Effect of phenobarbital and 3-methylcholanthrene treatment on NADPH- and NADH-dependent production of reactive oxygen intermediates by rat liver nuclei.

The effect of inducing the rat liver nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene on NADPH- and NADH-dependent production of reactive oxygen intermediates was evaluated. The inducing agents produced a 2-fold increase in cytochrome P-450, a 50 to 70% increase in NADPH-cytochrome c reductase activity, and a 20 to 30% increase in NADH-cytochrome c reductase activity. Associated with these increases was a corresponding increase in NADPH- and NADH-dependent production of hydroxyl radical (.OH)-like species and of H2O2. Rates of .OH production were inhibited by catalase and partially sensitive to superoxide dismutase. The increase in nuclear production of .OH-like species after drug treatment appears to be due a corresponding increase in H2O2 generation. In contrast to H2O2 and .OH generation, production of thiobarbituric acid-reactive material by nuclei was not increased by the phenobarbital or 3-methylcholanthrene treatment. Redox cycling agents such as menadione and paraquat increased oxygen radical generation to similar extents in the control and the induced nuclei. These results indicate that induction of the nuclear mixed-function oxidase system by phenobarbital or 3-methylcholanthrene can result in a subsequent increase in production of reactive oxygen intermediates in the presence of either NADPH or NADH.     

Induction of drug metabolizing enzymes in human liver cell line Hep G2

Human cytochrome P-450, UDP-glucuronosyltransferase and sulphotransferase activities have been measured in the cell line Hep G2 following treatment of cells with 3-methylcholanthrene or phenobarbital. 3-Methylcholanthrene treatment caused a 20-30-fold increase in the O-deethylation of 7-ethoxycoumarin. The glucuronidation and sulphation of the product 7-hydroxycoumarin were increased 36 and 7 fold, respectively. In comparison, phenobarbital treatment did not increase these activities significantly. However, phenobarbital-inducible proteins were identified on "Western blots' using antibodies to a rat liver phenobarbital inducible P-450 form. The molecular masses of the proteins did not coincide with those expected for cytochromes P-450. However, characteristic of P-450 forms, the synthesis of these proteins was suppressed by 3-methylcholanthrene treatment. The Hep G2 cell line represents a potentially useful model for studying the regulation of human P-450 genes.     

Histone acetylation and hormone action. Early effects of oestradiol-17β on histone acetylation in rat uterus

The effect of oestradiol treatment on the acetylation of histones of the immature rat uterus has been studied. A 10mug dose of oestradiol causes a 70% increase at 5min and a 140% increase at 10min after administration in the labelling of the histone fraction F2+F3. No effect of oestradiol is seen on the labelling of histones F1 or acidic non-histone chromatin proteins. The oestradiol stimulation is seen in animals pretreated with either cycloheximide or actinomycin D. The stimulation of labelling caused by oestradiol is completely abolished by pretreatment of the animals with the anti-oestrogen, nafoxidine. The stimulation is given by lower doses of oestradiol, by stilboestrol and oestriol, but is not given by testosterone. These results suggest that stimulation of histone acetylation in the uterus is the earliest known effect of the hormone on its target tissue.     

ADP-ribosylation of nuclear proteins is increased by phenobarbital. Identification of the ADP-ribosylated histone fractions in rat liver nuclei

Changes in the ADP-ribosylation of total proteins and purified histones of rat liver nuclei after phenobarbital treatment (80 mg/kg, 24 h) have been studied. The [32P]NAD incorporation into total trichloroacetic acid precipitated proteins, in histone Hl and in core histones was evaluated, the specific radioactivities increasing 150, 40 and 8%, respectively. Histones Hl and H2B were the best ADP-ribose acceptors. Histone H4 did not show any 32P incorporation, as revealed by autoradiography after SDS-PAGE of the purified histones, in either the control or phenobarbital treated rats. Possible involvement of ADP-ribosylation of nuclear proteins in the adaptative response of liver to phenobarbital is discussed.     

Differences in the binding of 3-methylcholanthrene and phenobarbital to rat liver cytosolic and nuclear protein fractions

The inducers of cytochrome P-450c and P-450b, 3-methylcholanthrene and phenobarbital, respectively, have been studied in their interaction with subcellular fractions from rat liver. 3-Methylcholanthrene bound to both nuclear and cytoplasmic components as demonstrated by DNA-cellulose chromatography. The binding of 3-methylcholanthrene to cytosolic proteins, on DNA-cellulose, was approximately 27 fmol/mg of applied protein, whereas the binding to nuclear proteins was 250–570 fmol/mg applied protein. Phenobarbital did not bind to proteins of rat serum, rat liver cytosol, or rat liver nuclei which could bind to DNA-cellulose. Further examination of the potential interaction of phenobarbital to rat liver cytosolic proteins was carried out using either DEAE A-50 Sephadex chromatography, charcoal dextran analysis, or sucrose density gradients. No binding of phenobarbital to rat liver cytosolic proteins was observed under these experimental conditions. In contrast, the binding of 3-methylcholanthrene to cytosolic proteins showed four peaks of radioactivity after DEAE A-50 Sephadex chromatography, two peaks by sucrose density gradient analysis, and specific binding (0.13 pmol/mg protein) was observed using the charcoal dextran technique. One of the peaks on sucrose gradients was labile in the presence of salt. The uptake and intranuclear distribution of 3-methylcholanthrene and phenobarbital were markedly different after incubation with whole nuclei: 64% of the available 3-methylcholanthrene but only 3% of the available phenobarbital radioactivity became associated with nuclei. Of this radioactivity, the highest specific activity of the 3-methylcholanthrene radioactivity was associated with the 2 m KCl-resistant nuclear pellet and the highest specific activity of the phenobarbital radioactivity was associated with the nuclear fraction soluble in the absence of salt. These results are interpreted in regard to the induction of cytochrome P-450c.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Electrophoretic study of plant histones: Comparison with vertebrate histones

The histones of seven plant species (barley, leek, onion, pea, radish, rye, and wheat) were isolated and compared to the histones of calf thymus and rat liver using electrophoresis on polyacrylamide-urea and polyacrylamide-SDS gels. It was found that the F1 histone of plants contains more subspecies and has generally higher molecular weights than their animal histone counterparts. Histones F3 of plants and animals have identical molecular weights and similar but not identical mobilities on polyacrylamide-urea gels. No histones were found in plants which have molecular weights and mobilities on polyacrylamide-urea gels which resemble the values for histones F2a2 and F2b of animals, but instead the series of histones observed differ from any of the animal histones. These plant histones may represent either substantially modified forms of F2a2 and F2b, or else may be a different class of histone molecules unique to plants. Fractions F2al in plants and animals are identical in electrophoretic behavior, but seem to differ in degree of acetylation.     

Turnover of chromatin proteins in rat liver during induction of RNA synthesis by electrostimulation of the hypothalamus

It has been shown that the induction of D-RNA synthesis in rat liver nuclei by electrostimulation of hypothalamus is accompanied by a decrease in chromatin protein synthesis and an increase in phosphorylation and acetylation of chromatin proteins. The decrease of the histone synthesis is mainly due to the decrease of [14C]lysine and [14C]alanine incorporation into histones H1 and H4. The relationship between H1, H2b-H3, H2a and H4 histone fractions remains unchanged. Electrostimulation of hypothalamus increases acetylation of H2a and H4 histone fractions and phosphorylation of all histones with the exception of histone H1.     

In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.     

Liver microsomal cytochromes P-450 and azoreductase activity.

Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.     

Induction of UDP glucuronyltransferase in the liver and extrahepatic organs of the rat

UDP glucuronyltransferase (4-methylumbelliferone) activity was enhanced in the liver of the rat after administering phenobarbital, cinchophen or 3-methylcholanthrene, in the lung after administration of cinchophen or 3-methylcholanthrene, and in the kidney after cinchophen. The UDP glucuronyltransferase of the spleen could not be induced with the dosage of the drugs used. Chlorpromazine was not able to induce either hepatic or extrahepatic UDP glucuronyltransferase. Chlorpromazine competitively inhibited hepatic UDP glucuronyltransferase $\text{in vitro}$.     

Comparison of phenobarbital-and carcinogen-induced aldehyde dehydrogenases in the rat

There is a genetically determined variation in the inducibility of a high-K_m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo-pdioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.     

Site-specific loss of acetylation upon phosphorylation of histone H3

Post-translational modification of histones is a central aspect of gene regulation. Emerging data indicate that modification at one site can influence modification of a second site. As one example, histone H3 phosphorylation at serine 10 (Ser(10)) facilitates acetylation of lysine 14 (Lys(14)) by Gcn5 in vitro (, ). In vivo, phosphorylation of H3 precedes acetylation at certain promoters. Whether H3 phosphorylation globally affects acetylation, or whether it affects all acetylation sites in H3 equally, is not known. We have taken a genetic approach to this question by mutating Ser(10) in H3 to fix either a negative or a neutral charge at this position, followed by analysis of the acetylation states of the mutant histones using site-specific antibodies. Surprisingly, we find that conversion of Ser(10) to glutamate (S10E) or aspartate (S10D) causes almost complete loss of H3 acetylation at lysine 9 (Lys(9)) in vivo. Acetylation of Lys(9) is also significantly reduced in cells bearing mutations in the Glc7 phosphatase that increase H3 phosphorylation levels. Mutation of Ser(10) in H3 and the concomitant loss of Lys(9) acetylation has minimal effects on expression of a Gcn5-dependent reporter gene. However, synergistic growth defects are observed upon loss of GCN5 in cells bearing H3 Ser(10) mutations that are reminiscent of delays in G(2)/M progression caused by combined loss of GCN5 and acetylation site mutations. Together these results demonstrate that H3 phosphorylation directly causes site-specific and opposite changes in acetylation levels of two residues within this histone, Lys(9) and Lys(14), and they highlight the importance of these histone modifications to normal cell functions.     

Multiplicity of in vitro glucuronidation of 2-hydroxyestriol

In vitro glucuronidation of 2-hydroxyestriol has been investigated by means of HPLC with dual-electrode coulometric detection. When incubated with rat or dog liver microsomal preparation in the presence of UDPGA, 2-hydroxyestriol was transformed into the 2-glucuronide together with a small amount of 16- and/or 17-glucuronides. In contrast, incubation of 2-hydroxyestriol with guinea-pig liver microsomal preparation yielded the 3-glucuronide and a trace amount of the 2-glucuronide, but no ring D glucuronides. Upon pretreatment with 3-methylcholanthrene male rat liver exhibited a marked increase in both 2- and 3-glucuronidation activities, whereas female rat liver showed an elevation only in 2-glucuronidation. On the other hand, in male and female rats pretreatment with phenobarbital caused a relatively small increase in the glucuronidation activity of the liver. In the male guinea-pig, glucuronidation was not affected by pretreatment with either of the two compounds. The present result demonstrates the multiplicity of hepatic 2-hydroxyestriol UDP-glucuronyl-transferase in the rat, guinea-pig and dog.     

Stereoselective metabolism of the optical isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene to bay-region diol epoxides by rat liver microsomes

Enantiomerically pure isomers of trans-1,2-dihydroxy-1,2-dihydrophenanthrene have been obtained by chromatographic separation of their diastereomeric bis esters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. Liver microsomes from control rats, as well as rats treated with phenobarbital or 3-methylcholanthrene, metabolize these dihydrodiols to a pair of diastereomerically related bay-region 1,2-diol-3,4-epoxides in which the benzylic hydroxyl group and the epoxide oxygen are either cis (isomer-1) or trans (isomer-2) to each other. In general, diol epoxide-1 was the major metabolite of the (+)-(1S,2S)-dihydrodiol, whereas diol epoxide-2 was the major metabolite of the (?)-(1R-2R)-dihydrodiol. The extent of this stereoselectivity is dependent on the source of the microsomes and is greatest for liver microsomes from 3-methylcholanthrene-treated rats; the ratio of diol epoxide-1 relative to diol epoxide-2 was 5.6 : 1 with the (+)-enantiomer as substrate and 1 : 5.5 with the (?)-enantiomer as substrate. For a given microsomal preparation, rates of metabolism were independent of the enantiomer composition of the substrate. Relative to microsomes from control animals, treatment of rats with 3-methylcholanthrene enhanced rates of metabolism by about 40%, whereas treatment with phenobarbital decreased rates to a similar extent when the amounts of metabolites formed per nanomole of cytochrome P?450 were compared. The failure of treatment by 3-methylcholanthrene to enhance markedly the rate of metabolism of a polycyclic aromatic hydrocarbon substrate is unusual.     

Effect of biotin on phosphorylation,acetylation, methylation of rat liver histones

Biotin deficient rat liver histones showed decreased phosphorylation and methylation, and increased acetylation rates as compared to normal rat liver histones: these alterations may be related to the observed lower stability of the interactions between histones and DNA. The modifications of the metabolic process might be the consequence of an alteration of the synthesis of the enzymes involved in histone phosphorylation, acetylation and methylation mechanisms and are presumably related to a biotin effect upon the synthesis of RNA and proteins.