Biomedical subjects
Publications and source records attributed to D W Nebert.
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Comparative EPR studies were made on two high-spin Fe(III) porphine model systems and mammalian liver microsomal cytochromes P-450, all of which exhibit approximately the same degrees of rhombicity in their EPR spectra. Comparison of g values and linewidths as a function of temperature, and of the microwave power saturation demonstrated that EPR characteristics of P-450 are more similar to the Fe(III) porphines having the thiolate axial ligand than in the other model systems, the mixed crystals of Fe(III) porphine with the corresponding free base porphine, in which no thiolate ligand is involved. There is, however, a discrepancy between P-450 and the model thiolates with respect to the size of the zero-field parameter D. These observations indicate that P-450 heme has essential structural features in common with thiolates but the Fe-S bond of P-450 may be modified from its normal orientation in model thiolates, probably as a result of the constraints imposed by the protein structure.
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The stimulation of reduced-NAD(P):menadione oxidoreductase (EC 1.6.99.2) activity in liver cytosol is highly correlated with the stimulation of hepatic microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity in 3-methylcholanthrene-, beta-naphthoflavone-, phenobarbital-, or pregnenolone-16alpha-carbonitrile-treated inbred C57BL/6N and DBA/2N mice and in eight other inbred strains treated with 3-methylcholanthrene. No oxidoreductase activity is detectable in mouse liver microsomes. Cytochrome c and 2,6-dichlorophenolindophenol are equally good electron acceptors for the oxidoreductase. There is no preferential in vitro inhibition of induced versus control oxidoreductase activities by either alpha-naphthoflavone or metyrapone. In 3-methylcholanthrene-treated F1 and F2 progeny and offspring from backcrosses between the F1 and either C57BL/6N or DBA/2N parent, however, there is not a strict correlation between induced or noninducible aryl hydrocarbon hydroxylase and oxidoreductase activities. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, at doses (80 mug kg-1) sufficiently high to induce the hydroxylase almost as well in DBA/2N as in C57BL/6N mice, induces the oxidoreductase about 3-fold in C57BL/6N and less than 50% in DBA/2N mice. All the data are consistent with an hypothesis that two loci (Ox-1 and Ox-2) regulate oxidoreductase induction by 3-methylcholanthrene, that one of the genes is linked to the Ah locus (with an estimated recombination frequency between 2% and 23%), and that the other gene is not linked to the Ah locus. These experimental data might be useful in the protein activator hypothesis of the Britten-Davidson model for gene regulation.
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Various polycyclic aromatic compounds induce certain monooxygenase activities, including aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2), and cytochrome P1-450 in the liver and many nonhepatic tissues of the mouse. This induction process is controlled by the Ah locus. Genetic differences that have been shown in the past to be associated with the Ah locus include an increased susceptibility to chemical carcinogenesis, mutagenicity in vitro, and drug toxicity--manifested as hepatic necrosis, aplastic anemia, or shortened survival time. Pregnant mice received a single injection of 3-methylcholanthrene or 7,12-dimethylbenz[a] anthracene between day 5 and day 13 of gestation, and the uterine contents were examined on day 18. Striking increases were observed in the incidence of MC-1 and DMBA-induced resorptions and congenital malformations in the aromatic hydrocarbon "responsive" C57BL/6N inbred strain, and of DMBA-induced resorptions in the "responsive" C3H/HeN and BALB/cAnN strains--when compared with the similarly treated genetically "nonresponsive" AKR/N strain. These data suggest but do not prove that an association exists between the Ah locus and developmental toxicity, i.e., teratogenesis. Although numerous teratogenic differences among inbred mouse strains have previously reported, this study is unique in that the genetic differences in teratogenicity observed were predicted in advance on the basis of known differences among these strains in polycyclic hydrocarbon metabolism regulated by the Ah locus.
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The genetic trait of "responsiveness," which refers to the capacity for induction of cytochrome P-448 and numerous monooxygenase activities by certain aromatic hydrocarbons, is known to segregate almost exclusively as a single autosomal dominant gene among progeny of appropriate crosses originating from the responsive C57BL/6 and the nonresponsive DBA/2 inbred mouse strains. In this report the allele for responsiveness is shown to be associated with (a) increases in the apparent KS values for metyrapone bound to reduced P-450; (b) increases in the ethylisocyanide difference ratio (deltaA455-490/deltaA430-490);(c) increases in the deltaA455-490 per mg of microsomal protein but not in the deltaA430-490 per mg of protein from the reduced P-450-ethylisocyanide complex; (d) an approximately 2-nm hypsochromic shift in the spectral maximum in the 446 nm region for the reduced P-450-metyrapone complex; (e) an approximately 2-nm hypsochromic shift of the absorption maximum in the 455 nm region, but not of the maximum in the 430 nm region, for the reduced P-450-ethylisocyanide complex; and (f) larger increases in the deltaA455-490 than in the deltaA430-490 per mg of microsomal protein for the reduced P450-ethylisocyanide complex as a function of increasing pH. All of these phenomena are felt to be associated with the genetically regulated induction of liver microsomal cytochrome P-448 by polycyclic aromatic compounds. Whereas increases in the total hepatic P-450 content appear to be expressed almost exclusively as a single autosomal dominant trait, the increase in apparent KS value for metyrapone bound to reduced P-450 appears to be expressed additively. The reason for this finding is unclear. The increase in apparent KS value for metyrapone in 3-methylcholanthrene-treated rats is known to occur even when the induction process is presumably blocked by treating the rat concomitantly with cycloheximide. Several lines of evidence in this report indicate that, although total P-450 content does not increase in C57BL/6N mice treated with 3-methylcholanthrene plus cycloheximide, hepatic P-448 induction does occur; P-448 induction does not occur in DBA/2N mice under these same conditions. These results indicate that cytochrome P-448 induction is relatively resistant to the inhibition of protein synthesis and that a responsive animal treated with 3-methylcholanthrene plus cycloheximide cannot be considered experimentally the same as a genetically nonresponsive animal treated with 3-methylcholanthrene alone.
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The Ah locus in the mouse controls the induction of cytochrome P1-450 and at least eleven associated monooxygenase activities. These enzyme systems metabolically potentiate and detoxify drugs, environmental pollutants, and other foreign chemicals, as well as numerous endogenous substrates. For certain substrates, it is known that cytochrome P1-450 produces different reactive intermediates and products that other forms of P-450. Alleles at the Ah locus can be identified in utero. Development toxicity (in the form of stillborns, resorptions, and malformations of the fetus) by 3-methylcholanthrene and 7,12-dimethylbenz[a]anthracene given to the pregnant mother is associated with genetically mediated aromatic hydrocarbon responsiveness in C5BL/6N mice, compared with that in nonresponsive AKR/N mice. Acetaminiphen-produced hepatic necrosis is associated with glutathione depletion in the liver, covalent binding of metabolite(s) of the drug to cellular macromolecules, and P1-450 induction controlled by the Ah locus. For reasons not known, the fetus and mice 10 days of age or less are relatively resistant to glutathione depletion and therefore hepatic necrosis by acetaminophen.
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Interindividual and intraindividual variations in aryl hydrocarbon hydroxylase (AHH) induction by 3-methylcholanthrene were studied in cultured lymphocytes from normal adult volunteers. Using eight pairs of monozygotic and eight pairs of dizygotic twins, we examined to what extent these variations are controlled by heritable factors and whether AHH inducibility correlations in an individual with the plasma half-lives of three drugs. Substantial overestimation of the induction ratio (fold inducibility) may occur if the nonlinearity of the assay standard curve is not considered. Fold inducibility remains relatively constant for an individual, but large intraindividual variations occur in absolute "control" and "induced" AHH activities. Fetal calf serum may contain inducers of AHH activity that vary with the particular lot of serum, thereby rendering the apparent induction ratio an imprecise indicator of genetic susceptibility to induction by 3-methylcholanthrene. The index of heritability for AHH fold inducibility in twins studied with different lots of fetal calf serum (0.80) or with a single lot of fetal calf serum (0.77) suggests nonetheless that genetic rather than environmental factors are mainly responsible for interindividual variations in AHH inducibility by 3-methylcholanthrene in human lymphocytes. In these twins a significant but poor correlation (r=-0.551; 0.03 less than p less than 0.05) occurs between AHH inducibility in culture and the plasma antipyring half-life, but not between AHH inducibility and phenylbutazone or bishdroxycoumarin half-lives.