Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens

In this study we describe the development of strain BMX100,a new Escherichia coli K12 tester strain, derived from MX100,a strain which was constructed for detection of mutagens andfor mechanistic studies of chemical carcinogens. We demonstratehere that strain BMX100 can be used for stable expression ofhuman CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochromeP450 reductase. Mutagenicity of precarcinogens known to be bioactivatedby CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin Bl (AFB1)and 2-amino-3-methylimidazo[4,5-f]quinoline (1Q), could be detected.The mutagenic activity of 2-AA using BMX100 expressing CYP1A2alone and in combination with rat CYP reductase was respectively10 and 20 times higher than in BMX100 with the standard metabolicactivation system, rat liver S9 fraction. Furthermore, the mutagenicityof 2-AA could be nullified by a-naphthoflavone, a known inhibitorof CYP1A2. IQ responded equally in BMX100 expressing the CYP1A2-reductasefusion protein as compared with usage of rat liver S9 fraction.Rat liver S9 fraction was much more potent in generating a mutagenicresponse to A FBI in BMX100 than in the strain expressing humanCYP1A2 aloneZ or CYP1A2 fused to rat reductase. The resultsdescribed in this study demonstrate that this new E.coli straincan function as a human CYPlA2-competent prokaryotic mutagenicitytest system and they seem to characterize BMX100 as a strainof interest for studies to identify individual human CYPs involvedin bioactivation and bloinactivation reactions of putative genotoxins. ⁴To whom correspondence should be addressed. Tel: +351 1 3610290; Fax: +351 1 3622018; Email: jose.rueff{at}gene.unl.mailpac.pt     

On the mechanisms of genotoxicity and metabolism of quercetin

Quercetin has been the subject of numerous studies on its genetictoxicity and carcinogenicity. Despite its well-proven geneticdamaging activity for various genetic end-points (reverse mutations,induction of SOS functions, induction of sister chromatid exchanges,chromosomal aberrations and micronuclei), the mechanisms ofgenetic damage by quercetin remain, by and large, unknown. Thepresent study aims to further extend the observations on thepossible active oxygen species mediated DNA-damaging activityof quercetin and the role of cytochrome P450-dependent metabolismon the genotoxicity of quercetin. The results reported in thiswork show that querceitn can produce the OH radical, as assessedby deoxyribose degradation in the presence of Fe^3³/EDTA(ethylenediaminetetraacetic acid), and that it induces strandbreakage in isolated plasmidic DNA (pUC18). The data supportthe hypothesis that the production of OH is mediated by H₂O₂.The results with genetically engineered V79 cells expressingrat cytochromes 1A1, 1A2 and 2B1 failed to demonstrate metabolismof quercetin, as indicated by the fact that neither an enhancementnor a decrease in the genotoxicity of quercetin was observed.Results obtained on the pH dependence of the induction of chromosomalaberrations by quercetin in V79 cells show that, as the valueof the medium is increased to 8.0, there is a significant increasein the number of aberrant cells, as expected if oxygen radicalsare responsible for the formation of chromosomal aberrations. ³To whom correspondence should be addressed     

Assay-specific genotoxicity of N-nitrosodibenzylamine to the rat liver in vivo

N-Nitrosodibenzylamine (NDBzA) is mutagenic to Salmonella typhimurium and induces DNA strand breaks in isolated rat hepatocytes, yet it is reported to be non-carcinogenic to the rat. Here we report that it is inactive in both the rat and mouse bone marrow micronucleus assays and in a rat liver autoradiographic assay for unscheduled DNA synthesis. It is, however, clearly active as a micronucleus-inducing agent and mitogen in the rat liver and is capable of inducing single-strand breaks in the DNA of rat liver. The origin and implications of this curious conflict of in vivo genotoxicity data are discussed. Irrespective of that discussion, it is concluded that NDBzA is genotoxic to the rat liver in vivo.     

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation

Cytochrome b₅ (b₅) is increasingly recognized to be of importancefor specific cytochrome P450 (CYP) activities. We developedhuman b₅/CYP-competent mutagenicity tester bacteria to studythe role of b₅ in the bioactivation activity of human CYP. Thesenew tester bacteria were derived from the previously engineeredhuman CYP-competent Escherichia coli K12 tester strain MTC,containing a bi-plasmid system for the co-expression of a specificCYP form (CYP1A2, 2A6 or 2E1) with human b₅, and human NADPHcytochrome P450 reductase (RED), resulting in the strain BTC-b₅-1A2,BTC-b₅-2A6 and BTC-b₅-2E1, respectively. The relative contentof b₅ with CYP and RED in these three BTC-b₅-CYP strains demonstratedphysiologically relevant co-expression levels and typical CYP-specificactivities could be determined with their specific chemicalprobes. These strains were applied in mutagenicity assays alongwith their corresponding b₅-void strains to determine the effectof b₅ on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivationof several promutagens. For CYP1A2, of the 5 compounds tested[2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinolineand 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], onlythe mutagenicity of 2AA was slightly increased (     

Role of cytochrome P4501A in biotransformation of the potential anticancer drug oracin.

Metabolic fate of the potential anticancer drug oracin (I), was studied at microsomal level in rat using enzyme induction and inhibition. One of the main metabolites arising during incubation of hepatic microsomal fraction with oracin is 3-hydroxyoracin (III). Cytochromes P450 non-specific inhibitors (carbon monoxide, aminobenzotriazole, 1-benzylimidazole, proadifen hydrochloride, n-octylamine) diminished amount of III. Among several specific inducers of rat cytochromes P450 isoforms used, only 3-methylcholanthrene, inducer of cytochrome P4501A, caused a significant stimulation of 3-hydroxyoracin production. The amount of III was decreased to the level of controls when the microsomes prepared from 3-methylcholanthrene treated rats were incubated with substrate in the presence of specific P4501A inhibitor alpha-naphthoflavone. From the above mentioned results we can assume that metabolite III is formed from oracin by cytochrome P450 belonging to subfamily 1A.     

Involvement of oxygen free radicals in the serum-mediated increase of benzoquinone genotoxicity

The genotoxicity of benzoquinone (BQ), a toxic benzene metabolite, is greatly enhanced by the presence of fetal calf serum (FCS) in the incubation medium. The FCS effect is abolished by heat denaturation of serum proteins and is slightly decreased by dialysis. In the present study, we have further investigated the serum effect on BQ genotoxicity by measuring DNA damage produced in peripheral blood mononuclear cells (PBMCs) using the Comet assay. We have also evaluated the effect of human serum and rat liver post-mitochondrial fraction (S9) on the DNA damage produced by BQ. Both human serum and a rat liver S9 enhanced the genotoxicity of BQ in a manner similar to FCS. Gel filtration experiments showed that all the enhancing activity of the serum eluted with the high molecular weight fractions, suggesting that low molecular weight serum constituents do not play an important role in modulating genotoxicity. The genotoxicity-enhancing activity of serum was inhibited by the iron chelator deferoxamine and by superoxide dismutase and catalase. Incubating PBMCs with BQ in the presence of FCS also resulted in the accumulation of intracellular peroxides as demonstrated by loading the cells with 2',7'-dichlorofluorescin diacetate and analyzing for peroxide formation by flow cytometry. These results indicate that oxygen free radicals are involved in the enhancement of BQ-induced DNA damage by serum. We hypothesize that enzyme activities that reduce BQ by transferring single electrons could be the source of the oxygen free radicals.     

Involvement of different pathways in the genotoxicity of nitropropanes in cultured mammalian cells

The metabolic pathways leading to genotoxicity of nitropropanes in mammalian cells were investigated by measuring the effects of 2-nitropropane (2-NP) and 1-nitropropane (1-NP) on various cell lines characterized for their expression of cytochrome P450-dependent mono-oxygenases. Cells used were the rat hepatoma cell lines 2sFou, H4IIEC3/G- and C2Rev7, which express various forms of cytochrome P450-dependent mono-oxygenases, and V79 Chinese hamster cells which lack these enzyme activities. Induction of DNA repair synthesis, micronuclei and, where assessable, mutations to 6-thioguanine (TG) resistance served as indicators of genotoxic effects. 2-NP elicited a positive response at all endpoints measured in the hepatoma lines after pretreatment of the cells with dexamethasone, an inducer of various liver-specific cytochrome P450 forms. Genotoxicity was much weaker or not detectable in cells not pretreated with the inducer. 1-NP was not genotoxic in the hepatoma cells irrespective of whether the cells were pretreated or not. Neither isomer elicited DNA repair synthesis in V79 cells, but both isomers caused mutations to TG resistance, and 1-NP increased the number of micronucleated and multinucleated cells. The findings show that there are different pathways in mammalian cells by which nitropropanes can be converted to genotoxic products. Presumably the induction of liver tumours by 2-NP is linked to the metabolic pathway which is characterized by the formation of genotoxic metabolites from 2-NP but not 1-NP. This pathway appears to depend on the presence of liver-specific, dexamethasone-inducible, cytochrome P450 forms. The relevance of the genotoxic effects of the nitropropanes observed in V79 cells for the situation in vivo is open to question.     

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.     

Respirable coal dust particles modify cytochrome P4501A1 (CYP1A1) expression in rat alveolar cells

Cytochrome P4501A1 (CYP1A1) metabolizes polycyclic aromatic hydrocarbons in cigarette smoke to DNA-binding reactive intermediates associated with carcinogenesis. Epidemiologic studies indicate that the majority of coal miners are smokers but have a lower risk of lung cancer than other smokers. We hypothesized that coal dust (CD) exposure modifies pulmonary carcinogenesis by altering CYP1A1 induction. Therefore, male Sprague Dawley rats were intratracheally instilled with 2.5, 10, 20, or 40 mg CD/rat or vehicle (saline); and 11 d later, pulmonary CYP1A1 was induced by intraperitoneal injection of beta-naphthoflavone (BNF; 50 mg/kg). Fourteen days after CD exposure, CYP1A1 protein and activity were measured by Western blot and 7-ethoxyresorufin-O-deethylase activity, respectively. CYP1A1 and the alveolar type II markers, cytokeratins 8/18, were localized and quantified in lung sections by dual immunofluorescence with morphometry. The area of CYP1A1 expression in alveolar septa and alveolar type II cells in response to BNF was reduced by exposure to 20 or 40 mg CD compared with BNF alone. CD exposure significantly inhibited BNF-induced 7-ethoxyresorufin-O-deethylase activity in a dose-responsive manner. By Western blot, induction of CYP1A1 protein by BNF was significantly reduced by 40 mg CD compared with BNF alone. These findings indicate that CD decreases BNF-induced CYP1A1 protein expression and activity in the lung.     

Immunological reactivity towards chondrocytes in rat and man: relevance to autoimmune arthritis

Immunological investigation of articular chondrocytes obtained from rat and man have shown the presence of unique differentiation antigens on the cell surface demonstrated by poly- or monoclonal antibodies in both species, and by the analysis of T cell reactivity in the rat. Both species of chondrocytes express Class I antigens in common with all nucleated mammalian cells and, in man, individual specificities of the MHC A, B, and C locus can be identified using standard histocompatibility testing. Class II antigens are strongly expressed in the rat but are expressed poorly in the human specimens we have analyzed. This finding is at variance with the reports of others and may depend upon the antisera we have used or the diseased state of our patient donors. In the rat, T cell reactivity to chondrocyte antigens is strong and can be demonstrated in both proliferation and cytotoxic assay. Moreover, syngeneic reactivity is present in naive animals, suggesting that rats are not tolerant of their own CSDA. The cross-reactivity found on analysis of cytotoxic killing suggests a sharing of differentiation antigens amongst the different strains of rats and possibly a limited polymorphism for this system. In man, conventional assays for T cell reactivity are hampered by the presence of a soluble inhibitor of lymphocyte proliferation released by chondrocytes and as yet unidentified. The poor representation of Class II antigens on our chondrocyte specimens may further contribute to the difficulty in producing proliferation. On the other hand, early success is reported in finding a significant number of T cell clones isolated from the inflammatory membrane of patients with rheumatoid arthritis, which respond to chondrocyte antigens. The possibility that these antigens may play a role in the pathogenesis of inflammatory arthritis may be more readily explored by exploring these approaches.     

Solid-phase extraction and gas chromatography-mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography-mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was +/-9.40% for kaempferol and +/-7.34% for quercetin (n = 6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with beta-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.     

Involvement of D1 dopamine receptors in the control of TSH secretion in the male rat

The effects of SCH 23390 (D1 dopamine receptor antagonist), SK&F 38393 (D1 dopamine receptor agonist), raclopride and remoxipride (D2 dopamine receptor antagonists) and ketanserin (5-hydroxytryptamine 2 receptor antagonist) on TSH serum levels (radioimmunoassay) and on brain catecholamine levels (Falck-Hillarp methodology in combination with quantitative histofluorimetry) were studied. SCH 23390 produced a dose-dependent increase in serum TSH levels in the lower dose range (0.01-0.03 mg kg-1, i.p.) administered 30 min before decapitation and in the higher dose range (1.0-3.0 mg kg-1) when given 2 h before decapitation. Following 30 min of treatment with the high doses of SCH 23390, reductions in serum TSH levels were found. The changes observed following SCH 23390 treatment occurred without affecting catecholamine levels in the median eminence and the peri- and paraventricular hypothalamic regions. Raclopride (0.1-10 mg kg-1, i.p.), remoxipride (1.0 mg kg-1, i.p.) or ketanserin (0.3 mg kg-1, i.p.) changed neither serum TSH levels nor brain catecholamine levels, SK&F 38393 (1.0-10 mg kg-1, i.p.) produced an increase in serum TSH levels. The results suggest the existence of inhibitory and facilitatory mechanisms regulating TSH secretion mediated via D1 dopamine receptors.     

Ultrastructural effects of Nifurtimox on rat adrenal cortex related to reductive biotransformation

Nifurtimox (Nfx) (4(5-nitrofurfurylidene)amino)-3-methylthiomorpholine-1, 1-dioxide) is a drug used against Chagas' disease, a parasitic sickness afflicting several million Latin Americans. Nfx administration to Sprague-Dawley male rats (220-250 g) at a dose of 100 mg/kg caused pronounced alterations in the adrenal cortex involving the fasciculata and reticularis zones but which were not evident in the glomerulosa. Alterations observed involved mitochondria, nuclei, Golgi apparatus, and the endoplasmic reticulum but were more intense in the mitochondria. There is Nfx nitroreductase activity in the adrenal microsomal, mitochondrial, and cytosolic-rich fractions but most of it is in the mitochondrial-rich fraction. Activity in the first two fractions requires NADPH and that in the cytosol is only observed in the presence of hypoxanthine as substrate. Enzymatic activity in all fractions is inhibited by oxygen. CO does not inhibit mitochondrial Nfx nitroreductase and inhibits only 10% of the microsomal enzyme activity. Hypoxanthine-dependent cytosolic activity is inhibited by allopurinol. Present results suggest that Nfx is activated to damage-producing reactive metabolites by nitroreductive biotransformation in rat adrenal organelles. Mitochondrial and microsomal bioactivation would occur at the level of the flavoenzyme P-450 reductase rather than at P-450 itself, and cytosolic bioactivation would be mediated by xanthine oxidase. Epidemiological studies on adrenal function in patients undergoing Nfx treatment would be necessary to establish the potential toxicological relevance of these findings.     

Involvement of local ischemia in endothelin-1 induced lesions of the neostriatum of the anaesthetized rat

Summary The present study examines the possibility that lesions induced by intrastriatal injections of endothelin-1 (ET-1, 0.43 nmol/0.5 µl) are ischemic in nature due to a vasoconstriction of the cerebral microvessels. In time course and dose-response experiments with ET-1 and in comparisons with ET-3, the volume of the lesions has been determined based mainly on the disappearance of striatal nerve cells, using a computer assisted morphometrical analysis. The blood flow in the neostriatum close to the site of injection of ET-1 was determined acutely by Laser-Doppler flowmetry. The acute metabolic effects of ET-1 were also studied on striatal superfusate levels of lactate, pyruvate, dopamine and its metabolites DOPAC (3,4-dihydroxyphenylacetic acid) and homovanilic acid (HVA) using an instrastriatal microdialysis probe. Dose related striatal lesions were observed with ET-1 (0.043–0.43 nmol) with a peak lesion volume after 24–48 h and the possible existence of a penumbra area. ET-3 showed a reduced potency to produce striatal lesions compared to ET-1. The lesions induced by ET-1 were prevented by coinjection with dihydralazine, a vasodilator drug. Acutely ET-1 (0.43 nmol/0.5 µl) produced a prolonged reduction of the cerebral blood flow down to 40% of control values and temporary increases of striatal lactate and DA efflux, the latter change being very marked. Also a significant reduction of DOPAC and HVA was observed. These neurochemical changes were all prevented by treatment with dihydralazine. These results indicate that ET-1 injected in the neostriatum may produce lesions by causing local ischemia, related to its vasoconstrictor activity and possibly also to an activation of ET-1 receptors in the astroglial-endothelial complex. Based on the present results it seems possible that ET-1 may participate in the multifactorial pathogenesis of cerebral ischemia.     

Involvement of cytochrome b-245 in the respiratory burst of human neutrophils.

The cytochrome b 245 of human neutrophils was reduced when the neutrophils under anaerobic conditions were stimulated by serum-treated zymosan, immune complexes, or phorbol myristate acetate. Reintroduction of oxygen rapidly reoxidized the cytochrome; azide was without effect on any of these reactions. This indicates that cytochrome b 245 participates in the respiratory burst of neutrophils.     

TGFB1 gene polymorphisms: their relevance in the susceptibility to Helicobacter pylori-related diseases

Recent studies have revealed elevated expression of transforming growth factor beta1 (TGF-beta1) in gastric mucosa of patients with gastric cancer (GC) and those undergoing ulcer repair. As production of TGF-beta1 is genetically regulated, we aimed to assess whether functional polymorphisms of the TGFB1 gene are involved in susceptibility to and clinical characteristics of Helicobacter pylori-related diseases. DNA from 142 unrelated Spanish patients with GC, 200 with peptic ulcer and 342 healthy controls was typed for the MspA1I T+869C, and the Sau96I G+915C polymorphisms of the TGFB1 gene using polymerase chain reaction and RFLP analysis. H. pylori infection and CagA/VacA antibody status were determined by Western blot in patients and controls. H. pylori infection (odds ratio (OR): 11.44; 95% confidence interval (CI): 4.45-29.42; P<0.001) and non-steroidal anti-inflammatory drugs (OR: 5.07; 95% CI: 2.53-10.16; P<0.001) were identified as independent risks factors for duodenal ulcer (DU), whereas the TGFB1+869(*)C/C genotype was associated with reduced risk of developing the disease (OR: 0.32; 95% CI=0.15-0.68; P=0.003). Our results show that the TGFB1 T+869C gene polymorphism is involved in the susceptibility to DU and provide further evidence that host genetic factors play a key role in the pathogenesis of H. pylori-related diseases.     

A new porous titanium-nickel alloy: Part 1. Cytotoxicity and genotoxicity evaluation

Porous titanium-nickel (PTN) alloys represent new biomaterials for long-term implantation. Their porosity properties might confer them the capacity to trigger fluid capillarity, tissue ingrowth, as well as good tissue-implant apposition and fixation. Before PTN materials are used as long-term implants, their biocompatibility level must be assessed. In this study, porous titanium-nickel was therefore extracted in a saline semi-physiological solution and materials were evaluated for potential cytotoxicity and genotoxicity reactions. The cytocompatibility elution test was performed in order to determine PTN toxic potential at the in vitro cellular level: no reactivity was detected in cell layers exposed to PTN extracts or the negative controls. In parallel, the genocompatibility of porous titanium-nickel was evaluated using three different assays in order to assess potential damage at the DNA level: the test for chemical induction of chromosome aberrations, the Salmonella typhimurium and Escherichia coli reverse mutation assay, and the mouse micronucleus test. No significant increase in the number of chromosomal aberrations, bacterian revertant colonies, or micronuclei was observed in presence of PTN extracts when compared to negative control exposition. Based on the above results, porous titanium-nickel can be considered completely cytocompatible and genocompatible, and therefore represents a good candidate for long-term implantation.     

Study of the biotransformation of benfluron using the isolated perfused rat liver

The isolated perfused rat liver method (IPRL) was used to find, isolate and identify further metabolites of Phase I and Phase II biotransformation of the potential cytostatic agent benfluron with special regard to the conjugation processes. Its pharmacokinetic profile during the perfusion was also estimated. The rat liver was isolated from the body and perfused in vitro using a recirculating perfusion system. Benfluron was added to the reservoir as a bolus in doses of 200, 100, 30 mg/kg of body weigh and 1 mg/perfusate volume and also as a continual infusion in a dose of 0.1 mg/min in separate series of experiments. The following metabolites formed during Phase I biotransformation were found in the perfusion liquid as well as in the bile: benfluron N-oxide, 9-hydroxy benfluron, demethylated 9-hydroxy benfluron, demethylated benfluron, and reduced benfluron. The major Phase II metabolite found in the bile samples was the glucuronide of 9-hydroxy benfluron. The pharmacokinetic profile of benfluron in IPRL indicated its main disposition and metabolic pathway, i.e. its rapid extraction from perfusate by the liver (t1/2 alpha = 3.76 min), 9-hydroxylation followed up O-glucuronidation and excretion to the bile. It was revealed that 12% of the total dose of the parent compound was excreted to the bile in the form of conjugates during the first hour of perfusion, 32% during 1.5 hour, and 70% during 2 hours after the administration of benfluron. The conjugates with glucuronic acid represented 96-98% of all metabolites found in the bile.