Biomedical subjects

D W Nebert

Publications and source records attributed to D W Nebert.

At least 37 records · Page 2Linked to original sources

PubMed1998

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↗

PubMed1998

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↗

PubMed1998

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↗

PubMed1998

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↗

PubMed1998

Phospholipase A2 activation and increases in specific prostaglandins in the oxidatively stressed 14CoS/14CoS mouse hepatocyte line.

This laboratory has previously shown that increases in the expression of several genes in SV40-transformed hepatocyte cultures derived from the untreated newborn c14CoS/c14CoS mouse, and in newborn mouse liver--when compared with the cch/cch wild-type--are associated with enhanced levels of reactive oxygenated metabolites (ROMs) and reduced glutathione (GSH). We show here that, in contrast to the ch/ch wild-type levels, the oxidatively stressed 14CoS/14CoS liver cell line displays 2- to 5-fold increases in 1) phospholipase A2 (PLA2) enzyme activity, 2) Ca2+ dependent Group II secreted PLA2 mRNA levels, 3) arachidonic acid release, and 4) arachidonic acid metabolites co-eluting with prostaglandins D2, E2, and F2 alpha. These findings suggest that the cyclooxygenase-2 (COX2) pathway, and possible involvement of the "inflammatory" and/or "acute phase response" signal transduction pathways, might be activated during the endogenous ROM-mediated oxidative stress response in 14CoS/14CoS cells.

Animals↗

PubMed1998

Role of CYP1A2 in the hepatotoxicity of acetaminophen: investigations using Cyp1a2 null mice.

Acetaminophen (APAP) is known to cause centrilobular hepatic necrosis under overdose conditions. This is thought to be mediated via the P450-generated reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). Initially, NAPQI is detoxified by conjugation with glutathione (GSH), but once GSH is depleted, NAPQI reacts more extensively with hepatic proteins leading to hepatocellular damage. The P450 isoforms thought to be responsible for APAP hepatotoxicity in humans are CYP2E1, CYP1A2, and CYP3A4, and thus, we have investigated the effect of murine Cyp1a2 on APAP hepatotoxicity using Cyp1a2 knockout mice (Liang et al., Proc. Natl. Acad. Sci. USA 93, 1671-1676, 1996). Doses of 250 mg/kg were markedly hepatotoxic in these mice, and surprisingly, deaths only occurred in the knock-out and heterozygote mice over a 24-h period after dosing. Furthermore, there were no significant differences among survivors of any genotype in serum ALT concentrations, a well correlated indicator of APAP hepatotoxicity in mice. Finally, no differences were observed in the urinary metabolites excreted ove the 24-h period, including those derived from GSH conjugation of the major reactive metabolite NAPQI. Consistent with the effects on hepatotoxicity and metabolism, 2 h after hepatotoxic doses (500 mg/kg, i.p.) of APAP no significant differences were observed in total whole liver homogenate nonprotein thiol concentrations among the three genotypes even though hepatic thiols were decreased compared to control animals (> 90%). In addition, when the liver cytosol and microsome samples were examined by immunoblotting for the presence of APAP-protein adducts using a specific antiserum, there were no observable differences in either the intensity of staining or in the spectrum of adducts formed between APAP-dosed mice of any genotype. The cumulative data suggest that Cyp1a2 doses not play a significant role in APAP hepatotoxicity in these mice.

Acetaminophen↗

PubMed1998

Regulation of mouse Ah receptor (Ahr) gene basal expression by members of the Sp family of transcription factors.

The aromatic hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of several drug-metabolizing enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxicities. The mouse Ahr gene 5' proximal promoter region, which contains four potential Sp1 motifs, is required for efficient basal expression. Using a fragment spanning the region from nt -174 to +70 of the Ahr promoter, we found that four regions corresponding to four Sp1 sites were protected from DNase I digestion using nuclear extracts from MLE-12 (lung), F9 (embryonal carcinoma), Hepa-1 (hepatoma), and 41-5a (epidermal) cells. The Hepa-1 and F9 cell lines were shown by reverse transcriptase-polymerase chain reaction and Western blot to contain mRNA and protein for Sp1 and Sp3, but not Sp2 and Sp4. In electrophoretic mobility shift assays using oligonucleotide probes corresponding to the four Ahr Sp1 sites, nuclear extracts from Hepa-1 and F9 cells formed complexes that were determined immunologically to contain both Sp1 and Sp3 protein. The two Ahr proximal Sp1 sites (A and B) were shown to bind both Sp1 and Sp3 proteins, whereas the more distal sites (C and D) bound only Sp1. Competition gel shift experiments showed that sites A and B had 10-fold higher affinity for Sp factors than did sites C and D. To determine the transactivation potential of each of the four Ahr Sp1 sites, we fused the Ahr promoter to a luciferase (LUC) reporter gene and transfected the construct into the Drosophila cell line Schneider-2, which contains no Sp1 or Sp1-like factors. Cotransfection of this construct with expression plasmids for each of the Sp factors revealed that Sp3 was approximately 1.6-fold more efficient than Sp1 in Ahr transactivation. Mutation of the four Sp1 sites individually and in combination demonstrated that each site contributes to the overall level of expression of the reporter gene and that interactions between these sites play a minor role in regulation of the Ahr-LUC construct. These results suggest that basal Ahr expression may be regulated by the expression and distribution of Sp1-like factors.

Animals↗

PubMed1998

Cytochrome P450 knockout mice: new toxicological models.

1. Despite the frenetic pace of cytochrome P450 (CYP) research, important questions remain about the possible role of xenobiotic-metabolizing CYP in development and cellular homeostasis. 2. The recent experimental development of CYP null mouse lines has provided valuable new models for addressing these issues, as well as providing novel in vivo models to study the precise role of CYP in chemical-mediated toxicity. 3. The present review summarizes results from the three Cyp gene knockout mouse lines generated so far and looks at likely future directions of this research field. 4. Also discussed are null mouse lines with targeted mutations in genes encoding receptors having primary roles in Cyp gene regulation.

Animals↗

PubMed1998

Aromatic hydrocarbon receptor polymorphism: development of new methods to correlate genotype with phenotype.

Differential CYP1A1 inducibility, reflecting variations in aromatic hydrocarbon receptor (AHR) affinity among inbred mouse strains, is an important determinant of environmental toxicity. We took advantage of the Ahr polymorphism in C57BL/6 and DBA/2 mice to develop an oligonucleotide-hybridization screening approach for the rapid identification of DNA sequence differences between Ahr alleles. Oligonucleotides containing single-base changes at polymorphic sites were immobilized on a solid support and hybridized with C57BL/6 or DBA/2 AHR cDNA radiolabeled probes. The observed hybridization patterns demonstrate that this approach can be used to detect nucleotide differences in the Ahr coding region with very high accuracy. In parallel experiments, we used a yeast two-hybrid system to assess phenotypic differences in AHR function. AHR activation, as measured by beta-galactosidase reporter activity in Saccharomyces cerevisiae strain SFY526, was determined following treatment with varying doses of the AHR ligand beta-naphthoflavone (BNF). We found that the C57BL/6 AHR has about a 15-fold higher affinity for BNF than the DBA/2 AHR, in much better agreement with results reported for whole-animal studies than the values observed by in vitro ligand-binding assays. Using C57BL/6 and DBA/2 AHR chimeric proteins, we also confirmed the previously reported observation that an A375V change is principally responsible for the high- to low-affinity AHR phenotype. There has been no straightforward method to reliably and reproducibly phenotype large numbers of humans for CYP1A1 inducibility or AHR affinity. Screening human AHR cDNAs by oligonucleotide-hybridization and yeast two-hybrid methodologies will be invaluable for the rapid and unequivocal determination of changes in DNA sequence and receptor-ligand affinities associated with human AHR polymorphisms.

Animals↗

PubMed1998

Nonallelism for the audiogenic seizure prone (Asp1) and the aryl hydrocarbon receptor (Ahr) loci in mice.

Previous studies showed an association between the Ahr locus on Chr 12 and a major gene, Asp1, that influences susceptibility to audiogenic seizures (AGS) in mice. Although the association was thought to involve close linkage, a pleiotropic effect of the Ahr locus on AGS susceptibility was not excluded. Two congenic strains, D2.B6N-Asp1b and the D2N.B6N-Ahrb1, were used to evaluate further the association between the Ahr and Asp1 loci. Both strains are genetically identical to the AGS susceptible DBA/2 (D2) strain except for a small amount of C57BL/6N (B6N) genome surrounding the Ahr locus and encompassing the Asp1 locus. The AGS susceptibility of both congenic strains is similar and significantly lower than that of the D2 strain. We found that the Ahr/Asp1 critical region encompasses 5.5-7.0 cM from the proximal microsatellite marker D12Mit153 to the distal marker D12Mitl12. The D2N.B6N-Ahrb1 expresses B6 alleles for all markers within the critical region, whereas the D2.B6N-Asp1b expresses the B6 allele only at the Asp1 locus. Furthermore, we determined that the D2.B6N-Asp1b mouse expresses both the D2 phenotype and genotype at the Ahr locus, i.e., zoxazolamine paralysis and T to C and G to A transition mutations in the Ahr cDNA at bp sites 3330 and 3336, respectively. We therefore conclude that the Ahr and Asp1 loci are nonallelic and that the Ahr gene is excluded as a candidate for Asp1.

Alleles↗

PubMed1998

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes.

The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.

Acetaminophen↗

PubMed1997

Sensitivity of CYP1A1 mRNA inducibility by dioxin is the same in Cyp1a2(+/+) wild-type and Cyp1a2(-/-) null mutant mice.

In mammals, the induction of experimental porphyria by halogenated aromatic hydrocarbons (HAHs) seems to be influenced by the levels of hepatic CYP1A2. The pharmacokinetics and relative rates of uptake and storage of HAHs in the liver are correlated with hepatic CYP1A2 concentrations. It is possible that these rates of HAH uptake and storage might affect the expression of other HAH-inducible genes. The differential inducibility of liver CYP1A1 mRNA by dioxin was therefore compared in Cyp1a2(+/+) wild-type mice, Cyp1a2(+/-) heterozygotes, and Cyp1a2(-/-) homozygous null mutants. Using doses of dioxin over eight orders of magnitude (from 10[-12] to 10[-4] g/kg), we could detect no differences in the sensitivity of CYP1A1 mRNA inducibility. These data indicate that the complete absence of the microsomal CYP1A2 enzyme has no measurable effect on hepatic expression of the Cyp1a1, gene, the only other known member of the mammalian CYP1A cytochrome P450 subfamily.

Animals↗

PubMed1997

Extrahepatic expression of NAD(P)H:menadione oxidoreductase, UDP glucuronosyltransferase-1A6, microsomal aldehyde dehydrogenase, and hepatic nuclear factor-1 alpha mRNAs in ch/ch and 14CoS/14CoS mice.

Oxidative stress-induced gene expression in liver of the untreated newborn c14CoS/c14CoS mouse, as compared with that in the cch/cch wild-type mouse, appears to be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on Chr 7 and absence of the FAH enzyme, which leads to increased levels of endogenous reactive oxygenated metabolites (ROMs) formed in the tyrosine degradative pathway. In these mice almost all studies to date have been carried out in liver. We have examined the extrahepatic expression of four genes. Two genes are members of the [Ah] battery and induced by ROM-mediated oxidative stress: NAD(P)H:menadione oxidoreductase (Nmo1) and UDP glucuronosyltransferase-1A6 (Ugt1a6). The other two genes are decreased in the livers of 14CoS/ 14CoS mice as compared with that in ch/ch mice: microsomal aldehyde dehydrogenase (Ahd3) and hepatocyte-specific nuclear factor-1 alpha HNF-1 alpha (Hnf1 alpha). In liver plus nine extrahepatic tissues of untreated newborn 14CoS/14CoS mutant and ch/ch wild-type mice, we compared NMO1, UGT1A6, AHD3 and HNF-1 alpha mRNA levels. Our results show a wide variation in extrahepatic tissue-specific expression of all four transcripts and indicate that numerous differences exist in the extrahepatic expression of these genes between 14CoS/14CoS and ch/ch mice.

Aldehyde Dehydrogenase↗

PubMed1997

How knockout mouse lines will be used to study the role of drug-metabolizing enzymes and their receptors during reproduction and development, and in environmental toxicity, cancer, and oxidative stress.

The dioxin-inducible mouse [Ah] battery contains at least six genes that "cross-talk" with one another and are believed to play important roles in reproduction and development, and in environmental toxicity, cancer, and oxidative stress. In addition to two P450 genes, Cyp1a1 and Cyp1a2, this laboratory has shown that the four Phase II [Ah] genes include: NAD(P)H:menadione oxidoreductase (Nmo1); a cytosolic "class 3" aldehyde dehydrogenase (Ahd4); a UDP glucuronosyltransferase having 4-methylumbelliferone as substrate (Ugt1a6); and a glutathione transferase having 2,4-dinitro-1-chlorobenzene as substrate (Gsta1, Ya). The Ah receptor-mediated coordinate induction is controlled positively in all six [Ah] battery genes. Oxidative stress up-regulates the four Phase II [Ah] genes. This laboratory is generating conventional, plus inducible, knockout mouse lines having homozygous disruptions in the above-mentioned genes; this novel methodology is described herein. If the conventional knockout is healthy and viable, the mouse line would be useful for studies involving environmental agents. If the conventional knockout is lethal during development, this model would be important for developmental biology, but the inducible (also called conditional) knockout can still be used--at selected ages and even in selected tissue or cell types--for studies designed to understand the mechanisms involved in reproduction and development, and in environmental toxicity, cancer, and oxidative stress.

Animals↗

PubMed1997

Perturbation of the mitosis/apoptosis balance: a fundamental mechanism in toxicology.

Perturbations of the balance between cell gain via mitosis and cell loss by apoptosis play a pivotal role in mediating and modifying the action of carcinogens and other toxicants in tissues such as liver, brain, the immune system, the gastrointestinal tract, and the reproductive organs. Apoptosis describes a highly conserved morphology associated with the death of many different cell types from diverse tissues. This symposium focused on induced changes in this critical balance as a key mechanism of action of a variety of diverse toxicants. In the colon, the "toxicology" of 5 fluorouracil (5FU) is entirely dependent on p53, since p53 knockouts lose the pathology of 5FU damage. Presumably, this is because DNA damage is not detected and there is no cell cycle arrest. In the testes, testicular germ cell survival is mediated by adjacent Sertoli cells via the Fas ligand (FasL)-Fas receptor (Fas) system. This system appears to mediate germ cell apoptosis after exposure to testicular toxicants such as the phthalate, mono(2-ethylhexyl) phthalate (MEHP). Interestingly, MEHP is a member of the peroxisome proliferator (PP) class of nongenotoxic carcinogens. PPs perturb both hepatocyte apoptosis and mitosis. This suppression of apoptosis occurs via activation of the peroxisome proliferator-activated receptor alpha (PPARalpha), providing a paradigm for the regulation of liver growth via activation of nuclear receptors. Similarly, the toxicological effects of dioxins are mediated via the Ah receptor (AHR), another ligand-activated nuclear receptor. This receptor upregulates a variety of genes (the Ah gene battery) associated with the toxicology of dioxins. Taken together, the data presented in this symposium illustrate to the toxicologist the need to quantitate and interpret modulations in apoptosis alongside more conventional assessments of S-phase. Although the toxicant may initiate cell damage, genes like Bcl-2, p53, Fas, PPARalpha, and AHR are final arbiters of the choice between death, survival, and proliferation.

Animals↗

PubMed1997

Human Ah receptor (AHR) gene: localization to 7p15 and suggestive correlation of polymorphism with CYP1A1 inducibility.

The mammalian aromatic hydrocarbon receptor (AHR) is a ubiquitous ligand-activated transcription factor. AHR ligands include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), benzo[a]pyrene, and polychlorinated and polybrominated biphenyls; the endogenous ligand is not yet known. Following ligand binding, the AHR transcriptionally activates genes encoding drug-metabolizing enzymes important in both the metabolic potentiation of substrates to genotoxic reactive intermediates and ultimate carcinogens, and the detoxification of toxic or carcinogenic drugs and other environmental pollutants. AHR-mediated gene expression is also involved in many critical life processes (e.g. cell type-specific differentiation, cell division, apoptosis) by signal transduction mechanisms. Similar to mice, human populations exhibit a > 20-fold range of the CYP1A1 inducibility/AHR affinity phenotype. In the present study, we localized the human AHR gene to chromosome 7p15, using fluorescence in situ hybridization (FISH). Performing linkage analysis in a three-generation family, we show with good probability that the high CYP1A1 inducibility phenotype segregates with the 7p15 region. Sequencing 93 nt (31 amino acids) of the human AHR gene's exon 9, which is the region correlated with the mouse A375V polymorphism responsible for the major portion of high vs low CYP1A1 inducibility/AHR affinity, we found no nucleotide differences; Val-381 was present in all five individuals examined (four related and one unrelated), two of whom show "high' and three of whom show "low' CYP1A1 inducibility. These data indicate that the "high' and "low' CYP1A1 inducibility trait, in the population studied, cannot be explained by a difference among these 31 amino acids in exon 9 of the AHR gene.

Amino Acid Sequence↗

PubMed1997

Genetic differences in alcohol drinking preference between inbred strains of mice.

Genetic factors are known to influence the preference for drinking alcohol-in humans as well as certain inbred strains of laboratory animals. Here we examined the possible role of the aromatic hydrocarbon receptor (AHR) in alcohol-preferring C57BL/6J (B6, high-affinity AHR) and alcohol-avoiding DBA/2J (D2, low-affinity AHR) inbred mouse strains, and in the two congenic lines B6.D2-Ahrd (> 99% B6 genome with the D2 low-affinity AHR) and D2.B6-Ahrb-1 (> 99% D2 genome with the B6 high-affinity AHR). This laboratory had previously shown an association between resistance to intraperitoneal ethanol-induced toxicity and the high-affinity AHR. Offering the choice between drinking water and 10% ethanol, we found that alcohol preference is three- to four-fold greater in B6 than D2 mice, as well as three- to four-fold greater in B6.D2-Ahrd than D2.B6-Ahrb-1 mice-indicating that alcohol preference is AHR-independent. The prototype AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) did not affect the rates of chronic alcohol consumption in B6 or D2 mice, suggesting that dioxin-inducible metabolism does not play a major role in alcohol drinking preference. In B6 mice, we found that oral treatment with the aldehyde dehydrogenase (ALDH) inhibitor disulfiram decreased alcohol preference by 50%, whereas oral treatment of the catalase inhibitor 3-amino-1,2,4-triazole increased alcohol drinking preference by 15-20%. Although liver and brain ALDH activities were both significantly higher in D2 than B6, these activities were not related to alcohol consumption. Hepatic and brain catalase activities, on the other hand, were two- to three-fold higher in D2 and D2.B6-Ahrb-1 mice, compared with that in B6 and B6.D2-Ahrd. Furthermore, brain acetaldehyde levels were inversely related to the quantity of alcohol voluntarily consumed. We conclude that the alcohol drinking preference between the B6 and D2 inbred mouse strains is independent of the Ah receptor-but is genetically determined, in part, by the level of brain catalase activity which, in turn, regulates brain acetaldehyde concentrations.

Acetaldehyde↗

PubMed1997

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Amino Acid Sequence↗