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D W Nebert

Publications and source records attributed to D W Nebert.

At least 217 records · Page 12Linked to original sources
PubMed1982

The Ah locus: correlation of intranuclear appearance of inducer-receptor complex with induction of cytochrome P1-450 mRNA.

The Ah locus regulates the induction of cytochrome P1-450 by foreign chemicals such as 3-methyl-cholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. The induction process is controlled by the cytosolic Ah receptor. The cytosolic and nuclear Ah receptors were studied in the liver from inbred C57BL/6N (Ahb/Ahb) mice, inbred DBA/2N (Ahd/Ahd) mice and heterozygotes (Ahb/Ahd) and homozygotes (Ahd/Ahd) derived from the (C57BL/6N X DBA/2N)F1 X DBA/2N backcross. After [3H-1,6]-2,3,7,8-tetrachlorodibenzo-p-dioxin (3H-TCDD) is given in vivo, the receptor in Ahb/Anb and Ahb/Ahd mice is detectable in the cytoplasm and nucleus; in Ahd/Ahd mice the receptor is not measurable in the cytosol, but is found in the nucleus at levels one fourth to one fifth of those in Ahb/Ahb mice. P1-450 (23S) mRNA content was estimated by Northern hybridization and by Rot analysis with a mouse P1-450 cloned cDNA. An excellent dose-response relationship (r = 0.99) was found between the amount of 3H-TCDD-Ah receptor complex appearing in the nucleus and the quantity of P1-450 mRNA induced in mice with all three possible Ah genotypes.

Animals↗
PubMed1982

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor.

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxine ([3H]TCDD) from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [3H]TCDD from the Ah receptor.

Animals↗
PubMed1982

Cytochrome P1-450 structural gene in mouse, rat, and rabbit: differences in DNA methylation and developmental expression of mRNA.

Clone 46 previously was shown to represent an 1100-bp cDNA clone of the mouse cytochrome P1-450 structural gene. Clone 46 [32P]DNA was hybridized to DNA and mRNA from mouse, rat, and rabbit of different ages. In Hpa II digests of DNA, two hybridizable fragments of less than 0.5 kb exist in adult "Ah-responsive" C57BL/6N mouse liver but not in C57BL/6N sperm or embryo or in adult "Ah-nonresponsive" DBA/2N mouse liver. The reason for this hypomethylation of adult C57BL/6N liver DNA is not known but might be related to the high degree of expressivity of this gene in adult C57BL/6N liver, compared with adult DBA/2N liver. No differences in Hpa II- or Msp I-digested DNA are seen in C57BL/6N or DBA/2N inbred strains treated with the P1-450 inducer, 3-methylcholanthrene (3-MC), versus untreated controls. The ontogenetic expression of 3-MC-induced P1-450 mRNA (23S) from mouse or rat liver corresponds well to previous developmental studies from this laboratory involving 3-MC-inducible aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) (EC 1.14.14.1) activity. P1-450 mRNA--induced transplacentally by 3-MC given to the mother--is readily detectable by clone 46 as early as gestational day 15. The cloned cDNA probe hybridizes to rat and rabbit DNA fragments of different sizes, and with less intensity, when compared with hybridization to mouse DNA. No hybridization of this DNA is observed with rabbit mRNA of all ages ranging from neonate to adult. These data suggest that sequence homology exists among the mouse, rat, and rabbit P1-450 structural genes and between mouse and rat P1-450 mRNA. The mouse cDNA probe is believed to hybridize to a segment of the rabbit P1-450 gene that is not transcribed.

Animals↗
PubMed1982

The Ah phenotype. Survey of forty-eight rat strains and twenty inbred mouse strains.

Forty-four inbred and four randombred rat strains and 20 inbred mouse strains were examined for their Ah phenotype by determining the induction of liver microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity (EC 1.14.14.1) by intraperitoneal treatment with either beta-naphthoflavone or 3-methylcholanthrene. All 48 rat strains were found to be Ah-responsive. The maximally induced hydroxylase specific activities of the ALB/Pit, MNR/Pit, MR/Pit, SHR/Pit, and Sprague-Dawley strains were of the same order of magnitude as the basal hydroxylase specific activities of the ACI/Pit, F344/Pit, OKA/Pit, and MNR/N strains. Six of the 20 mouse strains were Ah-nonresponsive (i.e. lacking the normal induction response and presumably lacking detectable amounts of the Ah receptor). The basal hydroxylase specific activities of the BDL/N, NFS/N, STAR/N, and ST/JN mouse strains were more than twice as high as the maximally induced hydroxylase specific activity of the CBA/HT strain.--To date, 24 Ah-nonresponsive mouse strains have been identified, out of a total of 68 known to have been characterized. The reasons for not finding a single Ah-nonresponsive inbred rat strain--as compared with about one Ah-nonresponsive inbred mouse strain found for every three examined--remain unknown.

Animals↗
PubMed1982

Genetic differences in drug metabolism associated with ocular toxicity.

The tissue localization and subcellular distribution of drug-metabolizing enzymes in the eye are described. With the use of inbred strains of mice, the [Ah] complex is shown to be an important experimental system for probing genetic differences in drug metabolism and related drug toxicities. Although the genetic system described in detail here involves mice, there is ample evidence that the same system operates in man. Genetic differences in acetaminophen- and naphthalene-induced cataract formation and and other ocular degeneration are shown to be related to the [Ah] complex. Because this toxicity appears similar to senile cataracts, we propose that certain types of drug-induced cataracts might exist among clinical populations of senile cataracts but that any cause-and-effect relationship would be very difficult to determine because of underlying interindividual differences in genetic predisposition. It is therefore suggested that genetic differences in drug metabolism be an important consideration in the clinical assessment of ocular toxicity caused by drugs and other environmental pollutants.

Acetaminophen↗
PubMed1982

Pharmacogenetics and human cancer.

Pharmacogenetics involves research into the hereditary basis for the different responses of different individuals to drugs or other environmental pollutants. Circumstantial evidence from a growing number of laboratory animal model systems suggests that human susceptibility to environmentally caused cancers may be influenced by an underlying genetic predisposition for metabolism of drugs and other foreign chemicals. The Ah locus is an example of a well-characterized model system for studying pharmacogenetics in mice. This complex controls the induction by polycyclic aromatic compounds of numerous drug-metabolizing enzyme 'activities'. Allelic differences at the Ah locus have been shown to be associated in the mouse with increased individual risk for cancer, mutation, DNA binding of reactive metabolites, drug toxicity, birth defects and enhanced detoxication. These differences appear to reflect changes in the steady-state level of reactive intermediates in target tissues. Tissue sites in direct contact with the carcinogen develop cancer more readily in responsive animals because of the presence of induced P1-450; tissues at distant sites of the body may develop malignancy more readily in nonresponsive animals because more carcinogen reaches that tissue, due to decreased P1-450 induction in proximal tissues and, therefore, a decrease in detoxication. Hence, not only the dose but the route of administration, the timing of the dosage, and the site of the tumour or toxicity--relative to the site of administered drug--are all very important in the interpretation of data from carcinogenicity or toxicity experiments involving P1-450 inducers, such as polycyclic hydrocarbons. These data emphasize the utility of studies in intact animals, in which differences in pharmacokinetics and enzyme induction may occur among various tissues; such subtleties cannot be detected in short-term testing with bacteria or tissue cultures. There is sufficient evidence that heritable variation of the Ah complex occurs in man. With the advent of recombinant DNA technology, this laboratory has recently cloned the P1-450 gene. In addition to a better understanding of P-450 induction and evolution of P-450 genes, we hope to develop a more sensitive assay for assessing the human Ah phenotype.

Aryl Hydrocarbon Hydroxylases↗
PubMed1981

Structural gene products of the Ah locus. Evidence for many unique P-450-mediated monooxygenase activities reconstituted from 3-methylcholanthrene-treated C57BL/6N mouse liver microsomes.

Cytochrome P-450 from cholate-solubilized liver microsomes prepared from 3-methylcholanthrene-treated genetically "responsive" C57BL/6N mice (Ahb/Ahb) was partially purified by aminooctyl-Sepharose 4B column chromatography with an elution buffer that included Emulgen 911, followed by hydroxylapatite column chromatography. The P-450 was separated into 16 fractions: 15 fractions principally associated with the Ahb allele and induction by 3-methylcholanthrene and 1 fraction not associated with the Ahb allele, i.e. predominantly constitutive form(s) of P-450. The metabolism of benzo[a]pyrene, 7-ethoxycoumarin, biphenyl, ethoxyresorufin, acetanilide, 2-acetylaminofluorene, phenacetin, estradiol-17 beta, and testosterone was investigated. With these nine substrates, we examined the rates of formation of 17 products, i.e. 17 P-450-mediated reconstituted monooxygenase "'activities," in each of the 16 fractions. Differing reductase/cytochrome and lipid/cytochrome requirements were observed for each activity; the reasons for these empirical differences are not known. By two-factor analysis of variance our data for 17 catalytic activities can be explained by a minimum of 19 unique groups of monooxygenase activities: 12 induced by 3-methylcholanthrene and 7 control (endogenous). At least one of these distinctly different 3-methylcholanthrene-induced groups and at least one of these constitutive groups is associated with a marked blue spectral shift (approximately 2.0 nm) in the Soret peak of the reduced hemoprotein.CO complex, suggesting multiple forms of inducible and control "P-448." Almost every reconstituted monooxygenase activity therefore appears to be unique and probably represents the aggregate activity from numerous forms of P-450.

Animals↗
PubMed1981

Isolation and characterization of a cloned DNA sequence associated with the murine Ah locus and a 3-methylcholanthrene-induced form of cytochrome P-450.

Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P(1)-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric "tailing" and cloned in E. coli LE392. Clone 46 hybridized with [(32)P]cDNA made from 23S mRNA from "Ah-responsive" C57BL/6N mice but did not hybridize with similarly prepared [(32)P]cDNA from "Ah-nonresponsive" DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [(32)P]cDNA probes; these two clones were therefore used as "positive" and "negative" control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-P(1)-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 (32)P-labeled nick-translated DNA to EcoRI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P(1)-450.

Animals↗