Human cytochrome P450 (CYP) genes: recommendations for the nomenclature of alleles.
Explore the source record for details and available documents.
Alleles↗
Biomedical subjects
D W Nebert
Publications and source records attributed to D W Nebert.
Refining the mouse chromosomal location of Cdm, the major gene associated with susceptibility to cadmium-induced testicular necrosis.
Cadmium (Cd++) is a widespread environmental pollutant and classifed as an IARC 'Category I' human carcinogen. Cd++ can also cause severe renal toxicity and may be involved clinically in cardiovascular disease and osteoporosis. Genetic differences in sensitivity to cadmium toxicity have been noted in humans, whereas, among inbred mouse strains, unequivocal genetic data exist. Resistance to cadmium-induced testicular damage was reported in 1973 to be associated with a single major recessive gene, named Cdm, which has now been localized to mouse chromosome (Chr) 3. Using polymorphic microsatellite markers and semiquantitative histological parameters, we have corroborated the original 1973 data concerning mendelian inheritance and have further refined the region containing the Cdm gene from more than 24 cM to 0.64 cM (estimated 40-80 genes). We phenotyped 26 recombinant inbred lines generated from C57BL/6J (B6, resistant) and DBA/2J (D2, sensitive) inbred mice, and determined that the Cdm gene maps between microsatellite markers D3Mit110 and D3Mit255. Although toxicity to numerous heavy metals is well known, virtually no molecular mechanisms have yet been uncovered either in humans or laboratory animals. Identification and characterization of the mouse Cdm gene should enhance our understanding of heavy metal toxicity by identifying and characterizing, for the first time, a major mammalian gene responsible for susceptibility to diseases caused by heavy metal toxicity.
Animals↗
Suggestions for the nomenclature of human alleles: relevance to ecogenetics, pharmacogenetics and molecular epidemiology.
The current number of 9422 symbols for human gene names (http://www.gene.ucl.ac.uk/nomenclature/) is expected to increase 7- to 15-fold over the next 2 years. In and around each gene, a tremendous degree of single-nucleotide polymorphism (SNP) heterogeneity is now realized to exist. This review is intended to be visionary, to point out some of the enormously complex nomenclature issues that we face, and to offer some reasonable solutions to these issues. For example, I believe that a 'gene' should be defined as that region from the furthest 5'-ward enhancer to at least 150 bases downstream of the last exon. Just as established rules are critically important for the systematic naming of all new genes, standardized nomenclature rules for the naming of allelic variants are also desperately needed. The evolving consensus for naming the alleles of all human genes (ideally based on evolutionarily diverging haplotype patterns) is described herein. Because of the anticipated explosion in finding new genes and allelic variants due to high-throughput resequencing and DNA-chip technologies, this excess of new knowledge will undoubtedly overwhelm their publication by scientific journals alone. I suggest that the best approach to this staggering 'information overload' is to place the data on appropriate web sites--with numerous links between sites, and frequent updates of all information--so that colleagues in all fields of medical and genetic research can remain knowledgeable. Examples of successful web sites to date include those for the cytochrome P450 (CYP) genes and human CYP alleles, UDP glycosyltransferase (UGT) genes and human alleles, human N-acetylaminotransferase (NAT2, NAT1) alleles, and aldehyde dehydrogenase (ALDH) genes and human alleles. Many more web sites will be necessary. For each site, the webmaster will need to be responsible, accurate, energetic, highly organized, and keen to keep the site current. I believe that interactive discussions on these sites should be encouraged, and advisory committees must be willing to check frequently to ensure that all new information is accurate. Lastly, for the field of molecular epidemiology, the importance of correlating an informative genotype with an unequivocal phenotype is emphasized, and the emerging realization that racial and ethnic groups are highly admixed is summarized and updated.
Alleles↗
Transgenic zebrafish as sentinels for aquatic pollution.
Using the golden mutant zebrafish having a decrease in interfering pigmentation, we are developing transgenic lines in which DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed. We have begun with three response elements that recognize three important classes of foreign chemicals. Aromatic hydrocarbon response elements (AHREs) respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. Soon, we will include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the effects of retinoids in the environment. Each of these substances is known to be bioconcentrated in fish to varying degrees; for example, 10(-17) M TCDD in a body of water becomes concentrated to approximately 10(-12) M TCDD in a fish, where it would act upon the AHRE motif and turn on the luciferase (LUC) reporter gene. The living fish as a sentinel will not only be assayed intact in the luminometer, but--upon several days or weeks of depuration--would be usable again. To date, we have established that zebrafish transcription factors are able to recognize both mammalian and trout AHRE, EPRE, and MRE sequences in a dose-dependent and chemical-class-specific manner, and that expression of both the LUC and jellyfish green fluorescent protein (GFP) reporter genes is easily detected in zebrafish cell cultures and in the intact live zebrafish. Variations in sensitivity of this model system can be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core consensus response element and flanking regions. This transgenic technology should allow for a simple, exquisitely sensitive, and inexpensive assay for monitoring aquatic pollution. We have already initiated studies using sentinel zebrafish to monitor a public drinking water source.
Animals↗
"Gene-swap knock-in" cassette in mice to study allelic differences in human genes.
Genetic differences in environmental toxicity and cancer susceptibility among individuals in a human population often reflect polymorphisms in the genes encoding drug-metabolizing enzymes (DMEs), drug transporters, and receptors that control DME levels. This field of study is called "ecogenetics", and a subset of this field--concerning genetic variability in response to drugs--is termed "pharmacogenetics". Although human-mouse differences might be 3- to perhaps 10-fold, human interindividual differences can be as great as 20-fold or more than 40-fold. It would be helpful, therefore, to study toxicokinetics/pharmacokinetics of particular environmental agents and drugs in mice containing these "high-" and "low-extreme" human alleles. We hope to use transgenic "knock-in" technology in order to insert human alleles in place of the orthologous mouse gene. However, the knock-in of each gene has normally been a separate event requiring the following: (a) construction of the targeting vector, (b) transfection into embryonic stem (ES) cells, (c) generation of a targeted mouse having germline transmission of the construct, and (d) backcross breeding of the knock-in mouse (at least 6-8 times) to produce a suitable genetically homogeneous background (i.e., to decrease "experimental noise"). These experiments require 1 1/2 to 2 years to complete, making this very powerful technology inefficient for routine applications. If, on the other hand, the initial knock-in targeting vector might include sequences that would allow the knocked-in gene to be exchanged (quickly and repeatedly) for one new allele after another, then testing distinctly different human polymorphic alleles in transgenic mice could be accomplished in a few months instead of several years. This "gene-swapping" technique will soon be done by zygotic injection of a "human allele cassette" into the sperm or fertilized ovum of the parental knock-in mouse inbred strain or by the cloning of whole mice from cumulus ovaricus cells or tail-snip fibroblasts containing the nucleus wherein each new human allele has already been "swapped." In mouse cells in culture using heterotypic lox sites, we and others have already succeeded in gene swapping, by exchanging one gene, including its regulatory regions, with a second gene (including its regulatory regions). It is anticipated that mouse lines carrying numerous human alleles will become commonplace early in the next millennium.
Alleles↗
The evolution of drug metabolism.
So-called 'drug-metabolizing enzyme' (DME) genes have existed on this planet for more than 2.5 billion years and would be more appropriately named 'effector-metabolizing enzymes'. Genes encoding DMEs have functioned in many fundamental processes in prokaryotes and, more recently, in countless critical life processes in plants and animals. DME genes exist in every eukaryotic cell and in most, if not all, prokaryotes. Over the past decade, it has become clear that each person has their own 'individual fingerprint' of unique alleles coding for DMEs. The underlying genetic predisposition of each patient reflects combinations of poor- and extensive-metabolizer phenotypes. If these enzymes cooperate in the same metabolic pathway for any given drug or environmental agent, such ecogenetic variability might be synergistic and could cause 30- to > 40-fold differences in activation or degradation. The end result can be large interindividual differences in risk of environmentally caused toxicity or cancer. Human DME gene polymorphisms often show high frequencies of variant alleles. Many factors contribute to persistence of these high frequencies, including a combination of selective pressures involving diet, climate and geography, as well as 'balanced polymorphisms' ('shared benefit' for the heterozygote). However, the extensive heterogeneity in the human genome currently being discovered suggests many more polymorphisms will occur not only in drug metabolism genes, but in all genes, and exhibiting large gene-by-gene variability.
Animals↗
Drug-metabolizing enzymes, polymorphisms and interindividual response to environmental toxicants.
Individual risk of toxicity or cancer reflects the amount of exposure to environmental agents, combined with one's underlying genetic predisposition. More than six dozen human ecogenetic polymorphisms have been described; whereas some of these have been demonstrated to be associated with altered risks of toxicity or cancer, others presently remain equivocal and require further study. Thus, genetic differences in the regulation, expression and activity of "environmental susceptibility genes" can be decisive in defining susceptibility to toxicity or cancer. "Drug-metabolizing enzymes" (DMEs) are regarded as one class of environmental susceptibility genes. DME genes have actually existed on this planet for more than 2.5 billion years, and might more appropriately be named "effector-metabolizing enzymes." Receptors controlling DME levels have been called "DME receptors." DMEs have functioned in many critical life processes in prokaryotes and, more recently, in countless basic functions in plants and animals - events that evolved long before the existence of pharmaceutical companies and apothecaries. DME genes exist in every eukaryotic cell and probably in all prokaryotes. Virtually all environmental agents act as either agonists or antagonists - in competing with endogenous ligands that bind to DME receptors and/or competing as substrates for the DMEs. Over the past decade it has become clear that each of us has our own "individual fingerprint" of unique alleles coding for DMEs. The underlying genetic predisposition of each patient will reflect combinations of poor- and extensive-metabolizer phenotypes; if these enzymes cooperate in the same metabolic pathway for any given drug or environmental agent, such ecogenetic variability might be synergistic and lead to as much as 30- or >40-fold differences in activation or degradation. The end result can be large interindividual differences in risk of environmentally caused toxicity or cancer.
Animals↗
Inhibition of CYP1A1 enzyme activity in mouse hepatoma cell culture by soybean isoflavones.
The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent Ki values of 325 microM and 140 microM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent Ki values of 55 microM and 50 microM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.
Animals↗
Tissue- and cell type-specific expression of cytochrome P450 1A1 and cytochrome P450 1A2 mRNA in the mouse localized in situ hybridization.
We used in situ hybridization to examine organ- and cell type-specific constitutive and 3-methylcholanthrene (3MC)-inducible cytochrome P450 (CYP)1A1 and CYP1A2 mRNA expression in various tissues of the C57BL/6N mouse. In situ hybridization was carried out 10 hr after the mice had received intraperitoneal 3MC, or vehicle alone. We detected levels of 3MC-induced CYP1A1 mRNA in: liver (centrilobular, more so than periportal, regions); lung (Clara Type II cells much more than Type I epithelial cells); brain, especially endothelial cells lining the vascular surface of the choroid plexus; the digestive tract (duodenum > jejunum > ileum > colon > esophagus > stomach--in particular, the villous epithelium, plus cells surrounding glands in the lamina propria); renal corpuscles of the kidney; the ovary (medulla more so than cortex); and the endothelial cells of blood vessels throughout the animal. Constitutive CYP1A1 mRNA was not detectable by in situ hybridization in any of these tissues. In contrast, constitutive CYP1A2 mRNA was measurable in liver, and 3MC-inducible CYP1A2 mRNA was observed only in liver, lung, and duodenum (having cell-type locations similar to those of CYP1A1); the other above-mentioned tissues were negative for CYP1A2 mRNA. These data demonstrate the striking differences in tissue- and cell type-specific expression between the two members of the mouse Cypla subfamily. Because of the ubiquitous nature of 3MC-inducible CYP1A1 throughout the animal rather than just "portals of entry," these results support our hypothesis that CYP1A1, induced by particular endogenous signals in various tissues and cell types, might participate in one or more critical life processes--in addition to its well-established role of metabolism of polycyclic hydrocarbons, certain drugs, and other environmental pollutants.
Animals↗
Pharmacogenetics and pharmacogenomics: why is this relevant to the clinical geneticist?
Adverse drug reactions, due at least in part to interindividual variability in drug response, rank between the 4th and 6th leading causes of death in the USA. The field of 'pharmacogenetics', which is 'the study of variability in drug response due to heredity', should help in reducing drug-caused morbidity and mortality. The recently coined term 'pharmacogenomics' usually refers to 'the field of new drug development based on our rapidly increasing knowledge of all genes in the human genome'. However, the two terms - pharmacogenetics and pharmacogenomics - are often used interchangeably. A classification of more than five dozen pharmacogenetic differences is presented here. Most of these variations occur in drug-metabolizing enzyme (DME) genes, with some presumed to exist in the DME receptor and drug transporter genes, and others have not yet been explained on a molecular basis. A method for unequivocally defining a quantitative phenotype (drug efficacy, toxicity, etc.) is proposed; this is where help from the clinical geneticist can be especially important. Our current appreciation of the degree of variability (including single-nucleotide polymorphisms, SNPs) in the human genome is described, with emphasis on the need to prove that a particular genotype is indeed the cause of a specific phenotype; this topic has been termed 'functional genomics'. Furthermore, the current amount of admixture amongst almost all ethnic groups will obviously make studies of gene-drug interactions more complicated, as will the withholding of ethnic information about DNA samples during any molecular epidemiologic study. DME genes and DME receptor and drug transporter genes can be regarded as 'modifier genes', because they influence disorders as diverse as risk of cancer, bone marrow toxicity resulting from occupational exposure, and Parkinson's disease; for this reason, the clinical geneticist, as well as the medical genetics counselor, should be knowledgeable in the rapidly expanding fields of pharmacogenetics and pharmacogenomics.
Enzymes↗
Genetic epidemiology of environmental toxicity and cancer susceptibility: human allelic polymorphisms in drug-metabolizing enzyme genes, their functional importance, and nomenclature issues.
Pharmacogenetics is the study of idiosyncratic drug responses that have an hereditary basis and usually reflect differences in drug-metabolizing enzymes (DMEs) and the receptors that control DME levels. The purpose of this review is to provide a brief overview of recent findings concerning more than a dozen clinically important polymorphisms and to emphasize the need to standardize the nomenclature of these alleles in each polymorphism, as quickly as possible. This nomenclature system should be consistent with the Human Gene Nomenclature Guidelines. Because DMEs have existed before divergence of prokaryotes and eukaryotes more than 2 billion years ago, it is clear that DME genes first must have evolved for critical life functions and that, more recently in animals, DME genes expanded to include the role of detoxification of dietary products, evolving plant metabolites, and, of course, pharmaceutical drugs. Many human DME polymorphisms are relevant to clinical problems in that they represent the basis of risk factors in the development of cancer, toxicity, and other diseases associated with drug, chemical, or dietary exposure. The study of the relationship among human genetic polymorphisms, cancer susceptibility, toxicity, and environmental exposure is a new and exciting area of research--which will undoubtedly have increasingly important implications for risk assessment and the prevention, early diagnosis, and intervention of clinical disease.
Cytochrome P-450 Enzyme System↗
Genetic differences in spatial learning between Dark Agouti and Sprague-Dawley strains: possible correlation with the CYP2D2 polymorphism in rats treated neonatally with methamphetamine.
Following neonatal exposure to d-methamphetamine, adult rats have previously been shown to exhibit augmented acoustic startle and spatial learning deficits. d-Methamphetamine is structurally similar to several phenylethylamines that are metabolized by CYP2D6. In humans, allelic differences in the CYP2D6 confer the extensive or poor metabolizer phenotype for the more than three dozen drugs that are members of the CYP2D6-mediated 'debrisoquine/sparteine panel.' An analogous genotype exists with the CYP2D2 gene in rats. Female Dark Agouti rats show the poor metabolizer phenotype, whereas Sprague-Dawley rats show the extensive metabolizer phenotype; male Dark Agouti rats are intermediate. We sought to test the possibility that these strains might exhibit altered d-methamphetamine-induced developmental neurotoxicity. Dark Agouti and Sprague-Dawley litters (11-20 days of age) were given d-methamphetamine or vehicle alone subcutaneously twice daily (15 mg/kg). Offspring were assessed as adults (beginning at 50 days of age) on acoustic startle, straight-channel swimming, and spatial learning and memory in a Morris hidden platform maze. Increases in d-methamphetamine-induced acoustic startle were found in both male and female Dark Agouti rats, but not Sprague-Dawley rats. In the Morris maze, d-methamphetamine-induced spatial navigation deficits were found in both strains among males, suggesting some mechanism other than the CYP2D2 polymorphism. In contrast, among females only the d-methamphetamine-treated Dark Agouti rats showed deficits in spatial navigation. The maze deficits in Dark Agouti females, and enhanced acoustic startle in Dark Agouti females and males, support the hypothesis that the CYP2D2 poor metabolizer phenotype confers increased vulnerability to d-methamphetamine-induced developmental neurotoxicity, indicating that the parent drug rather than a CYP2D2-mediated metabolite is responsible for this behavioural defect--which occurs in adults who had been exposed to d-methamphetamine during the neonatal period.
Animals↗
Mouse cytosolic class 3 aldehyde dehydrogenase (Aldh3a1): gene structure and regulation of constitutive and dioxin-inducible expression.
The mouse cytosolic aldehyde dehydrogenase ALDH3A1 (encoded by the Aldh3a1 gene) has previously been shown in cell culture to be markedly inducible by 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), downregulated by the metabolism of functional CYP1A1/1A2 enzymes, and upregulated by a gene on Chr 7 that leads to endogenous oxidative stress. In order to study the regulation of Aldh3a1 gene expression, we isolated two overlapping genomic sequences from a B6/CBA mouse genomic library that included the entire Aldh3a1 gene, along with considerable 5' and 3' flanking sequences. The Aldh3a1 gene was shown to span approximately 10 kb and comprise 11 exons including a noncoding first exon. The sequence of 3.18 kb upstream of exon 1 reveals numerous consensus transcription factor-binding sites, some of which were shown to be important in the positive and negative control of Aldh3a1 gene expression; these include seven aromatic hydrocarbon response elements (AHREs), an electrophile response element (EPRE), and AP-1, C/EBP beta, c/EBP alpha, NF-kappaB, Sp1, and NF-1 putative binding sites. Deletion fusion constructs containing regions of the Aldh3a1 gene 5' flanking sequence, ligated to chloramphenicol experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor. acetyltransferase (CAT) or luciferase (LUC) reporter genes, were studied. Transient transfection experiments suggested that the 5' flanking region of the gene contains a strong promoter, at least four functional AHREs appear to act cooperatively in causing dioxin-mediated upregulation, and a putative negative regulatory element (NRE) controls basal gene expression independent of dioxin inducibility. The dioxin-mediated upregulation of Aldh3a1 expression in mouse hepatoma Hepa-1c1c7 cell cultures was shown to depend exclusively on the aromatic hydrocarbon receptor.
Aldehyde Dehydrogenase↗
Eukaryotic aldehyde dehydrogenase (ALDH) genes: human polymorphisms, and recommended nomenclature based on divergent evolution and chromosomal mapping.
As currently being performed with an increasing number of superfamilies, a standardized gene nomenclature system is proposed here, based on divergent evolution, using multiple alignment analysis of all 86 eukaryotic aldehyde dehydrogenase (ALDH) amino-acid sequences known at this time. The ALDHs represent a superfamily of NAD(P)(+)-dependent enzymes having similar primary structures that oxidize a wide spectrum of endogenous and exogenous aliphatic and aromatic aldehydes. To date, a total of 54 animal, 15 plant, 14 yeast, and three fungal ALDH genes or cDNAs have been sequenced. These ALDHs can be divided into a total of 18 families (comprising 37 subfamilies), and all nonhuman ALDH genes are named here after the established human ALDH genes, when possible. An ALDH protein from one gene family is defined as having approximately < or = 40% amino-acid identity to that from another family. Two members of the same subfamily exhibit approximately > or = 60% amino-acid identity and are expected to be located at the same subchromosomal site. For naming each gene, it is proposed that the root symbol 'ALDH' denoting 'aldehyde dehydrogenase' be followed by an Arabic number representing the family and, when needed, a letter designating the subfamily and an Arabic number denoting the individual gene within the subfamily; all letters are capitalized in all mammals except mouse and fruit fly, e.g. 'human ALDH3A1 (mouse, Drosophila Aldh3a1).' It is suggested that the Human Gene Nomenclature Guidelines (http://++www.gene.ucl.ac.uk/nomenclature/guidelines.h tml) be used for all species other than mouse and Drosophila. Following these guidelines, the gene is italicized, whereas the corresponding cDNA, mRNA, protein or enzyme activity is written with upper-case letters and without italics, e.g. 'human, mouse or Drosophila ALDH3A1 cDNA, mRNA, or activity'. If an orthologous gene between species cannot be identified with certainty, sequential naming of these genes will be carried out in chronological order as they are reported to us. In addition, 20 human ALDH variant alleles that have been reported to date are listed herein and are recommended to be given numbers (or a number plus a capital letter) following an asterisk (e.g. 'ALDH3A2*2, ALDH2*4C'). It is anticipated that this eukaryotic ALDH gene nomenclature system will be extended to include bacterial genes within the next 2 years and that this nomenclature system will require updating on a regular basis; an ALDH Web site has been established for this purpose (http://++www.uchsc.edu/sp./sp./alcdbase/a ldhcov.html) and will serve as a medium for interaction amongst colleagues in this field.
Aldehyde Dehydrogenase↗
Dioxin causes a sustained oxidative stress response in the mouse.
Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) is the prototype for environmental agonists of the aromatic hydrocarbon receptor (AHR) that are known to produce multiple adverse effects in laboratory animals as well as humans. Although not directly genotoxic, dioxin is known to increase transformation and mutations in mammalian cell culture and to cause an exaggerated oxidative stress response in the female rat. In humans and mice, however, dioxin-mediated oxidative stress appears to be more subtle, causing a response that has been poorly characterized. Using the female C57BL/6J inbred mouse, we show here that intraperitoneal treatment of 5 micrograms TCDD per kilogram on 3 consecutive days produces a striking, prolonged oxidative stress response: hepatic oxidized glutathione levels increase 2-fold within 1 week, and these effects persist for at least 8 weeks despite no further dioxin treatment. Urinary levels of 8-hydroxydeoxyguanosine--a product of DNA base oxidation and subsequent excision repair--remain elevated about 20-fold at 8 weeks after dioxin treatment, consistent with chronic and potentially promutagenic DNA base damage. These results demonstrate that dioxin exposure does produce a sustained oxidative stress response in the mouse.
8-Hydroxy-2'-Deoxyguanosine↗
Markedly increased constitutive CYP1A1 mRNA levels in the fertilized ovum of the mouse.
Using a highly sensitive RT-PCR technique that measures mRNA (cDNA)-to-DNA ratios, we are able to detect constitutive CYP1A1 mRNA in adult mouse liver as well as in the oocyte. Twelve hours after fertilization of the ovum, there is a more than 100-fold increase in constitutive CYP1A1 mRNA levels; this dramatic increase completely disappears by the 2-cell stage at gestational day 1.5 (GD1.5), as well as in the blastocyst at GD3.5. The CYP1A1 enzyme has been shown to remove an endogenous ligand for the Ah receptor (AHR), and the AHR is known to play a role in cell cycle regulation and apoptosis. We therefore postulate that this striking abundance of constitutive CYP1A1 mRNA in the fertilized ovum at GD0.5 might be important for maintaining sufficient amounts of the CYP1A1 enzyme during the transition from maternal to zygotic control (GD0.5 to GD1.5 embryo); availability of catalytically active CYP1A1 would ensure that any (exogenous or endogenous) AHR ligand be rapidly degraded, so that all undesirable AHR-mediated gene transcription would be prevented during these very critical moments of early mammalian embryogenesis.
Animals↗
Role of CYP2A5 and 2G1 in acetaminophen metabolism and toxicity in the olfactory mucosa of the Cyp1a2(-/-) mouse.
Acetaminophen (AP) is a widely-used analgesic agent that has been linked to human liver and kidney disease with prolonged or high-dose usage. In rodents, the target organs that are affected include liver, kidney, and the olfactory mucosa. AP toxicity requires cytochrome P450(CYP)-mediated metabolic activation, and the isozymes CYP1A2, 2E1, and 3A are known to activate AP in the human. In the present study, we determined that olfactory mucosal toxicity of AP was not different between the Cyp1a2(+/+) wild-type and the Cyp1a2(-/-) knockout mouse, whereas the hepatic toxicity of AP was significantly diminished in Cyp1a2(-/-) mice. Western blots of olfactory mucosa revealed that CYP2E1 and CYP3A levels are similar between untreated Cyp1a2(+/+) and Cyp1a2(-/-) mice. Diallyl sulfide (DAS), a known inhibitor of CYP2E1 and of CYP2A10/2A11 (the rabbit orthologue of mouse CYP2A5), completely eliminated olfactory toxicity of AP in both the Cyp1a2(-/-) and wild-type mouse olfactory mucosa. We found that heterologously expressed mouse CYP2A5 and CYP2G1 enzymes (known to be present in olfactory mucosa) form 3-hydroxyacetaminophen (3-OH-AP) and 3-(glutathion-S-yl)acetaminophen (GS-AP); CYP2A5 is considerably more active than 2G1. Addition of GSH caused increases in GS-AP proportional to decreases in 3-OH-AP, suggesting that these two metabolites arise from a common precursor or are formed by way of competing pathways. We also found that both CYP2A5 and CYP2G1 are inhibitable by DAS in vitro. These studies provide strong evidence that, in addition to CYP2E1, CYP2A5 and 2G1 are important in AP bioactivation in the mouse olfactory mucosa and that CYP1A2 appears to be of minor importance for AP olfactory toxicity.
Acetaminophen↗
Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice.
In the present study we have investigated the putative requirement for the cytochrome P-450 isoform CYP1A2 in murine uroporphyria, by comparing Cyp1a2(-/-) knockout mice with Cyp1a2(+/+) wild-type mice. Uroporphyria was produced by injecting animals with iron-dextran and giving the porphyrin precursor 5-aminolaevulinic acid in the drinking water. Some animals also received 3-methylcholanthrene (MC) to induce hepatic CYP1A2. In both protocols, uroporphyria was elicited by these treatments in the Cyp1a2(+/+) wild-type mice, but not in the null mutant mice. Uroporphyrinogen oxidation activity in hepatic microsomes from untreated Cyp1a2(+/+) mice was 2.5-fold higher than in Cyp1a2(-/-) mice. Treatment with MC increased hepatic CYP1A1 in both mouse lines and hepatic CYP1A2 only in the Cyp1a2(+/+) line, as determined by Western immunoblotting. MC increased hepatic ethoxy- and methoxy-resorufin O-dealkylase activities in both mouse lines, but increased uroporphyrinogen oxidation activity in the Cyp1a2(+/+) wild-type mice only. These results indicate the absolute requirement for hepatic CYP1A2 in causing experimental uroporphyria under the conditions used.
Aminolevulinic Acid↗