Human Cytochrome P450 Genes Research Articles

Amplified and distinctive genotoxicity of titanium dioxide nanoparticles in transformed yeast reporters with human cytochrome P450 (CYP) genes

Titanium dioxide nanoparticles (nTiO2) have been considered a possible carcinogen to humans, but most existing studies have overlooked the role of human enzymes in assessing the genotoxicity of nTiO2. Here, a toxicogenomics-based in vitro genotoxicity assay using a GFP-fused yeast reporter library was employed to elucidate the genotoxic potential and mechanisms of nTiO2. Moreover, two new GFP-fused yeast reporter libraries containing either human CYP1A1 or CYP1A2 genes were constructed by transformation to investigate the potential modulation of nTiO2 genotoxicity in the presence of human CYP enzymes. This study found a lack of appreciable nTiO2 genotoxicity as indicated by the yeast reporter library in the absence of CYP expression but a significantly elevated indication of genotoxicity in either CYP1A1- or CYP1A2-expressing yeast. The intracellular reactive oxygen species (ROS) measurement indicated significantly higher ROS in yeast expressing either enzyme. The detected mitochondrial DNA damage suggested mitochondria as one of the target sites for oxidative damage by nTiO2 in the presence of either one of the CYP enzymes. The results thus indicated that the genotoxicity of nTiO2 was enhanced by human CYP1A1 or CYP1A2 enzyme and was associated with elevated oxidative stress, which suggested that the similar mechanisms could occur in human cells.

Global perspectives on CYP2D6 associations with primaquine metabolism and Plasmodium vivax radical cure.

Clinical trial and individual patient treatment outcomes have produced accumulating evidence that effective primaquine (PQ) treatment of Plasmodium vivax and P. ovale liver stage hypnozoites is associated with genetic variation in the human cytochrome P450 gene, CYP2D6. Successful PQ treatment of individual and population-wide infections by the Plasmodium species that generate these dormant liver stage forms is likely to be necessary to reach elimination of malaria caused by these parasites globally. Optimizing safe and effective PQ treatment will require coordination of efforts between the malaria and pharmacogenomics research communities.

Exploring the Kinh Vietnamese genomic database for the polymorphisms of the P450 genes towards precision public health

Background Human cytochrome P450 (CYPs) genes are essential in metabolising drugs. Due to their high polymorphism, population-specific studies are of great interest. Aim This research examined the six CYP genes, including CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, and CYP4F2 in the Kinh Vietnamese (KHV) for population-scale precision medicine. Subjects and methods We processed data from a genomics database of 206 healthy and unrelated KHV individuals to calculate CYP allele frequencies. First, we compared the CYP genes of the KHV to six other populations retrieved from the 1000 Genomes Project. Second, we searched the PharmGBK database for drug-CYP interaction data to compile a drug dosage recommendation for the KHV. Results We observed the diverging trends in genetic variations of CYP2B6, CYP2D6, and CYP3A5 in the KHV. Regarding phenotypic drug responses in the KHV, CYP2C19 exhibited all metabolic phenotypes at a non-trivial frequency. In addition, CYP3A5 metabolised drugs at a lower rate compared to the other five CYPs. Conclusion This is the first large-scale study to investigate multiple CYP genes in the KHV for precision medicine from a public health perspective. Differences found in the distributions of metabolizers for the KHV suggest careful prescriptions for CYP2C19 and CYP3A5-metabolised drugs.

Pharmacogenetics of the cytochromes P450: Selected pharmacological and toxicological aspects.

With the availability of detailed genomic data on all 57 human cytochrome P450 genes, it is clear that there is substantial variability in gene product activity with functionally significant polymorphisms reported across almost all isoforms. This article is concerned mainly with 13 P450 isoforms of particular relevance to xenobiotic metabolism. After brief review of the extent of polymorphism in each, the relevance of selected P450 isoforms to both adverse drug reaction and disease susceptibility is considered in detail. Bleeding due to warfarin and other coumarin anticoagulants is considered as an example of a type A reaction with idiosyncratic adverse drug reactions affecting the liver and skin as type B. It is clear that CYP2C9 variants contribute significantly to warfarin dose requirement and also risk of bleeding, with a minor contribution from CYP4F2. In the case of idiosyncratic adverse drug reactions, CYP2B6 variants appear relevant to both liver and skin reactions to several drugs with CYP2C9 variants also relevant to phenytoin-related skin rash. The relevance of P450 genotype to disease susceptibility is also considered but detailed genetic studies now suggest that CYP2A6 is the only P450 relevant to risk of lung cancer with alleles associated with low or absent activity clearly protective against disease. Other cytochrome P450 genotypes are generally not predictors for risk of cancer or other complex disease development.

Pharmacogenetic testing in psychiatry and neurology: an overview of reviews.

Pharmacogenetic testing is available to healthcare professionals to guide drug selection and prevent adverse events. However, its implementation in the clinical practice of psychiatry/neurology still has barriers, mainly due to a lack of evidence. We conducted a literature search on Cochrane Library, Embase and Pubmed, from their inception to 18 June 2020. We included 16 published systematic reviews. The most studied drug categories were anticonvulsants and selective serotonin reuptake inhibitors associated with human leukocyte antigen and cytochrome P450 genes (HLA-A, HLA-B, CYP2C9, CYP2D6, CYP2C19), classified as critically lowquality/low quality. There is a need for more robust studies with adequate design to assess the potential benefits of adopting pharmacogenetics in health systems and services.

Biotransformation, Using Recombinant CYP450-Expressing Baker's Yeast Cells, Identifies a Novel CYP2D6.10A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme.

CYP2D6, a cytochrome P450 (CYP) enzyme, metabolizes codeine to morphine. Within the human body, 0–15% of codeine undergoes O-demethylation by CYP2D6 to form morphine, a far stronger analgesic than codeine. Genetic polymorphisms in wild-type CYP2D6 (CYP2D6-wt) are known to cause poor-to-extensive metabolism of codeine and other CYP2D6 substrates. We have established a platform technology that allows stable expression of human CYP genes from chromosomal loci of baker’s yeast cells. Four CYP2D6 alleles, (i) chemically synthesized CYP2D6.1, (ii) chemically synthesized CYP2D6-wt, (iii) chemically synthesized CYP2D6.10, and (iv) a novel CYP2D6.10 variant CYP2D6-C (i.e., CYP2D6.10A122V) isolated from a liver cDNA library, were cloned for chromosomal integration in yeast cells. When expressed in yeast, CYP2D6.10 enzyme shows weak activity compared with CYP2D6-wt and CYP2D6.1 which have moderate activity, as reported earlier. Surprisingly, however, the CYP2D6-C enzyme is far more active than CYP2D6.10. More surprisingly, although CYP2D6.10 is a known low metabolizer of codeine, yeast cells expressing CYP2D6-C transform >70% of codeine to morphine, which is more than twice that of cells expressing the extensive metabolizers, CYP2D6.1, and CYP2D6-wt. The latter two enzymes predominantly catalyze formation of codeine’s N-demethylation product, norcodeine, with >55% yield. Molecular modeling studies explain the specificity of CYP2D6-C for O-demethylation, validating observed experimental results. The yeast-based CYP2D6 expression systems, described here, could find generic use in CYP2D6-mediated drug metabolism and also in high-yield chemical reactions that allow the formation of regio-specific dealkylation products.

Association of CYP2B6 (G15631T) polymorphism with Clopidogrel resistance and genetic predisposition to Acute Coronary Syndromes (ACSs) in Morocco

Despite its well-known efficacy as an antiplatelet agent, inter-patient variability of response to Clopidogrel has been documented among patients from different ethnic origins. As polymorphisms in drug metabolizing enzymes contribute to wide heterogeneity in drug pharmacokinetics, response and toxicity, we have tried to investigate the potential association of CYP2B6 (G15631T) polymorphism as one of the less well studied human cytochrome P450 genes, with inter-patient variability of response to Clopidogrel, and to examine its effect on genetic susceptibility to the disease, among a sample of ACS Moroccan patients compared to healthy controls.We found a higher frequency of the mutant allele among resistant patients compared to non-resistant ones (70% and 56% respectively) (P = 0.04). The allelic and genotypic distribution of the polymorphism showed no big difference between ACS cases and healthy controls (P > 0.05).Genetic profiling before adoption of a therapeutic strategy appears to be very useful to identify patients at increased risk for developing cardiovascular events, and may help to choose the most reliable antiplatelet strategy for each patient. The current findings might be preliminary but promising in such field not yet well developed, especially in Moroccan population.

Diversity and distribution of CYP gene family in Bactrian camel

Cytochrome P450 (CYP) enzymes belong to a superfamily of monooxygenases which are phase I enzymes responsible for the first pass metabolism of about 90% of drugs in animals. However, these enzymes are often polymorphic and metabolism of the same drug in different species or different individuals is influenced by genetic and non-genetic factors. Bactrian camels are capable of survival in harsh living environments, being able to consume diets that are often toxic to other mammals and can tolerate extreme water and food deprivation. The aim of this study was to investigate whether the Bactrian camel’s special metabolic pathways and unique detoxification capabilities are attributable to particularities of the CYP gene family. The Bactrian camel’s whole genome sequencing data were systemically analyzed and annotated, and then, CYP gene family was searched from the whole protein database and compared with CYP gene families of cattle, horse, chicken, and human. The total of 63 CYP gene copies were found in Bactrian camel’s whole genome and were classified into 17 families and 38 subfamilies. Among them, 9 multi-gene families were found, and CYP2, CYP3, and CPY4 have 27, 6, and 7 subfamilies, accounting for 43, 10, and 11% in camel CYP gene, respectively. In comparison with cattle, chicken, horse, and human, the distribution of CYP gene subfamilies in camel is different, with more CYP2J and CYP3A copies in the Bactrian camel, which may contribute to the Bactrian camel’s specific biological characteristics and metabolic pathways. Comparing to the cow, horse, chicken, and human CYP genes, the distribution of CYP gene subfamilies is distinct in the Bactrian camel. The higher copy number of CYP2J gene and CYP3A gene in Bactrian camel may be the important factors contributing to the distinct biological characteristics and metabolic pathways of Bactrian camels for adaptation to the harsh environments.

Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery.

The human cytochrome P450 (CYP) superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA (“Orthologous MAtrix”) Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

Genetic variation in the human cytochrome P450 supergene family.

Single nucleotide variations (SNVs) in the cytochrome P450 (CYP) gene family are a primary cause of interindividual differences in therapeutic effects and adverse reactions to drugs. However, we still lack important information on the extent of CYP polymorphisms at the population level. Here, we developed a comprehensive data set of SNVs in all 57 human CYP genes by integrating data from two extensive population sequencing projects and analyzed the distribution of SNVs in different subpopulations. CYP genetic variants derived from the NHLBI and 1000 Genomes project were classified by variant type, frequency, and ethnic origins. The genetic variability of CYP genes was normalized on the basis of nonlinear regression and the total number of genetic variations was estimated by the derived formulas. In total, we detected 6165 SNVs, of which many were novel. The vast majority (83.2%) of all SNVs in coding regions were very rare (minor allele frequency <0.1%). On the basis of the regression analysis, the total number of genetic variations in human CYP genes was calculated to be 3.4 × 10 and 4.8 × 10 for a population size of one million in Europeans and Africans, respectively. Our results suggest that the variant spectrum of human CYP genes is extensive and only a fraction of SNVs has been characterized to date. Moreover, the multitude of very rare novel sequence variants indicates that the commonly used SNV platforms are not satisfactory for determining the true genotype, which is critical information for personalized treatment with drugs influenced by CYP polymorphisms.

The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish.

A clinical and molecular genetics study of primary congenital glaucoma in South Korea

ObjectivesTo investigate the clinical manifestations associated with the mutation spectrums of the human cytochrome P450 (CYP1B1) and myocilin (MYOC) genes in South Korean patients with primary congenital glaucoma (PCG).MethodsEighty-five unrelated...

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

BackgroundYarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase.ResultsFor the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system.Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains.ConclusionsAlkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.

Use of transgenic animals in understanding molecular mechanisms of toxicity.

Understanding molecular mechanisms of chemical toxicity and the potential risks of drugs to man is a pivotal part of the drug development process. With the dramatic increase in the number of new chemical entities arising from high throughput screening, there is an urgent need to develop systems for the rapid evaluation of potential drugs so that those agents which are most likely to be free of adverse effects can be identified at the earliest possible stage in drug development. The complex mechanisms of action of chemical toxins has made it extremely difficult to evaluate the precise toxic mechanism and also the relative role of specific genes in either potentiating or ameliorating the toxic effect. This problem can be addressed by the application of genetic strategies. Such strategies can exploit strain differences in susceptibility to specific toxic agents or, with the rapidly developing technologies, can exploit the use of transgenic animals where specific genes can be manipulated and subsequent effects on chemical toxicity evaluated. Transgenic animals can be exploited in a variety of ways to understand mechanisms of chemical toxicity. For example, a human gene encoding a drug metabolizing enzyme can be directly introduced and the effects on toxic response evaluated. Alternatively, specific genes can be deleted from the mouse genome and the consequences on toxicological response determined. Many toxic chemical agents modulate patterns of gene expression within target cells. This can be used to screen for responses to different types of toxic insult. In such experiments the promotor of a stress-regulated gene can be ligated to a suitable reporter gene, such as lacZ, or green fluorescent protein, and inserted into the genome of an appropriate test species. On administration of a chemical agent, cells which are sensitive to the toxic effects of that chemical will express the reporter, which can then be identified using an appropriate assay system. This latter strategy provides the potential for screening a large number of compounds rapidly for their potential toxic effects and also provides information on tissue and cellular specificity. Experiments using transgenic animals can be complex, and care must be taken to ensure that the results are not affected by background activities within the species being used. For example, the introduction of a specific human cytochrome P450 gene may have no effect on the metabolic disposition of a drug or toxin because of the background activity within the mouse. As the toxicity of a chemical agent is determined by a wide range of different factors including drug uptake, metabolism, detoxification and repair, differences between man and the species being used could potentially generate a toxic response in the animal model whereas no toxicity may be observed in man. In spite of these confounding factors, the application of transgenic animals to toxicological issues has enormous potential for speeding up the drug discovery process and will undoubtedly become part of this process in the future.

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

BackgroundIncreasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development.ResultsZebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters.ConclusionsTranscript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

Identification of a Transcriptional Regulatory Factor for Human Aromatase Cytochrome P450 Gene Expression as Nuclear Factor Interleukin-6 (NF-IL6), a Member of the CCAAT/Enhancer-Binding Protein Family

Human aromatase cytochrome P450 catalyzes the ultimate reaction in the estrogen biosynthetic pathway by coupling with another enzyme, NADPH-cytochrome P450 reductase, in the endoplasmic reticulum. The expression of the gene encoding the enzyme (CYP19) is regulated, in part, by tissue-specific promoters through the use of alternative-splicing mechanisms. Recently, we have localized a transcriptional activating element at positions –2141 to –2115 relative to the major cap site of the gene, by transient expression analyses in human BeWo choriocarcinoma cells using the bacterial chloramphenicol acetytransferase reporter gene ligated with CYP19 promoter sequences which regulate expression in this tissue. Here, we report the isolation of a cDNA encoding a DNA-binding protein which binds specifically to the regulatory element. The deduced amino-acid sequence of the insert is identical to that corresponding to the DNA-binding domain and the dimerization domain of a transcription factor, nuclear factor interleukin-6 (NF-IL6), a member of the CCAAT/enhancer-binding protein (C/EBP) family. Studies using specific antibodies against members of the C/EBP family demonstrate that NF-IL6 is the major nuclear factor binding to the regulatory element in BeWo cells; nevertheless, C/EBP α also seems to be involved. Disruption of the NF-IL6-binding site within the regulatory element resulted in the disappearance of the transcriptional enhancing activity of the element, indicating that NF-IL6 is at least one of the nuclear factor(s) which enhances transcription through binding to the cis-acting element. These results indicate the intrinsic importance of NF-IL6 in the transcriptional regulation of CYP19 expression.

Applications and Limitations of Genetically Modified Mouse Models in Drug Discovery and Development

Genetically modified mouse models in which a specific gene is removed or replaced have proven to be powerful tools for identification/validation of target gene and scientific understanding of molecular mechanisms underlying drug-induced toxicity through mechanistic studies. In spite of the advantage, there are significant limitations of genetically modified mouse models. Modification of a given gene does not always result in the anticipated phenotype. In some instances, phenotypes of targeted mouse mutants were not those predicted from the presumed function of the given genes, while other null mutants revealed no apparent defects. Furthermore, the phenotypic outcome can be influenced by many environmental and genetic factors. Therefore, interpretation of the significance of the findings from studies using genetically modified mouse models is not always as straightforward as one would expect, especially when desire is to extrapolate the findings to humans. Interestingly, many humanized mouse models have been generated for evaluating the function and regulation of cytochrome P450 (CYP) enzymes. Our fascination with humanized animals dates back to ancients. For example, the Great Sphinx of Giza, a large half-human and half-lion statue, is believed to have been built by Egyptians about 4500 years ago. Although the creation of humanized animals that carry a particular human CYP gene provides useful tools for scientific understanding of the function and regulation of the CYP enzyme, these humanized mouse models are not so useful in prediction of human pharmacokinetics in a quantitative sense. Accordingly, it is important to keep in mind that an animal engineered to express a human gene and its protein is still an animal.

Revealing the paradox of drug reward in human evolution

Neurobiological models of drug abuse propose that drug use is initiated and maintained by rewarding feedback mechanisms. However, the most commonly used drugs are plant neurotoxins that evolved to punish, not reward, consumption by animal herbivores. Reward models therefore implicitly assume an evolutionary mismatch between recent drug-profligate environments and a relatively drug-free past in which a reward centre, incidentally vulnerable to neurotoxins, could evolve. By contrast, emerging insights from plant evolutionary ecology and the genetics of hepatic enzymes, particularly cytochrome P450, indicate that animal and hominid taxa have been exposed to plant toxins throughout their evolution. Specifically, evidence of conserved function, stabilizing selection, and population-specific selection of human cytochrome P450 genes indicate recent evolutionary exposure to plant toxins, including those that affect animal nervous systems. Thus, the human propensity to seek out and consume plant neurotoxins is a paradox with far-reaching implications for current drug-reward theory. We sketch some potential resolutions of the paradox, including the possibility that humans may have evolved to counter-exploit plant neurotoxins. Resolving the paradox of drug reward will require a synthesis of ecological and neurobiological perspectives of drug seeking and use.

Direct Assessment of Promoter Activity of Human Cytochrome P450 Genes Using Optimized Transfection in Vitro and in Vivo

Although transcription regulation of human cytochrome P450 enzymes (CYP) is known to play an important role in drug metabolism and homeostasis, factors influencing the expression of various CYP genes in humans remain largely undefined. We used three cell lines and CD-1 mice to assess the activity of genomic promoter sequences of human CYP2D6, 1A2, 3A4, 2C9, 2C18, and 2E1 genes. CYP promoter sequences were amplified by PCR using human liver genomic DNA as the template and cloned into pGL3-Basic vectors that contain a luciferase reporter gene but lack promoter or enhancer sequences. Each plasmid construct was transfected into cells in vitro using polyethylenimine (PEI) as the transfection reagent and into mice using the recently developed hydrodynamics-based procedure. Relative promoter strength was determined by the level of luciferase expression in transfected cells. All six human CYP promoters are active in driving reporter gene expression in cultured hepatic HepG2 cells and non-hepatic cells such as human embryonic kidney fibroblasts (293 cells) and murine melanoma cells (BL-16) as well as cells in intact mouse liver, lung, heart, kidney and spleen. The order of strength among CYP promoters examined was found to be 2D6 > 1A2 > 3A4 > 2C9 > 2C18 > 2E1.

Phytoremediation of the Herbicides Atrazine and Metolachlor by Transgenic Rice Plants Expressing Human CYP1A1, CYP2B6, and CYP2C19

This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.