Biomedical subjects

D W Nebert

Publications and source records attributed to D W Nebert.

At least 199 records · Page 11Linked to original sources

PubMed1984

Effects of cimetidine on theophylline, acetaminophen, and zoxazolamine toxicity in the intact mouse.

3-Methylcholanthrene treatment of C57BL/6N mice induces significant amounts of cytochromes P1-450, whereas P1-450 levels in 3-methylcholanthrene-treated DBA/2N mice are no different from those in control C57BL/6N or DBA/2N mice. Comparison of 3-methylcholanthrene-treated C57BL/6N and DBA/2N mice thus provides a convenient means of determining the role of P1-450 metabolism in two strains of mice following identical drug treatment regimens. 3-Methylcholanthrene-induced P1-450 is shown to be more effective than other forms of P-450 in detoxifying theophylline and zoxazolamine and in enhancing the toxicity of acetaminophen. Cimetidine in vivo blocks these metabolic pathways, resulting in increased toxicity of theophylline and zoxazolamine and protection against acetaminophen toxicity. These data illustrate the double-edged sword nature of P1-450 metabolism and the possibility of a paradoxical effect of cimetidine during drug-drug interactions in vivo. Cimetidine is shown to inhibit in vivo and in vitro the metabolism by both 3-methylcholanthrene-induced P1-450 and control forms of P-450; these data suggest that cimetidine may be acting at the level of P-450 reduction by NADPH-P-450 oxidoreductase. This same mechanism of action has been previously suggested for ellipticine.

Acetaminophen↗

PubMed1984

Variations in aryl hydrocarbon hydroxylase activities in mitogen-activated human and nonhuman primate lymphocytes.

A fluorometric assay for the cytochrome P-450-dependent enzyme system, aryl hydrocarbon hydroxylase (AHH), was performed in mitogen-activated human lymphocytes from over 300 different humans and from 64 baboons. Results reveal: a) an average interindividual variation in AHH activity of approximately 0.25 (coefficient of variation); range of activities among humans and baboon subjects of approximately 40-fold; c) both genetic and environmental determinants of interindividual variation, and d) high AHH activity in humans associated with primary lung cancer. Confirmation of these results awaits the development of improved methods for phenotyping humans and for prospective cancer patient studies. DNA probes might be employed in future studies to determine specific mRNA content, and to search for DNA polymorphisms in and near the human cytochrome P-450 gene.

Animals↗

PubMed1984

An investigation of genetic variation within a series of congenic strains of mice.

2 congenic strains of mice, B6N.AKN-Ahk and D2N.B6N-Ahb, imported from the USA, were found to be either segregating or fixed for an incorrect allele at a number of biochemical loci. B6N.AKN-Ahk, supposedly congenic with C57BL/6N, had the wrong genotype at 6 out of 12 biochemical loci; D2N.B6N-Ahb, supposedly congenic with DBA/2N, was segregating at 3 out of 9 loci. There was genetic variation in mandible shape within the 2 strains but no abnormal coat colours were found and no hybrid vigour in breeding performance was detected. Analyses in the USA confirmed these results and showed that 2 other congenic strains, C3N.D2N-Ahd and AKN.B6J-Ahb, were also segregating at a number of loci. Some of the alleles found in the C3N.D2N-Ahd mice must be the result of a genetic contamination. The simplest explanation for this breakdown in the backcrossing programme is genetic contamination with other congenic strains or recombinant inbred lines under development in the same laboratory. These findings emphasize the importance of continual genetic monitoring of all genetic stocks at regular intervals and in particular during the development of congenic and recombinant lines.

Alleles↗

PubMed1984

Aryl hydrocarbon hydroxylase inducibility among primary relatives of children with leukemia or solid tumors.

In mice, there is a correlation between genetically regulated levels of inducible aryl hydrocarbon hydroxylase (AHH) activity and the risk of polycyclic hydrocarbon-induced leukemia or solid tumors. Recent clinical studies suggest a relationship between high AHH activity and lung cancer associated with cigarette smoking (Kouri, R.E., McKinney, C.E., Slomiany, D.J., Snodgrass, D.R., Wray, N.P., and McLemore, T.L. Cancer Res. 42: 5030-5037, 1982). To determine whether there is a similar genetic relationship in humans between inducible AHH and the occurrence of pediatric cancers, we examined AHH activity in mitogen-stimulated benzo(a)anthracene-treated lymphocyte cultures from primary relatives of children with leukemia or solid tumors. Control families (parents and siblings with no history of cancer) comprised friends or neighbors of the proband families. By comparing variance among family members with variance among nonrelated individuals, we conclude that a small, but real, genetic component is detectable. Adjusting for age, smoking history, and the length of time during which the lymphocytes had been cryopreserved, however, we find no difference among 77 leukemia, 71 solid tumor, and 100 control family members with regard to median units (+/- median S.E.) of maximally induced AHH activity per unit of reduced nicotinamide adenine dinucleotide-cytochrome c reductase activity: 0.31 +/- 0.03; 0.28 +/- 0.03; and 0.28 +/- 0.03, respectively. Thus, benzo(a)anthracene-induced AHH activity in cultured mitogen-activated lymphocytes in our study population does not appear to be associated with the risk of occurrence of childhood leukemia or solid tumors.

Aryl Hydrocarbon Hydroxylases↗

PubMed1984

Structural gene products of the Ah locus. Transcriptional regulation of cytochrome P1-450 and P3-450 mRNA levels by 3-methylcholanthrene.

Mouse liver cytochromes P1-450 and P3-450 represent those forms of polycylic hydrocarbon-induced P-450 most closely associated with induced aryl hydrocarbon (benzo[a] pyrene) hydroxylase and acetanilide 4-hydroxylase activity, respectively. These two proteins are controlled by the Ah receptor: C57BL/6N mice possess the high-affinity receptor; DBA/2N mice, the poor-affinity receptor. 3-Methylcholanthrene at the highest dose technically possible induces both proteins in C57BL/6N but not DBA/2N mice, whereas sufficiently high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce both proteins in both inbred mouse strains. Plasmids containing DNA complementary to P1-450 and P3-450 mRNA, respectively, were used in an in vitro nuclear transcription assay to determine the mechanism of the induction response. In C57BL/6N mice, transcriptional rates of the P1-450 and P3-450 genes increase dramatically as early as 3 hr after 3-methylcholanthrene treatment and at 12 hr reach maximal levels of 20- and 15-fold, respectively, above control values. In contrast, no increase in either gene is found in 3-methylcholanthrene-treated DBA/2N mice. Following TCDD administration, both P1-450 and P3-450 gene transcription rates are elevated in DBA/2N mice. There is a 3- to 6-hr lag period between the early onset of enhanced transcription rates and the later rise in P1-450 and P3-450 mRNA. Basal and induced levels of P3-450 mRNA are about 5-fold greater than those of P1-450 mRNA. These data confirm that the 3-methylcholanthrene and TCDD induction responses, governed by the Ah receptor, are mediated principally through an increase in specific gene transcription.

Animals↗

PubMed1983

Association between susceptibility to dibenzanthracene-induced fibrosarcoma formation and the Ah locus.

The relationship between the Ah locus and the induction of subcutaneous fibrosarcomas by dibenz[a,h]anthracene and dibenz[a,c]anthracene was investigated in C57BL/6J (Ahb/Ahb), (C57BL/6J)(DBA/2J)F1 (Ahb/Ahd) and (Ahd/Ahd) mice. Ahb/Ahb and Ahb/Ahd mice have the high-affinity Ah receptor and therefore the polycyclic hydrocarbon induction of aryl hydrocarbon hydroxylase activity (cytochrome P1-450) proceeds with ease; Ahd/Ahd mice have the poor-affinity Ah receptor and this induction process proceeds more poorly, by a factor of at least 10-fold. Dibenz[a,c]anthracene proved to be a relatively weak carcinogen, producing less than 3% tumor incidence at doses up to 300 micrograms per mouse. In contrast, dibenz[a,h]anthracene caused an almost 50% tumor incidence in Ahb/Ahb and Ahb/Ahd mice, while causing approximately 2% tumor incidence in Ahd/Ahd mice. Both isomers bind avidly to the cytosolic Ah receptor, and both chemicals induce aryl hydrocarbon hydroxylase activity in Ahb/Ahb and Ahd/Ahd animals. Among progeny of the (C57BL/6J) (DBA/2J) F1 X DBA/2J backcross, 63 of 100 Ahb/Ahd mice and none of 75 Ahd/Ahd mice developed tumors. These data demonstrate a strict correlation between susceptibility to dibenz[a,h]anthracene-induced subcutaneous tumors and expression of the Ahb allele, i.e. presence of the high-affinity Ah receptor and therefore readily inducible P1-450.

Alleles↗

PubMed1983

Biological effects of the Sudan dyes. Role of the Ah cytosolic receptor.

The hepatic induction of two cytochrome P1-450-mediated activities [aryl hydrocarbon hydroxylase (AHH) and ethoxyresorufin O-deethylase (ETR)] was studied following the administration of the azo dyes Sudan I, II, III, and IV. When using Ah-responsive C57BL/6J mice, Sudan dye II proved to be quite potent as an inducer causing almost maximal induction at doses as low as 40 mg/kg (1.4 mumoles/kg body weight); Sudan dyes I, III and IV caused one-half the maximal induction at four times that dose. In contrast, none of these compounds caused induction of AHH or ETR in the Ah-nonresponsive DBA/2J animals. When the dyes were given to B6D2F1 X D2 backcross progeny, a strict correlation with the presence of the Ahb allele and the inducibility of AHH and ETR was observed. When these compounds were examined as agonists for the Ah cytosolic receptor by their capacity to replace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding, Sudan dye II was substantially more effective than Sudan dyes I, III and IV. When four repeated doses of Sudan dye II were administered intraperitoneally to Ah-responsive C57BL/6J mice and Ah-nonresponsive DBA/2J mice, Sudan dye II-induced immunotoxicity was markedly greater in C57BL/6J compared to DBA/2J mice.

Animals↗

PubMed1983

Bone marrow toxicity induced by oral benzo[a]pyrene: protection resides at the level of the intestine and liver.

The Ah locus encodes a cytosolic receptor that regulates the induction of certain drug-metabolizing enzymes by polycyclic aromatic hydrocarbons such as benzo[a]pyrene. Some inbred mouse strains such as C57BL/6N have the high-affinity Ah receptor (Ahb/Ahb), others such as DBA/2N, the poor-affinity receptor (Ahd/Ahd). Presence of the high-affinity receptor leads to greater cytochrome P1-450 induction by benzo[a]pyrene; in turn, enhanced benzo[a]pyrene metabolism can result in more toxic intermediates or greater detoxication, depending upon the test system studied. Benzo[a]pyrene in the growth medium, in direct contact with cultured myeloid cells, is more toxic to C57BL/6N than DBA/2N cultured cells. Oral benzo[a]pyrene induces P1-450 (measured by benzo[a]pyrene trans-7,8-dihydrodiol formation determined by high-performance liquid chromatography) in C57BL/6N but not DBA/2N intestine and liver. In the bone marrow of oral benzo[a]pyrene-treated C57BL/6N and DBA/2N mice, the magnitude of P1-450 induction is about the same. WB/ReJ (Ahd/Ahd), C57BL/6J (Ahb/Ahb), or (WB/ReJ)(C57BL/6J)F1 (Ahb/Ahd) marrow was transplanted into lethally irradiated (WB/ReJ)(C57BL/6J)F1 mice. DBA/2J (Ahd/Ahd) marrow was transplanted into lethally irradiated BALB/cByJ (Ahb/Ahb) mice and vice versa. Mice having the Ahd/Ahd intestine and liver died in less than 3 weeks of benzo[a]pyrene feeding (120 mg/kg/day), irrespective of the source of transfused marrow. All the data are consistent with pharmacokinetic differences in the tissue distribution of benzo[a]pyrene: mice having the high-affinity receptor, and therefore the P1-450 induction process in the intestine and liver, are protected from oral benzo[a]pyrene-induced myelotoxicity.

Administration, Oral↗

PubMed1983

Effects of cytochrome P1-450 inducers on the cell-surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells.

Hepa-1c1c7, a mouse hepatoma cell line, was used to study the effect of cytochrome P1-450 inducers on the binding of 125I-epidermal growth factor (EGF), 125I-insulin, or [20-3H]phorbol 12,13-dibutyrate each to its specific cell-surface receptor. After a 24-h exposure to the cultured cells, several polycyclic hydrocarbon P1-450 inducers decrease the binding of EGF to EGF receptors much more than phenobarbital does. There appears to be a selectivity in the inhibitory effects: whereas EGF binding to EGF receptors is blocked, the binding of either phorbol ester or insulin each to its specific cell-surface receptors remains unaffected. The rank order of binding affinities of these chemicals to the cytosolic Ah receptor (2,3,7,8-tetrachlorodibenzo-p-dioxin much greater than benzo[a]pyrene greater than benzo[a]anthracene greater than 6-aminochrysene much greater than phenobarbital) is not correlated with their effects on EGF binding capacity. The effect of polycyclic hydrocarbons on EGF binding takes 24 h at 37 degrees C to be maximal, whereas phorbol 12-myristate 13-acetate, a potent tumor-promoting compound, inhibits EGF binding in less than 30 min. Removal of benzo[a]anthracene from the growth medium after 24 h results in a gradual recovery in EGF binding, indicating that the effect is reversible. Benzo[a]pyrene and benzo[a]anthracene are relatively ineffective at decreasing EGF binding to the EGF receptors in Hepa-1 mutant clones c2 and c4, which lack a normally functioning Ah receptor and inducible aryl hydrocarbon hydroxylase activity (P1-450). The very toxic metabolite (+)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, when added directly to the growth medium of c4 cells, however, is effective at decreasing EGF binding. These data suggest that electrophilic metabolites of polycyclic aromatic compounds, formed by P1-450 induced during the exposure of Hepa-1 cells to these chemicals, are important in decreasing EGF binding to the EGF cell-surface receptor. Occupancy of the Ah receptor per se does not affect EGF binding.

Animals↗

PubMed1983

Characterization of cytochrome P2-450 (20-S) mRNA. Association with the P1-450 genomic gene and differential response to the inducers 3-methylcholanthrene and isosafrole.

Mouse liver cytochrome P2-450 is defined as the major isosafrole-inducible form of P-450 which is most specific for isosafrole metabolism. lambda AhP-1 represents a 15.5 X 10(3)-base-pair segment of mouse genomic DNA having the cytochrome P1-450 gene (approximately equal to 4600 base pairs) located in the middle portion. Using various subclones as probes, we investigated the differential expression of P1-450 mRNA and P2-450 mRNA induction as a function of association with the Ah locus, 3-methylcholanthrene or isosafrole dosage, tissue specificity, and developmental age. Both P1-450 (23-S) mRNA and P2-450 (20-S) mRNA induction processes are regulated by the Ah receptor. P2-450 mRNA is about 10-fold more sensitive than P1-450 mRNA to induction by either 3-methylcholanthrene or isosafrole. Phenobarbital pretreatment has no effect at all on either P1-450 mRNA or P2-450 mRNA. Whereas both P1-450 mRNA and P2-450 mRNA are induced by 3-methylcholanthrene in C57BL/6N liver, P1-450 (23-S) mRNA but not P2-450 (20-S) mRNA is induced by 3-methylcholanthrene in C57BL/6N kidney. P1-450 mRNA induction by 3-methylcholanthrene is measurable in C57BL/6N liver at day 15 of gestation, and the expression becomes enhanced with increasing age. P2-450 mRNA induction by 3-methylcholanthrene in C57BL/6N liver appears about 7 days later during development than 3-methylcholanthrene-inducible P1-450 mRNA. Both 3-methylcholanthrene-induced P1-450 mRNA and P2-450 mRNA are detectable in DBA/2N liver; their appearance is later in development, however, and at lower concentrations than that seen with C57BL/6N liver. P1-450 (23-S) mRNA and P2-450 (20-S) mRNA appear to hybridize to a common 5' fragment of the P1-450 gene.

Animals↗

PubMed1983

Structure of the mouse cytochrome P1-450 genomic gene.

Clone 46 was previously shown to represent mouse cytochrome P1-450 cDNA by both translation arrest experiments and segregation of induced P1-450 mRNA with induced aryl hydrocarbon hydroxylase activity among individual 3-methylcholanthrene-treated offspring of the (C57BL/6N)(DBA/2N)F1 X DBA/2N backcross. With clone 46 as a probe, a MOPC 41 mouse genomic-DNA library was screened. lambda 3NT12, a 16 X 10(3)-base-pair insert of genomic DNA grown in a recombinant Charon 4A lambda vector phage, was isolated and characterized. It was determined that clone 46 hybridizes to the extreme 5' end of lambda 3NT12. pMJE12, a 3.0 X 10(3)-base-pair fragment in the 5' region of lambda 3NT12, was subcloned in plasmid pBR322 and used as a probe to screen again the same mouse-DNA library; recombinant phages lambda 3NT13, lambda 3NT14, and lambda AhP-1 were isolated and characterized. The relative orientation of each of the four genomic clones on the mouse chromosome was determined. Only lambda AhP-1 contains the entire P1-450 genomic gene, which by R-loop analysis spans about 46 X 10(2) base pairs and contains at least five exons. Clone 46 is shown to be a 3' unique sequence of the genomic P1-450 gene. The lambda AhP-1 genomic-DNA clone from the MOPC 41 plasmocytoma is shown by a series of restriction enzymes to be the same as genomic DNA from normal mouse liver. With a subclone in the 5' portion of the P1-450 gene, two and three hybridizable fragments are found with mouse genomic DNA that has been digested with EcoRI and BamHI, respectively.

Animals↗

PubMed1983

Sulfobetaine derivatives of bile acids: nondenaturing surfactants for membrane biochemistry.

The syntheses of four new sulfobetaine derivatives of bile salts are presented, along with a general set of criteria for useful detergents in membrane biochemistry. Physical properties including the critical micelle concentration, aggregation number, partial specific volume, critical micellar temperature, uv-vis spectrum, and circular dichroism spectrum are examined for the new compounds. To examine the interaction of this class of compounds with macromolecules, one of these (CHAPS) was further studied. Circular dichroism spectra of apolipoprotein C-III2 were measured in the presence of varying concentrations of CHAPS to determine the effect of this compound on secondary structure. Gel-exclusion chromatography and sedimentation equilibrium studies of cytochrome P-450 in the presence of CHAPS were also performed to establish the ability of this detergent to disaggregate cytochrome P-450 to a monomeric/dimeric state.

Apolipoprotein C-III↗

PubMed1983

Dibenz[a,h]anthracene-induced subcutaneous tumors in mice. Strain sensitivity and the role of carcinogen metabolism.

Four inbred strains of mice (C3H/HeJ, C57BL/6J, AKR/J, and DBA/2J) were injected s.c. with 150 micrograms of dibenz[a,h]anthracene and followed for 9 months. Strains C3H/HeJ and C57BL/6J were most susceptible to dibenz[a,h]anthracene-induced carcinogenesis with 80% and 53% incidence of tumors, respectively. Strains AKR/J and DBA/2J were much less sensitive with only one tumor observed out of a total of 60 treated mice. Dibenz[a,h]anthracene was shown to induce hepatic aryl hydrocarbon hydroxylase activity in the two sensitive strains, C3H/HeJ and C57BL/6J, but not in the two resistant strains, AKR/J and DBA/2J. When 3-methylcholanthrene-treated liver microsomes from the four strains were studied for dibenz[a,h]anthracene metabolism in vitro, the two sensitive strains not only demonstrated a 3- to 4-fold greater overall rate of metabolism than the two resistant strains, but also showed a quantitative shift with a greater percentage of the total metabolites being the 3,4-diol of dibenz[a,h]anthracene. This diol is the presumed precursor to the apparent ultimate carcinogen, dibenz[a,h]anthracene 3,4-diol-1,2-epoxide.

Animals↗

PubMed1983

Screening of 16 common therapeutic drugs. Possible association with the Ah locus.

16 common therapeutic agents were screened for differences in sedation or lethality between C57BL/6N and DBA/2N inbred mouse strains that had been previously treated with beta-naphthoflavone. No differences were observed for meprobamate, valium, promethazine, valproic acid, lincomycin, imipramine, terbutaline, propoxyphene, nitrofurantoin, amphotericin B, or diphenhydramine. C57BL/6N mice appeared to be more resistant than DBA/2N mice to the lethal effects of isoxsuprine, niridazole, pentazocine, isoniazid, and hydralazine. None of these latter five drugs had any capacity to displace [3H-1,6]2,3,7,8-tetrachlorodibenzo-p-dioxin from the liver cytosolic Ah receptor in C57BL/6N mice. With the use of beta-naphthoflavone-pretreated offspring from the (C57BL/6N) (DBA/2N)F1 X DBA/2N backcross, a strict correlation (100% of 24 individuals in each case) was found between the Ahb allele and resistance to the lethal effects of isoxsuprine or niridazole. No correlation between the Ah locus and pentazocine, hydralazine, or isoniazid lethality was apparent. These results indicate that presence of the Ahb allele is associated with increased protection against isoxsuprine and niridazole lethality. This increased protection may reflect enhanced detoxication metabolic pathways (e.g., induced cytochrome P1-450 and/or uridine diphosphate glucuronosyltransferase controlled by the Ah locus). The increased protection is not related to interaction of these drugs with the Ah receptor. It should be kept in mind that gene-environment interactions involving the Ah locus and isoxsuprine or niridazole may be important in certain clinical instances.

Animals↗

PubMed1983