PhIP, beta-naphthoflavone (BNF) or saline i.p. at 50 mg/kg body wt on three consecutive days. On the fourth day the rats were killed and lungs, kidneys, liver and intestines taken. The microsomal fraction from each organ was prepared as well as 9000 g supernatant from the liver. The induction of cytochrome P450IA was measured at the protein level by enzymatic assays (ethoxyresorufin-O-deethylation, Ames' mutagenicity test) and immunoassays (Western blot) and at the pretranslational level by RNA hybridization (Northern blot). The binding affinities of MeIQx, PhIP and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-receptor were studied by evaluation of the competition with 3H-labelled TCDD for specific binding. Ethoxyresorufin-O-deethylase (EROD) activity was significantly increased in the liver (males 2.1-fold, females 3.3-fold), kidneys (males 2.1-fold, females 1.8-fold) and lungs (males 4.3-fold, females 3-fold) of the MeIQx-treated rats. Furthermore, the levels of cytochrome P450IA proteins were increased in these animals. It was not possible, however, to detect the corresponding mRNA. In the case of the PhIP-treated animals a significantly increased EROD activity (2.7-fold) and an increased cytochrome P450IA protein level were seen only in the male lungs. Only a very weak TCDD-receptor affinity was observed for PhIP, whereas MeIQx or IQ did not appear to compete significantly with [3H]TCDD for binding to the TCDD-receptor. It is concluded that MeIQx is a weak inducer of cytochrome P450IA in several organs of the rat, while PhIP induced these isoenzymes only in the male lungs. More work is needed to clarify the mechanism(s) whereby this induction occurs.     

Preferential inhibitions of hepatic P450IA2 expression and induction by lead nitrate in the rat

Male F344 rats were treated with lead nitrate and changes inthe expression and induction of P450IA subfamily enzymes anda placental form of glutathione-S-transferase (GST-P) in theliver were assessed by means of a bacterial mutation test, immunoblottingwith a monoclonal antibody reactive to P450IA1/IA2 and anti-GST-Psera and Northern blotting with P450IA2 cDNA as a probe. Treatmentof rats with lead nitrate (20, 50 or 100 µmol/kg bodywt) decreased P450IA2 mRNA and protein in the liver in the dose-dependentfashion and also decreased the microsomal activity for P450IA2-dependentmutagenization of aromatic amines. Pretreatment of rats withlead nitrate suppressed the inductions of both P450IA2 mRNAand protein by an inducer of P450IA subfamily enzymes in theliver. In addition, amount of the induced P450IA2 was decreasedalong with increase in that of the induced GST-P.     

Stable expression of human cytochrome P450IA2 cDNA in a human lymphoblastoid cell line: role of the enzyme in the metabolic activation of aflatoxin B1

A human lymphoblastoid cell line stably expressing a human cytochrome P450IA2 cDNA was developed. This recombinant cell line displayed P450IA2 protein and estradiol 2-hydroxylase activity, neither of which was detected in the parental cell line. The recombinant cell line was also approximately 1000-fold more sensitive to the cytotoxicity and mutagenicity of the carcinogenic mycotoxin aflatoxin B1 than was the parent cell line. The increase in mutagenicity was supported by a corresponding increase in the level of aflatoxin B1 binding to DNA in cells expressing P450IA2 relative to control cells.     

RNA expression of cytochrome P450 in Mexican women with breast cancer

Involvement of cytochrome P450 genes (CYPs) in breast cancer (BCa) may differ between populations, with expression patterns affected by tumorigenesis. This may have an important role in the metabolism of anticancer drugs and in the progression of cancer. The aim of this study was to determine the mRNA expression patterns of four cytochrome P450 genes (CYP2W1, 3A5, 4F11 and 8A1) in Mexican women with breast cancer. Real- time PCR analyses were conducted on 32 sets of human breast tumors and adjacent non-tumor tissues, as well as 20 normal breast tissues. Expression levels were tested for association with clinical and pathological data of patients. We found higher gene expression of CYP2W1, CYP3A5, CYP4F11 in BCa than in adjacent tissues and only low in normal mammary glands in our Mexican population while CYP8A1 was only expressed in BCa and adjacent tissues. We found that Ki67 protein expression was associated with clinicopathological features as well as with CYP2W1, CYP4F11 and CYP8A1 but not with CYP3A5. The results indicated that breast cancer tissues may be better able to metabolize carcinogens and other xenobiotics to active species than normal or adjacent non-tumor tissues.     

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17 beta-estradiol and 2-aminofluorene in V79 Chinese hamster cells.

In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.     

Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction.

The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp.