Acute sodium arsenite treatment induces Cyp2a5 but not Cyp1a1 in the C57Bl/6 mouse in a tissue (kidney) selective manner

Modulation of hepatic and extrahepatic detoxication enzymes Cyp1a1, Cyp2a5, glutathione S-transferse Ya (GSTYa) and NAD(P)H:quinone oxidoreductase (QOR) dependent catalytic activity and mRNA levels were investigated at 1, 2, or 4 days in liver, lung, or kidney of male, adult CD57 Bl/6 mice treated sc with a single dose (85 micromol/kg) of sodium arsenite (As3+). Maximum decreases of total hepatic cytochrome P450 (CYP) monooxygenase content and catalytic activities, occurring at 24 h, corresponded with maximum increases of heme oxygenase (HO-1) in all tissues, as well as maximum plasma total bilirubin. Extrahepatic increases in CYP were observed only in non-AHR dependent isozymes in the kidney, where both Cyp2a5 mRNA and catalytic activity increased maximally 24 h after treatment. In contrast, no significant changes in Cyp2b1/2-dependent PROD or mRNA activity and decreases in Cyp1a1-dependent-EROD activity were noted 1, 2, or 4 days after treatment. Increases in QOR catalytic activities were observed in all tissues examined with increased mRNA in kidney. On the other hand, GSTYa catalytic activity and mRNA increases were only detected in kidney. This study demonstrates the differential modulation of CYP, QOR, and GST-Ya, important drug metabolizing enzymes after acute As3+ administration. The induction of Cyp2a5, QOR, and GSTYa catalytic activity and gene expression occurred primarily in kidney during or shortly after conditions of oxidant stress.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

Sudan III dye strongly induces CYP1A1 mRNA expression in HepG2 cells

Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 μM Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III--treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III.     

3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.     

The effect of glycerol on cytochrome P450scc (CYP11A1) spin state,activity, and hydration

We examined the effects of glycerol, a stabilizing agent commonly used in cytochrome P450scc purification and analysis, on the spin state, catalytic activity, and molecular volume of the cytochrome. Glycerol induced a sigmoidal low-spin response. The binding of hydroxycholesterol reaction intermediates, but not cholesterol, increased the concentration of glycerol required for the spin transition to be 50% complete (K(1/2)). Glycerol weakened adrenodoxin binding to P450scc but had no effect on CO or 20alpha,22R-dihydroxycholesterol binding. Cytochrome P450scc activity was inhibited by glycerol with the K(1/2) for inhibition being substrate-dependent. The osmotic stress exerted by glycerol on P450scc resulted in decreases in P450scc molecular volume for both the transition to low spin state and the inhibition of activity. From this we determined that two dissociative water molecules are involved in the inhibition of activity with cholesterol as substrate and five or six dissociative waters are involved in the low-spin transition. The dehydration of P450scc by osmotic stress provides an explanation for the effects of glycerol on P450scc spin transition and activity.     

Upregulation of CYP2e1 and CYP3a activities in histamine-deficient histidine decarboxylase gene targeted mice

Histamine is a biogenic amine with multiple physiological functions. Its importance in allergic inflammation is well characterized; moreover, it plays a role in the regulation of gastric acid production, various hypothalamic functions, such as food uptake, and enhancing TH2 balance during immune responses. Using histidine decarboxylase gene targeted (HDC(-/-)) BALB/c mice, we studied the effect of the absence of histamine on four cytochrome p450 enzyme activities. Their selective substrates were measured: ethoxyresorufin O-dealkylase activity of CYP1A, pentoxyresorufin O-dealkylase activity of CYP2B, chlorzoxazone 6-hydroxylase activity of CYP2E1 and ethylmorphine N-demethylase activity of CYP3A.The results indicate a significant elevation of CYP2E1 and CYP3A activities, however, no change in CYP1A and CYP2B activities was seen in HDC targeted mice compared to wild type controls with identical genetic backgrounds.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

Possible differences in the mechanism of malignant transformation of HaCaT cells by arsenite and its dimethyl metabolites,particularly dimethylthioarsenics

Background: As a confirmed human carcinogen, arsenic can cause skin cancer, lung cancer, etc. However, its carcinogenic mechanism is still unclear. In recent years, the oxidative stress hypothesis has become widely accepted. In mammals it has been found that arsenic can be converted to dimethylarsinous acid (DMA^III) and dimethylmonothioarsinic acid (DMMTA^V) through a series of methylation and redox reactions. DMA^III and DMMTA^V are highly toxic.Methods: Human keratinocytes (HaCaT) were exposed to different concentrations of NaAsO₂ (IAs^III), DMMTA^V and DMA^III for 24 h. Reactive oxygen species (hydrogen peroxide and superoxide), oxidative damage markers (8-hydroxydeoxyguanosine and malondialdehyde), and antioxidant markers (glutathione and superoxide dismutase) were measured. In addition, sulfane sulfurs were measured in HaCaT cells and a cell-free system.Results: In the DMMTA^V and DMA^III treatment groups, the levels of hydrogen peroxide and superoxide in HaCaT cells were higher than in the IAs^III treatment groups at the same dose. Levels of 8−OHdG and MDA in the DMMTA^V and DMA^III treatment groups were also higher than those in the IAs^III treatment groups at the same dose. However, in the DMMTA^V and DMA^III treatment groups, the levels of GSH and SOD activity were lower than that in the IAs^III treatment groups. In DMMTA^V-treated HaCaT cells, sulfane sulfurs were produced. Further, it was found that DMMTA^V could react with DMDTA^V to form persulfide in the cell-free system, which may explain the mechanism of the formation of sulfane sulfurs in DMMTA^V-treated HaCaT cells.Conclusions: DMMTA^V and DMA^III more readily induce reactive oxygen species (ROS) and cause oxidative damage in HaCaT cells than inorganic arsenic. Further, the persulfide formed by the reaction of DMMTA^V and DMDTA^V produced from the metabolism of DMMTA^V may induce a stronger reductive defense mechanism than GSH against the intracellular oxidative stress of DMMTA^V. However, the cells exposed to arsenite are transformed by the continuous nuclear translocation of Nrf2 due to oxidative stress, and the persulfide from dimethylthioarsenics may promote Nrf2 by the combination with thiol groups, especially redox control key protein, Keap1, eventually cause nuclear translocation of sustained Nrf2.     

Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards beta-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 degrees C. In this system, beta-carotene was hydroxylated to beta-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low beta-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated beta-carotene at 3- and also 3'-positions, resulting in beta-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol beta-cryptoxanthin produced per nmol P450 per min.     

Comparing the electronic properties and docking calculations of heme derivatives on CYP2B4

Cytochrome P-450 is a group of enzymes involved in the biotransformation of many substances, including drugs. These enzymes possess a heme group (1) that when it is properly modified induces several important physicochemical changes that affect their enzymatic activity. In this work, the five structurally modified heme derivatives 2–6 and the native heme 1 were docked on CYP2B4, (an isoform of P450), in order to determine whether such modifications alter their binding form and binding affinity for CYP2B4 apoprotein. In addition, docking calculations were used to evaluate the affinity of CYP2B4 apoprotein-heme complexes for aniline (A) and N-methyl-aniline (NMA). Results showing the CYP2B4 heme 4- and heme 6-apoprotein complexes to be most energetically stable indicate that either hindrance effects or electronic properties are the most important factors with respect to the binding of heme derivatives at the heme-binding site. Furthermore, although all heme-apoprotein complexes demonstrated high affinity for both A and NMA, the CYP2B4 apoprotein-5 complex had higher affinity for A, and the heme 6 complex had higher affinity for NMA. Finally, surface electronic properties (SEP) were calculated in order to explain why certain arginine residues of CYP2B4 apoprotein interact with polarizable functionalities, such as ester groups or sp ² carbons, present in some heme derivates. The main physicochemical parameter involved in the recognition process of the heme derivatives, the CYP2B4 apoprotein and A or NMA, are reported. Figure Scheme of steps to be followed for obtaining five new CYP2B4 apoprotein-heme complexes by docking     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Induced CYP1A mRNA, protein and catalytic activity in the liver of feral fish, leaping mullet, Liza saliens

In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens, and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5'-dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3' was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positions 291 to 301 among the various fish species. Results of Northern blot analysis revealed that RNA isolated from the liver of mullet collected from the highly contaminated region of Izmir Bay with a dissolved and dispersed petroleum hydrocarbon content of 12.45 microg l(-1) gave a strong hybridization signal, whereas only a weak hybridization signal was detected in the liver RNA of fish caught from the reference site containing less than 1 microg l(-1) of petroleum hydrocarbons. Similarly, fish from the contaminated site had approximately 80 times more EROD activity than the feral fish captured from the reference site. Studies using polyclonal antibodies produced against purified mullet CYP1A also showed the similar trend. In conclusion, feral leaping mullet caught from contaminated water displayed induction of CYP1A at three levels of expression, namely, mRNA, apoprotein and catalytic activity.     

Detection of microwave radiation of cytochrome CYP102 A1 solution during the enzyme reaction

Microwave radiation at 3.4–4.2 GHz frequency of the cytochrome P450 CYP102 A1 (BM3) solution was registered during the lauric acid hydroxylation reaction. The microwave radiation generation was shown to occur following the addition of electron donor NADPH to a system containing an enzyme and a substrate. The radiation occurs for the enzyme solutions with enzyme concentrations of 10^?8 and 10^?9 М. The microwave radiation effect elicited by the aqueous enzyme solution was observed for the first time. The results obtained can be used to elaborate a new approach to enzyme systems research, including studying of the mechanism of interaction of a functioning enzyme system with microenvironment.     

A Revised Evolutionary History of the CYP1A Subfamily: Gene Duplication, Gene Conversion, and Positive Selection

Members of cytochrome P450 subfamily 1A (CYP1As) are involved in detoxification and bioactivation of common environmental pollutants. Understanding the functional evolution of these genes is essential to predicting and interpreting species differences in sensitivity to toxicity caused by such chemicals. The CYP1A gene subfamily comprises a single ancestral representative in most fish species and two paralogs in higher vertebrates, including birds and mammals. Phylogenetic analysis of complete coding sequences suggests that mammalian and bird paralog pairs (CYP1A1/2 and CYP1A4/5, respectively) are the result of independent gene duplication events. However, comparison of vertebrate genome sequences revealed that CYP1A genes lie within an extended region of conserved fine-scale synteny, suggesting that avian and mammalian CYP1A paralogs share a common genomic history. Algorithms designed to detect recombination between nucleotide sequences indicate that gene conversion has homogenized most of the length of the chicken CYP1A genes, as well as the 5′ end of mammalian CYP1As. Together, these data indicate that avian and mammalian CYP1A paralog pairs resulted from a single gene duplication event and that extensive gene conversion is responsible for the exceptionally high degree of sequence similarity between CYP1A4 and CYP1A5. Elevated nonsynonymous/synonymous substitution ratios within a putatively unconverted stretch of ∼250 bp suggests that positive selection may have reduced the effective rate of gene conversion in this region, which contains two substrate recognition sites. This work significantly alters our understanding of functional evolution in the CYP1A subfamily, suggesting that gene conversion and positive selection have been the dominant processes of sequence evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

The genetic regulation of liver microsomal CYP2E1 activity among strains of the viviparous fish Poeciliopsis

CYP2E1 expression was examined within, among, and in F(1) and backcross progeny of strains (P. monacha S68-5; P. viriosa M65-23) of the viviparous fish Poeciliopsis. CYP 2E1 activity varied dramatically in P. monacha, and P. viriosa (3.9+/-0.8 and 9.6+/-1.3 microg/min/mg) as well as the temperature which gave maximal activity (T(0)=25 degrees C and 31 degrees C). F(1) individuals from a crosses between P. monacha and P. viriosa, produced progeny whose CYP2E1 activity segregated into three different groups: (1) phenotypically the same as P. viriosa; (2) intermediate between the two parental strains; and (3) phenotypically the same as P. monacha. When a male P. monacha was crossed with a female P. viriosa 25% of the offspring had an intermediate phenotype and 65% the maternal P. viriosa phenotype. From the same cross, 85% of the females progeny had the maternal phenotype, while 80% of male progeny had the intermediate and paternal phenotype, suggesting an effect of the maternal genome on the F(1) phenotype. Among F(1) fish the T(0) was evenly distributed between parental values. In the backcross of a F(1) female to a male P. viriosa, CZX-6-hydroxylase activity segregated into the same three phenotypes with 60% of the progeny expressing the P. monacha phenotype. From the same cross, 70% of females and 40% of males expressed the P. monacha phenotype. The T(0) in the backcross were evenly distributed between the two parental values and the sex ratio among progeny was different than expected.     

19F-NMR reveals substrate specificity of CYP121A1 in Mycobacterium tuberculosis

Cytochromes P450 are versatile enzymes that function in endobiotic and xenobiotic metabolism and undergo meaningful structural changes that relate to their function. However, the way in which conformational changes inform the specific recognition of the substrate is often unknown. Here, we demonstrate the utility of fluorine (¹⁹F)-NMR spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis. CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicyclotyrosine. The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone was used to label an S171C mutation of the enzyme FG loop, which is located adjacent to the homodimer interface. Substrate titrations and inhibitor-bound ¹⁹F-NMR spectra indicate that ligand binding reduces conformational heterogeneity at the FG loop in both the dimer and in an engineered monomer of CYP121A1. However, only the dimer was found to promote a substrate-bound conformation that was preexisting in the substrate-free spectra, thus confirming a role for the dimer interface in dicyclotyrosine recognition. Moreover, ¹⁹F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a dicyclotyrosine specificity criterion. This study demonstrates the utility of ¹⁹F-NMR as applied to a multimeric cytochrome P450, while also revealing mechanistic insights for an essential M. tuberculosis enzyme.     

Comparison of the biotransformation of the 14C-labelled insecticide carbaryl by non-transformed and human CYP1A1-, CYP1A2-, and CYP3A4-transgenic cell cultures of Nicotiana tabacum

Transgenic tobacco-cell-suspension cultures expressing separately the human cytochrome P450 monooxygenases CYP1A1, CYP1A2, and CYP3A4 were utilized to study the biotransformation of the 14C-labelled insecticide carbaryl (=naphthalen-1-yl methylcarbamate). The resulting data were compared to similar data from the corresponding non-transformed (NT) tobacco-cell culture and commercially available membrane preparations (Bactosomes) of genetically modified bacteria separately containing the same human P450s. A rapid conversion rate of carbaryl was observed with the CYP1A1 and CYP1A2 cells, where only 49.7 and 0.2% of applied carbaryl (1 mg/l), respectively, remained after 24 h, as compared to 77.7% in the non-transformed culture. Unexpectedly, the corresponding results obtained from the CYP3A4 cultures were not definite. With 25 mg/l of carbaryl and 96 h of incubation, it was proven that the insecticide is also substrate of CYP3A4. This finding was supported by GC/EI-MS analysis of the primary metabolite pattern produced by the isozyme. This consisted of naphthalene-1-ol, N-(hydroxymethyl)carbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl, whereas the main product in non-transformed cells was N-(hydroxymethyl)carbaryl. Data obtained from the CYP1A1, CYP1A2, or CYP3A4 Bactosomes agreed with those of the P450-transgenic tobacco cells. Problems with GC/EI-MS analysis of carbaryl and its metabolites are discussed.     

Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.     

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 μmol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato. Received: 15 March 2001 / Accepted: 14 June 2001