Target validation of cytochrome P450 CYP1B1 in prostate carcinoma with protein expression in associated hyperplastic and premalignant tissue

PURPOSE: To investigate the localization and distribution of cytochrome P450 CYP1B1 protein expression in patients diagnosed with prostate carcinoma compared to those with bladder carcinoma. To validate CYP1B1 as a molecular target for the development of selective cancer therapeutics for use in combination with radiation. METHODS AND MATERIALS: Prostatectomy specimens (n = 33) of moderate Gleason grade (3 + 3 and 3 + 4) were analyzed immunohistochemically for CYP1B1 protein expression using a specific monoclonal antibody for the enzyme. The intensity of CYP1B1 staining was assessed both semiquantitatively using visual scoring and quantitatively by spectral imaging microscopy using reference spectra and compared with bladder carcinoma (n = 22). RESULTS: CYP1B1 protein expression was present in 75% of prostate carcinomas (n = 27) compared to 100% of bladder carcinomas (n = 22). In both cases, CYP1B1 protein expression was heterogeneous and localized in the cytoplasm of the tumor cells but absent from the surrounding stromal tissue. CYP1B1 was also detected in premalignant prostatic intraepithelial neoplasia (n = 2, 100%), as well as noncancerous tissues, including benign prostatic hyperplasia (n = 27, 82%), metaplastic prostatic urothelium (n = 8, 100%), and hyperplastic prostatic urothelium (n = 14, 100%). Higher CYP1B1 protein expression in bladder vs. prostate carcinoma was confirmed by their corresponding average normalized absorbances (+/- standard deviation), measured as 1.40 +/- 0.44 and 0.55 +/- 0.09, respectively. Overall CYP1B1 staining intensity in prostate carcinoma was similar to that in prostatic intraepithelial neoplasia, benign prostatic hyperplasia, and hyper-/metaplastic urothelial tissue. No CYP1B1 was detected in normal prostate tissue. CONCLUSIONS: CYP1B1 is overexpressed in prostate carcinoma at a high frequency and is also detectable in the associated premalignant and hyperplastic tissue, implicating a possible link with malignant progression and CYP1B1 as a suitable target for therapy. Spectral imaging microscopy has highlighted differences in CYP1B1 protein expression between different cancers.     

Altered Expression of Hepatic CYP1A Enzymes in Rat Hepatocarcinogenesis

Hyperplastic nodules of the liver were induced by treating male F344 rats with a combination of diethylnitrosamine and partial hepatectomy. The livers were examined for the expression of cytochrome P450 (CYP) enzymes, mainly CYP1A1 and CYP1A2; the amount and activity of the enzymes in the nricrosomes were assessed by enzymatic and immunological methods. Levels of CYP1A mRNAs were assayed by Northern blotting. In the liver bearing hyperplastic nodules, the total amount of microsomal CYP enzymes decreased to about 50% of the control. The microsomal activities for the CYP1A2-mediated activation of carcinogenic heterocyclic amines decreased to about 20% of the corresponding controls, in association with decreases in the levels of mRNA and protein of CYP1A2. Furthermore, the inducibility of CYP1A2 by CYP1A inducers such as 3-methoxy-4-aminoazobenzene and 3-methylcholanthrene was also decreased at the mRNA, protein and activity levels. On the other hand, CYP1A1 enzyme, which was undetectable in control rat liver, appeared in the liver bearing hyperplastic nodules, but its inducibility by a CYP1A inducer decreased slightly. The present findings indicated that individual CYP1A enzymes are differently regulated, and the expression of CYP1A2 is reduced preferentially in the liver bearing hyperplastic nodules.     

Profiling cytochrome P450 expression in ovarian cancer: identification of prognostic markers.

PURPOSE: The cytochromes P450 are a multigene family of enzymes with a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs and biologically active endogenous compounds. The purpose of this study was to define the cytochrome P450 profile of ovarian cancer and identify novel therapeutic targets and establish the prognostic significance of expression of individual cytochrome P450s in this type of cancer. EXPERIMENTAL DESIGN: Immunohistochemistry for a panel of 23 cytochrome P450s and cytochrome P450 reductase was done on an ovarian cancer tissue microarray consisting of 99 primary epithelial ovarian cancers, 22 peritoneal metastasis, and 13 normal ovarian samples. The intensity of immunoreactivity in each sample was established by light microscopy. RESULTS: In primary ovarian cancer, several P450s (CYP1B1, CYP2A/2B, CYP2F1, CYP2R1, CYP2U1, CYP3A5, CYP3A7, CYP3A43, CYP4Z1, CYP26A1, and CYP51) were present at a significantly higher level of intensity compared with normal ovary. P450 expression was also detected in ovarian cancer metastasis and CYP2S1 and P450 reductase both showed significantly increased expression in metastasis compared with primary ovarian cancer. The presence of low/negative CYP2A/2B (log rank = 7.06, P = 0.008) or positive CYP4Z1 (log rank = 6.19, P = 0.01) immunoreactivity in primary ovarian cancer were each associated with poor prognosis. Both CYP2A/2B and CYP4Z1 were also independent markers of prognosis. CONCLUSIONS: The expression profile of individual P450s has been established in ovarian cancer. Several P450s show increased expression in ovarian cancer and this provides the basis for developing P450-based therapeutics in ovarian cancer. Expression of CYP2A/2B or CYP4Z1 in primary ovarian cancer were independent markers of prognosis.     

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B₁ and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci. Mol. Carcinog. 22:229–234, 1998. © 1998 Wiley-Liss, Inc.     

Cytochrome P450 isoenzyme mRNA expression pattern in human urinary bladder malignancies and normal urothelium

This study was designed to analyze the cytochrome P450 isoenzyme mRNA expression pattern of transitional cell carcinomas of the bladder (N = 19) and normal urothelium (N = 10). In addition, biopsies from normal urothelium (N = 32) taken at the time of transurethral resection of bladder cancer in eight patients from surrounding histologically normal urothelium also were characterized concerning their specific cytochrome P450 mRNA expression pattern. A total of 13 of 19 of the analyzed tumor specimens (68%) revealed expression of cytochrome P450 1B1. Cytochrome P450 4B1 and 1A1 mRNA expression were detected in 79% (15 of 19) and 53% (10 of 19) of the tumor specimens, with no correlation between tumor stage and grade of the neoplasm. Biopsies from macroscopically and histologically normal urothelium from tumor-invaded bladders also showed expression of cytochrome P450 1B1 in 75% (24 of 32), 4B1 in 62.5% (20 of 32), and 1A1 in 50% (16 of 32). Furthermore, a 75% homology concerning cytochrome P450 1B1 and 4B1 mRNA expression was observed between the bladder tumor and the biopsies from this bladder. The polymerase chain reaction analysis of normal urothelium from normal bladders that do not harbor a neoplasm revealed CYP450 mRNA expression for CYP450 1A1 in 6 of 10; 1B1 in 5 of 10; 4B1 in 6 of 10; 2D6 in 2 of 10; and 2E1 in 2 of 10. According to our data, CYP450 1B1 mRNA expression is not tumor-specific. The present findings are the first to compare CYP450 expression in bladder cancer with biopsies from the same tumor-bearing bladder, and they indicate that, from the enzymatic point of view, bladder cancer also is a panurothelial field disease present in even normal urothelium.     

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein

Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism.     

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.     

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1- 12-3 recognized only one protein of identical mobility on the two- dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.      

Cytochrome P450 CYP1B1 activity in renal cell carcinoma

Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.     

Effect of modifying agents on the phenotypic expression of cytochrome P-450, glutathione S-transferase molecular forms, microsomal epoxide hydrolase, glucose-6-phosphate dehydrogenase and {gamma}-glutamyltranspeptidase in rat liver preneoplastic lesions

The expression of A and P forms of glutathione S-transferase(GST-A and P), two cytochrome P-450 isoenzymes (P-450 PB_3a andP-450 MC₂), microsomal epoxide hydrolase (mEHb), glucose-6-phosphatedehydrogenase (G6PD) and -glutamyltranspeptidase (-GT) was comparedin preneo-plastic liver lesions and background parenchyma ofF344 rats post-treated with butylated hydroxyanisole (BHA),ethoxyquin (EQ) or acetaminophen (AAP). These latter three compoundshave been shown to inhibit hepatocarcinogenesis after initialtreatment with N-ethyl-N-hydroxyethylnitrosamine (EHEN) anda significant decrease in the number of enzymealtered foci andnodules positive for GST-P, GST-A, G6PD and -GT and negativefor P-450 PB_3a, P-450 MC₂ was associated with their administration.Whereas in the foci case the decrease was most prominent fornon-discrete (heterogeneous) type lesions, the results of quantitationof nodules revealed a most significant alteration in the discretehomogeneously staining population. This indicates that BHA,EQ and AAP have the potential to inhibit the growth of the phenotypicallystable lesions thought most likely to be the immediate precursorsof hepatocellular carcinomas. The two anti-oxidants were associatedwith periportal increase of all enzymes investigated, whereasAAP induced GST species and mEHb in the perivenular zone. Irrespectiveof slightly elevated enzyme levels in surrounding parenchyma,mEHb antibody binding levels within lesions showed a reciprocalshift from positive to negative in rats treated with BHA, EQand AAP.     

Alpha-benzene hexachloride exerts hormesis in preneoplastic lesion formation of rat hepatocarcinogenesis with the possible role for hepatic detoxifying enzymes

Recently there has been a shift in the prevailing paradigm regarding the dose dependence of carcinogen action with increasing acceptance of hormesis phenomenon, although underlying mechanisms remain to be established. To ascertain whether alpha-benzene hexachloride (alpha-BHC) might act by hormesis, rats were initiated with diethylnitrosamine and then alpha-BHC ranging from 0.01 to 500 ppm was administered in the diet for 10 weeks. The highest concentration of alpha-BHC significantly increased the number and area of glutathione S-transferase placental form (GST-P) positive foci, preneoplastic lesions in the liver, but its low dose, 0.05 ppm, caused significant reduction, showing a J-shape dose-response curve. The proliferating cell nuclear antigen positive index for GST-P positive foci in the low dose-treated group was significantly reduced. The dose response curves of CYP450 content, NADPH-P450 reductase activity and 8-hydroxydeoxyguanosine formation revealed the same pattern as GST-P positive foci data. The response curves of CYP2B1 and 3A2 in their activities, protein and mRNA expression showed a threshold but CYP2C11 activity exhibited an inverted J-shape. These results might suggest the possibility of hormesis of alpha-BHC at early stages of rat hepatocarcinogenesis. The possible mechanism involves induction of detoxifying enzymes at low dose, influencing free radical production and oxidative stress, and consequently pathological change in the liver.     

Altered regulation of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines

The cytochrome P450 (CYP) systems catalyze the metabolic transformation of a wide variety of xenobiotics including procarcinogens present in cigarette smoke condensate as well as atmospheric pollutants. The CYP1A1 isoenzyme is of particular interest because it has been implicated as a risk factor in the etiology of lung cancer in heavy cigarette smokers. The identification and expression of the structural CYP1A1 gene in either normal human lung or lung cancer cells has not been reported. Because of its potential significance in human lung cancer, we investigated the expression of the CYP1A1 structural gene in 24 established human lung cancer cell lines including 15 non-small cell (eight adenocarcinomas, three large cell undifferentiated carcinomas, two bronchioloalveolar cell carcinomas, and two squamous cell carcinomas) and nine small cell lung carcinomas. CYP1A1 mRNA was detected in 14 of 15 (93%) of the non-small cell lung carcinoma cell lines examined following 24-hour treatment with benz[a]anthracene (BA) and in nine of 15 (60%) of the non-small cell lines cultured without an inducer in the medium. When the small cell lung cancer lines were evaluated for CYP1A1 gene expression, two of nine (22%) expressed detectable CYP1A1 mRNA in both BA-induced cell cultures and constitutive (control) cultures. A positive correlation was noted between BA-induced CYP1A1 mRNA levels and the corresponding aryl hydrocarbon hydroxylase activity expressed as absolute BA-induced enzyme activity (r = 0.74; P less than .01; n = 24), which further demonstrated that CYP1A1 mRNA expression reflects CYP1A1 enzyme activity in the individual cell lines. These observations represent the first known demonstration of constitutive (non-induced) CYP1A1 gene expression in human cells and suggest altered regulation of the CYP1A1 gene in selected lung cancer cell lines. These human pulmonary carcinoma cell lines, which have documented regulatory defects, could be useful for further identification of the mechanisms associated with CYP1A1 gene regulation.     

CYP1A2 is not the primary enzyme responsible for 4-aminobiphenyl-induced hepatocarcinogenesis in mice.

4-Aminobiphenyl (4-ABP), a potent carcinogen in rodents (liver cancer) and human (bladder cancer), is found as an environmental contaminant and in tobacco smoke. Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.     

Immunohistochemical analysis of CYP2A13 in various types of human lung cancers

Human CYP2A13, which is expressed in the respiratory tract, is the most efficient enzyme for the metabolic activation of tobacco‐specific nitrosamines such as 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK). The relevance of CYP2A13 in carcinogenicity and toxicity in the respiratory tract has been suggested, but the expression of CYP2A13 protein in lung cancer tissues remains to be determined. We first prepared a mouse monoclonal antibody against human CYP2A13. The antibody showed no cross reactivity with the other CYP isoforms including CYP2A6. Using the specific antibody, we performed immunohistochemical analysis for human lung carcinomas. In adenocarcinomas (n = 15), all specimens were positive for the staining and five samples showed strong staining. In squamous cell carcinomas (n = 15) and large cell carcinomas (n = 15), each 14 samples were positive for the staining and two and three samples showed strong staining, respectively. In small cell carcinoma samples (n = 15), eight samples were negative for the staining and five samples showed weak or moderate staining. In conclusion, we first found that the expression of CYP2A13 was markedly increased in non‐small cell lung carcinomas. The high expression might be associated with the tumor development and progression in non‐small cell lung carcinomas. (Cancer Sci 2010; 101: 1024–1028)     

Development of a human lymphoblastoid cell line constitutively expressing human CYP1A1 cDNA: substrate specificity with model substrates and promutagens

AHH-1 TK+/–cell derivatives were developed that stablyexpress human CYP1A1 cDNA, and an AHH-1 TK+/–derivativeexpressing higher levels of CYP1A2 cDNA in extrachromosomalvectors which confer resistance to I-histidinol. The CYP1A1-expressingcell lines, designated h1A1 and h1A1v2, differ by containingone and two CYP1A1 cDNA expression units per vector. The CYP1A2-expressingcell line, designated h1A2v2, also has two CYP1A2 cDNA expressionunits per vector. Microsomes prepared from CYP1A1 cDNA expressingcells exhibit high, constitutive levels of 7-ethoxyresorufindeethylase (EROD), 7-ethoxycoumarin deethylase (ECD), 7-ethoxy-4-trifluoromethylcoumarindeethylase (EFCD), benzo[a]-pyrene hydroxylase (BPH) activitiesand spectrally quantifiable cytochrome P450. Kinetic comparisonsbetween cDNA-expressed CYP1A1 and CYP1A2 indicate that CYP1A1is more active than CYP1A2 for EROD, ECD, EFCD and BPH. CYP1A2was more active than CYP1A1 for acetanilide hydroxylation andactivation of aflatoxin B₁ (AFB₁). The mutagenicity of selectedpromutagens were examined in h1A1 cells and control cells. Relativeto control cells, the h1A1 cell line exhibits increased sensitivityto the mutagenicity of benzo[a]pyrene, cyclopenta[c,d]pyrene,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and AFB₁.     

High promutagen activating capacity of yeast microsomes containing human cytochrome P-450 1A and human NADPH-cytochrome P-450 reductase

Yeast Saccharomyces cerevisiae strains have been constructedthat co-express cDNAs coding for the human cytochrome P-450enzymes CYP1A1 or CYP1A2 in combination with human NADPH-cytochromeP-450 reductase (oxidoreductase). Microsomal fractions preparedfrom the strains were able to efficiently activate various drugsto Salmonella mutagens. These experiments demonstrated thata functional interaction occurred between the respective humanenzymes in the yeast microsomes. For every drug tested, themicrosomes containing CYP enzymes and oxidoreductase were 2-to 4-fold better in activation than the corresponding microsomesthat contained CYP alone. Interestingly, co-expression of CYP1A2with oxidoreductase resulted in a decrease of 7-ethoxyresorufin-O-deethylaseactivity, a problem which is related to this specific substrate.Using the microsomes, it was demonstrated that aflatoxin B₁,was activated to a mutagen not only by CYP1A2 but also by CYP1A1.In contrast, benzo[a]pyrene was exclusively activated by CYP1A1whereas CYP1A2 was inactive. The drug 3-amino-1-methyl-5H-pyrido[4,3-b]indole(Trp-P-2) was activated by CYP1A2 and to a lesser extent byCYP1A1. A strong substrate specificity was observed with thetwo structurally related heterocyclic arylamines 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQ_x).MeIQ_x was activated efficiently by both CYP enzymes, whereasMeIQ was only activated by CYP1A2 and not by CYP1A1. The factthat microsomes from vector transformed control strains wereunable to activate any of the drugs studied underlines the suitabilityof these microsomes for metabolic studies. Moreover, the presenceof suitable marker genes in the yeast strains will enable usto study mitotic recombination and gene conversion events inducedby drugs that require metabolic activation.     

Lack of carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in cynomolgus monkeys.

The carcinogenic potential of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was evaluated in cynomolgus monkeys. The animals received MeIQx, beginning at the age of one year, at doses of 10 or 20 mg/kg body weight by gavage five times a week for 84 months and were autopsied 8 months thereafter. Although sporadic development of aberrant crypt foci in the colon and glutathione S-transferase pi-positive foci in the liver as well as hyperplastic changes of the lymphatic tissue in the lung and gastro-intestinal tract were observed in several monkeys, this was not treatment-related. No neoplastic or preneoplastic lesions were found in other organs. Serum chemistry data and organ weights were also within the normal ranges. From these data, it is concluded that MeIQx is not carcinogenic in the cynomolgus monkey under the conditions examined. This lack of carcinogenicity is probably related to the poor activation of MeIQx due to the lack of constitutive expression of CYP1A2 as well as an inability of other cytochrome P450s to catalyze N-hydroxylation of MeIQx in the cynomolgus monkey.