Xenobiotic-metabolizing P450 Research Articles

A transposon-introduced G-quadruplex motif is selectively retained and constrained to downregulate CYP321A1.

Insects utilize xenobiotic compounds to up- and downregulate cytochrome P450 monooxygenases (P450s) involved in detoxification of toxic xenobiotics including phytochemicals and pesticides. G-quadruplexes (G4)-forming DNA motifs are enriched in the promoter regions of transcription factors and function as cis-acting elements to regulate these genes. Whether and how P450s gain and keep G4 DNA motifs to regulate their expression still remain unexplored. Here, we show that CYP321A1, a xenobiotic-metabolizing P450 from Helicoverpa zea, a polyphagous insect of economic importance, has acquired and preserved a G4 DNA motif by selectively retaining a transposon known as HzIS1-3 that carries this G4 DNA motif in its promoter region. The HzIS1-3 G4 DNA motif acts as a silencer to suppress the constitutive and induced expression of CYP321A1 by plant allelochemicals flavone and xanthotoxin through folding into an intramolecular parallel or hybrid-1 conformation in the absence or presence of K+ . The G4 ligand N-methylmesoporphyrin IX (NMM) strengthens the silencing effect of HzIS1-3 G4 DNA motif by switching its structure from hybrid-1 to hybrid-2. The enrichment of transposons in P450s and other environment-adaptation genes implies that selective retention of G4 DNA motif-carrying transposons may be the main evolutionary route for these genes to obtain G4 DNA motifs.

Identification of Wild-Type CYP321A2 and Comparison of Allelochemical-Induced Expression Profiles of CYP321A2 with Its Paralog CYP321A1 in Helicoverpa zea.

Simple SummaryPlant toxin- and insecticide-detoxifying genes known as P450s are often involved in insect resistance to xenobiotics. For polyphagous generalists, investigation of ecologically significant P450s and their induced expression profiles by allelochemicals is of particular importance to understand the roles of these genes in detoxification of allelochemicals and insecticides, and the adaptation of generalists to their chemical environment. Previous studies identified an allelochemical-inducible P450 gene CYP321A1 in polyphagous Helicoverpa zea, which is associated with detoxification of its inducers including plant allelochemicals and insecticides. Our study represents the identification, features of CYP321A2 (a duplicated paralog of CYP321A1), as well as the responses of CYP321A paralogs to allelochemicals and plant signal molecules in H. zea. Moreover, xanthotoxin- and flavone-responsive regulatory elements of CYP321A1 were also detected in the promoter region of CYP321A2. Our results enrich the P450 inventory by identifying an allelochemical broadly induced CYP321A2 in H. zea. Our data also suggest that the CYP321A2/CYP321A1 paralogs are a pair of duplicated genes of multigene families and CYP321A2 has the potential to detoxify plant allelochemicals and adapt to its chemical environment.One possible way to overcome the diversity of toxic plant allelochemicals idiosyncratically distributed among potential host plants is to have more counterdefense genes via gene duplication or fewer gene losses. Cytochrome P450 is the most important gene family responsible for detoxification of the diversity of plant allelochemicals. We have recently reported the identification and cloning of the transposon (HzSINE1)-disrupted non-functional CYP321A2, a duplicated paralog of the xenobiotic-metabolizing P450 CYP321A1 from a laboratory colony of Helicoverpa zea. Here we report the identification of the wild-type intact allele of CYP321A2 from another H. zea colony. This CYP321A2 allele encodes a deduced protein of 498 amino acids and has the P450 signature motifs. Quantitative RT-PCR experiments showed that this CYP321A2 allele was highly expressed in midgut and fat body and achieved the highest expression level in the developmental stage of 5th and 3rd instar larvae. CYP321A2 and CYP321A1 were constitutively expressed in low levels but can be differentially and significantly induced by a range of the plant allelochemicals and plant signal molecules, among which xanthotoxin, flavone, and coumarin were the most prominent inducers of CYP321A2 both in midgut and fat body, whereas flavone, coumarin, and indole-3-carbinol were the prominent inducers of CYP321A1 in midgut and fat body. Moreover, xanthotoxin- and flavone-responsive regulatory elements of CYP321A1 were also detected in the promoter region of CYP321A2. Our results enrich the P450 inventory by identifying an allelochemical broadly induced CYP321A2, a paralog of CYP321A1 in H. zea. Our data also suggest that the CYP321A2/CYP321A1 paralogs are a pair of duplicated genes of multigene families and CYP321A2 could potentially be involved in the detoxification of plant allelochemicals and adaptation of H. zea to its chemical environment.

Diversifying selection detected in only a minority of xenobiotic-metabolizing CYP1-3 genes among primate species

1. Primates exhibit a high degree of among-species dietary diversity, which likely exposes them to varying levels of xenobiotic compounds. Here, we examined the evolution of primate CYP1-3 gene families, and we classified the 15 CYP1-3 gene subfamilies as either xenobiotic-metabolizing (XM) or endogenous-metabolizing (EM) based on sources in the P450 literature. 2. We predicted that XM P450s would show (1) greater variability in gene-copy number and (2) more evidence of diversifying selection and, especially on codons that encode the substrate-recognition sites (SRSs) for the final enzymes. 3. Counter to our first prediction, EM and XM P450s showed similar levels of variation in gene-copy number. We did find, however, that four XM P450 subfamilies (CYP2C, CYP2D, CYP2E, and CYP3A) showed evidence of diversifying selection while no EM subfamilies demonstrated any consistent signal of diversifying selection. Of these four, CYP2C, CYP2D, and CYP3A showed significant links between SRSs and diversifying selection. 4. These results reveal an amount of evolutionary dynamism that would not be expected when viewing P450 subfamilies along a simple binary EM/XM spectrum. We recommend that comparative studies of cytochrome P450 evolution should focus on the CYP2C, CYP2D, CYP2E, and CYP3A subfamilies.

Three Patterns of Cytochrome P450 Gene Expression during Liver Maturation in Mice

The neonatal period of liver development is an often overlooked phase of development. For instance, ontogeny of xenobiotic-metabolizing enzymes can markedly affect biotransformation as the liver matures. To systematically examine the ontogenic gene expression patterns of cytochrome P450 genes (P450) in mice, the gene expression profiles of 19 xenobiotic-metabolizing P450 in Cyp1 to 4 families were determined. The mRNA levels in C57BL/6 mouse livers were quantified using branched DNA technology at the following ages: gestational day 17 (2 days before birth) and postnatal days 0, 1, 3, 5, 10, 15, 20, 30, and 45. Among the 13 P450 genes expressed in mouse livers, three distinct ontogenic expression patterns were identified by cluster analysis. Genes in group 1 (Cyp3a16 as well as 3a41b in male) were expressed in the perinatal period, but they were essentially nondetectable by 30 days of age. Genes in group 2 (Cyp2e1, 3a11, and 4a10 as well as 3a41b in female) quickly increased after birth and reached maximal expression levels by day 5. Genes in group 3 (Cyp1a2, 2a4, 2b10, 2c29, 2d22, 2f2, 3a13, and 3a25) were expressed at low levels until days 10 to 15, but they markedly increased at day 20 to a high and stable level. In conclusion, the developmental expression of P450 in mouse liver can be divided into three patterns, suggesting that different mechanisms are responsible for the expression of P450 during liver maturation.

Molecular characterization of the first intact Transib transposon from Helicoverpa zea

Transib is a superfamily of DNA transposons recently reconstructed in silico from degenerate elements in the genomes of Drosophila melanogaster and Anopheles gambiae. Here we report characterization of the first intact Transib transposon designated Hztransib from Helicoverpa zea. At least two Hztransib copies (one full-length, one internally-deleted) exist in the genomes of a midgut cell line and a laboratory strain of H. zea. The full-length Hztransib has 3518 bp including a 5′ terminal inverted repeat (TIR) of 552 bp, a promoter sequence of 381 bp, an intact open reading frame encoding 507 amino acids, and a 3′ TIR of 502 bp, and is flanked by 5-bp (CGTCG) target site duplications. The full-length Hztransib is transcriptionally active, producing an 3′-truncated mRNA lacking a termination codon (known as nonstop mRNA) due to alternative polyadenylation in two somatic tissues (midgut and fat body) and one germline tissue (ovary). A BLAST search with the deduced Hztransib transposase identified 51 novel Transib elements from 11 insect genomes. While the full-length Hztransib inserts into the 5′-flanking region of a xenobiotic-metabolizing P450 gene CYP6B8 in the midgut cell line, it does not insert into the 5′-flanking region of CYP6B8 in the laboratory strain. Such an insertion dimorphism, together with its complete structural features and mRNA transcripts, demonstrates that the full-length Hztransib not only represents the first known intact Transib element in any organism, but has been recently transposed in H. zea. The fact that the intact Hztransib is transcribed into a 3′-truncated nonstop mRNA which may encode a non-functional transposase or be blocked from further translation via nonstop mRNA decay suggests that it is silenced at the translational or transpositional level.

Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

BackgroundTransposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search.ResultsTwelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions.ConclusionThese results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes.

Characterisation of the xenobiotic-metabolizing Cytochrome P450 expression pattern in human lung tissue by immunochemical and activity determination

The lung represents an important target for the toxic effects of chemicals. Many of the chemicals require enzymatic activation to exert their adverse effects, which is mostly catalysed by Cytochrome P450 (CYP) enzymes. Although there is considerable evidence that individual members of the xenobiotic-metabolizing P450 family are expressed in human lung tissue at the mRNA level, there is conflicting evidence concerning the following issues: (I) the qualitative expression pattern of CYP isoenzymes; (II) CYP expression at the protein and/or activity level; and (III) interindividual variability of CYP enzymes in human lung. The latter can be the basis for individual susceptibility towards the adverse effects of lung toxicants. In preparing for studying factors to explain interindividual variability of CYP expression in lung tissue, we investigated the qualitative pulmonary expression pattern of xenobiotic-metabolizing CYP enzymes and elaborated the optimal conditions for quantification at the protein and activity level. By using either individual human lung samples or pooled microsomes from different individuals, immunoreactive bands specific for the following CYP enzymes could be determined by Western blotting: CYP1A1, CYP1A2, CYP2E1 and CYP3A5. Western blotting experiments were also supportive of the presence of CYP2A, CYP2B6, CYP2D6 and CYP3A4 in human lung. By using antibodies specific for CYP2C enzymes and CYP1B1, respectively, immunoreactive bands, which differed slightly in mobility from corresponding standards, were detectable. In addition, we measured methoxy- and ethoxyresorufin dealkylase activities and chlorzoxazone (CLX)-hydroxylase activity in human lung and confirmed the specifities of the latter two activities by inhibition experiments. In summary, we have established methodologies to quantify a panel of CYP enzymes in human lung samples among which there are CYP enzymes whose expression at the protein and activity level has not been evidenced so far.

Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach

Lung is a target organ for the toxicity of inhalated compounds. The respiratory tract is frequently exposed to elevated concentrations of these compounds and become the primary target site for toxicity. Occupational, accidental or prolonged exposure to a great variety of chemicals may result in acute or delayed injury to cells of the respiratory tract. Nevertheless, lung has a significant capability of biotransforming such compounds with the aim of reducing its potential toxicity. In some instances, the biotransformation of a given compound can result in the generation of more reactive, and frequently more toxic, metabolites. Indeed, lung tissue is known to activate pro-carcinogens (i.e. polycyclic aromatic hydrocarbons or N-nitrosamines) into more reactive intermediates that easily form DNA adducts. Lungs express several enzymes involved in the metabolising of xenobiotics. Among them, cytochrome P450 enzymes are major players in the oxidative metabolism as well metabolic bioactivation of many organic toxicants, including pro-carcinogens. Xenobiotic-metabolising P450 enzymes are expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages Individual CYP isoforms have different patterns of localisation within pulmonary tissue. With the aid of sensitive techniques (i.e. reverse transcriptase-polymerase chain reaction, RT-PCR) it has become possible to detect CYP1A1, CYP1B1, CYP2A6, CYP2B6, CYP2E1 and CYP3A5 mRNAs in lung cells. Less conclusive results have been obtained concerning CYP2Cs, CYP2D6 and CYP3A4. CYP3A5 protein appears to be widely present in all lung samples and is localised in the ciliated and mucous cells of the bronchial wall, bronchial glands, bronchiolar ciliated epithelium and in type I and type II alveolar epithelium. Lung cells also express Phase II enzymes such as epoxide hydrolase, UGT1A (glucuronyl transferase) and GST-P1 (glutathione S-transferase), which largely act as detoxifying enzymes. A key question concerning organ-specific chemical toxicity is whether the actual target has the capacity to activate (or efficiently inactivate) chemicals. Results of several studies indicate that the different xenobiotic-metabolising CYPs, present in the human lung and lung-derived cell lines, likely contribute to in situ activation of pulmonary toxins, among them, pro-carcinogens. Some CYPs, in particular CYP1A, are polymorphic and inducible. Interindividual differences in the expression of these CYPs may explain the different risk of developing lung toxicity (possibly cancer), by agents that require metabolic activation. Few cell lines, principally A549, have been used with variable success as an experimental model for investigating the mechanisms of toxicity. Although RT-PCR analysis has evidenced the presence of the major human pulmonary CYP mRNAs, the measurable P450 specific activities are, however, far below those present in human lungs. Detection of the toxicity elicited by reactive metabolites requires the use of metabolically competent cells; consequently, better performing cells are needed to ensure realistic in vitro prediction of toxicity. Genetic manipulation of lung-derived cells allowing them to re-express key biotransformation enzymes appear to be a promising strategy to improve their functionality and metabolic performance.

Repression of phenobarbital-dependent CYP2B1 mRNA induction by reactive oxygen species in primary rat hepatocyte cultures.

Xenobiotic-metabolizing cytochrome P-450 (P-450) enzymes not only play a pivotal role in elimination of foreign compounds but also contribute to generation of toxic intermediates, including reactive oxygen species, that may elicit cellular damage if produced excessively. Expression of several xenobiotic-metabolizing P-450 enzymes is induced by phenobarbital (PB). Pronounced induction is observed for the rat CYP2B1 isoform. A primary rat hepatocyte culture system was used to investigate whether reactive oxygen species might modulate PB-dependent CYP2B1 induction. In cells cultivated for 3 days with 1.5 mM PB, substantial CYP2B1 mRNA induction was observed (100%). Addition of H(2)O(2) or of the catalase inhibitor 3-amino-1,2,4-triazole (AT) to the medium repressed induction to approximately 30% (at 1 mM H(2)O(2) and 2 mM AT, respectively). Accordingly, treatment of hepatocytes with PB and the glutathione precursor N-acetylcysteine (NAC) led to enhanced PB-dependent induction (to over 1000% at 10 mM NAC). In primary hepatocyte cultures transfected with a CYP2B1 promoter-luciferase construct containing approximately 2.7 kilobase pairs of the native CYP2B1 promoter sequence, PB-dependent reporter gene activation was repressed by AT and stimulated by N-acetylcysteine. Furthermore, a 263-base pair CYP2B1 promoter fragment encompassing the phenobarbital-responsive enhancer module conferred suppression of PB-dependent luciferase expression by AT and activation by NAC in a heterologous SV40-promoter construct. In summary, these data demonstrate a regulatory mechanism that is dependent on the cellular redox status, which modulates CYP2B1 mRNA induction by PB on the transcriptional level, thus representing a feedback mechanism preventing further P-450-dependent production of reactive oxygen intermediates under oxidative stress.

Identification of a meander region proline residue critical for heme binding to cytochrome P450: implications for the catalytic function of human CYP4B1.

Alignment of xenobiotic-metabolizing P450 protein sequences highlights an invariant proline residue in the meander region two amino acids N-terminal to the distal arginine of the putative ERR triad thought to be important for heme binding. This occurs as a serine in the sequences derived from human CYP4B1 gDNA and both human lung and placental CYP4B1 cDNAs. Reversion of this serine to the conserved proline residue (Ser427 --> Pro) by site-directed mutagenesis conferred the ability to incorporate heme on the human placental enzyme. Mutation of the corresponding proline in rabbit CYP4B1 (Pro422 --> Ser) abolished heme incorporation. Membrane preparations of human CYP4B1(Pro) and rabbit CYP4B1(Pro), but not the corresponding CYP4B1(Ser) variants, supported lauric acid hydroxylation preferentially at the omega-position. Purified, reconstituted human CYP4B1(Pro) and rabbit CYP4B1(Pro) formed 12-hydroxylauric acid at rates of 17-21 min-1, and both enzymes were also C-8 to C-10 fatty acid omega-hydroxylases preferentially, with total rates of hydroxylation decreasing in the order C-12 > C-10 > C-9 > C-8. Finally, neither human nor rabbit CYP4B1(Pro) formed detectable levels of any hydroxylated testosterone metabolites. Therefore, the presence of a consensus Pro-X-Arg motif is critical for incorporation of the heme prosthetic group in human and rabbit CYP4B1 proteins expressed in insect cells. Native human CYP4B1, expressed in vivo, is likely to be functionally impaired if Pro427 is required for holoenzyme expression in mammalian cells.

Strain differences in adrenal microsomal steroid metabolism in guinea pigs

We recently reported that CYP2D16, a xenobiotic-metabolizing P450 isozyme, was expressed at higher levels in adrenal microsomes from inbred Strain 13 guinea pigs than in those from outbred English Short Hair (ESH) animals. Studies were done to determine if there also were strain differences in adrenal microsomal steroid metabolism. In both inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zone preparations of the adrenal cortex, 21-hydroxylase activities were greater in microsomes from ESH than from Strain 13 guinea pigs. By contrast, 17 α-hydroxylase activities were similar in the two strains. In both strains, 21-hydroxylase activities were greater in inner than outer zone microsomes, but the opposite was found for 17 α-hydroxylase activities (outer>inner). Northern and Western analyses revealed higher levels of CYP21 mRNA and protein in adrenals from ESH than Strain 13 guinea pigs, but there were no strain differences in CYP17 mRNA or protein concentrations. Despite the zonal differences in adrenal 17 α-hydroxylase and 21-hydroxylase activities, CYP17 and CYP21 mRNA and protein levels were similar in the inner and outer zones within each strain of guinea pig. The results demonstrate strain differences in microsomal steroid metabolism that are explained by differences in CYP21 expression. By contrast, the zonal differences in steroid hydroxylase activities may be attributable to post-translational mechanisms.

A review of developmental aspects of cytochrome P450.

This article surveys the development of human hepatic P450 cytochromes (CYPs) involved in xenobiotic metabolism from the fetus through the life span and explores possible clinical consequences of developmental issues. These hepatic P450 CYPs come "on line" at different times during fetal and infant development, and each one is discussed in that temporal sequence. CYP3A7. the major fetal hepatic cytochrome, is present during organogenesis, and it is involved in steroid metabolism. Variably expressed in some fetuses, CYP3A5 is also present at significant levels in about half of all children. In adults, CYP3A4 is the major functional member of the CYP3A subfamily. CYP1A1 is also present during organogenesis, and it metabolizes exogenous toxins, some of which are procarcinogens. CYP2E1 may be present in some second-trimester fetuses, and it may be involved in prenatal alcohol metabolism. After birth, hepatic CYP2D6 and CYP2C8/9 and CYP2C18/19 become active. Both CYP2D6 and CYP2C19 have genetic polymorphisms that can bring about differing capacities to metabolize exogenous drugs, including psychotropic drugs. CYP1A2 becomes active in the fourth to fifth postfetal months. It provides the best current examples of the importance of developmental changes in xenobiotic-metabolizing P450 CYPs through its metabolism of caffeine and theophylline in premature infants, neonates, and adolescents.

Inhibition of adrenal steroid metabolism by administration of 1-aminobenzotriazole to guinea pigs.

Prior in vitro investigations demonstrated that the P450 suicide substrate, 1-aminobenzotriazole (ABT), was a potent inhibitor of xenobiotic metabolism but had no effect on steroidogenic enzymes in the guinea pig adrenal cortex. Studies were done to determine if ABT administration of guinea pigs in vivo also selectively inhibited adrenal xenobiotic metabolism. At single doses of 25 or 50 mg/kg, ABT effected rapid decreases in spectrally detectable adrenal P450 concentrations. The higher dose caused approx. 75% decreases in microsomal and mitochondrial P450 levels within 2 h. The decreases in P450 were sustained for 24 h but concentrations returned to control levels within 72 h. Accompanying the ABT-induced decreases in adrenal P450 content were proportionately similar decreases in P450-mediated xenobiotic and steroid metabolism. Microsomal benzo(a)pyrene hydroxylase, benzphetamine N-demethylase, 17 alpha-hydroxylase and 21-hydroxylase activities were decreased to 20-25% of control values by the higher dose of ABT. Mitochondrial 11 beta-hydroxylase and cholesterol sidechain cleavage activities were similarly diminished by ABT treatment. Adrenal 3 beta-hydroxysteroid dehydrogenase activity, by contrast, was not affected by ABT, indicating specificity for P450-catalyzed reactions. The results demonstrate that ABT in vivo is a non-selective inhibitor of adrenal steroid- and xenobiotic-metabolizing P450 isozymes. The absence of ABT effects on steroid metabolism in vitro suggests that an extra-adrenal metabolite may mediate the in vivo inhibition of steroidogenesis.

Inhibition of adrenal cytochromes P450 by 1-aminobenzotriazole in vitro selectivity for xenobiotic metabolism

Studies were done to determine the effects of a P450 suicide inhibitor, 1-aminobenzotriazole (ABT), on adrenal steroid and xenobiotic metabolism. Incubation of guinea pig adrenal microsomes with ABT plus an NADPH-generating system caused a time-dependent decline in total P450 concentrations. The maximal decrease in P450 levels was approximately 35% and was accompanied by an equimolar decrease in heme content. Western blot analyses indicated that ABT had no effect on P450 apoprotein levels. Benzphetamine (BZ) N-demethylase and benzo[ a]pyrene (BP) hydroxylase activities were inhibited almost completely by microsomal incubations with ABT. In contrast, neither steroid 17 α-hydroxylase nor 21-hydroxylase activity was affected by ABT. The steroid-induced type I spectral change in adrenal microsomes also was not affected by ABT, whereas that induced by BZ was eliminated. Similar studies with adrenal mitochondria indicated that ABT had no effect on mitochondrial P450 concentrations or on mitochondrial steroid metabolism. The results demonstrate that the in vitro actions of ABT on adrenal cytochromes P450 are highly selective for those isozymes that catalyze xenobiotic metabolism. Therefore, ABT should serve as a useful probe for further characterization of adrenal xenobiotic-metabolizing P450 isozymes.