Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate β-mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A⁺)-RNA. Poly (A⁺)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A⁺)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.     

Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

Selenium and hepatic microsomal hemoproteins

The microsomal share of liver homogenate ⁷⁵Se after injection of a tracer dose of ⁷⁵SeO₃^2? was three times greater in rats fed a selenium-deficient diet than in rats fed a selenium-adequate diet. Basal levels of microsomal cytochromes P-450 and b₅ were unaffected by selenium deficiency. However, induction of these cytochromes by phenobarbital was markedly inpaired in selenium-deficient rats, whereas liver weight increase and NADPH cytochrome $\text{c}$ reductase induction were not impaired. These data indicate that selenium is essential for phenobarbital induction of microsomal hemoproteins.     

Hydroxylation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by rat liver microsomes

Ring hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur in the presence of liver microsomes prepared from both normal and phenobarbital induced rats. The metabolite was identified by mass spectrometry after selective extraction and purification by liquid chromatography. The microsomal catalyzed reaction was oxygen and NADPH dependent, inhibited by carbon monoxide and induced 4–5 fold by $\text{in vivo}$ phenobarbital pre-treatment. Phenobarbital induced microsomes hydroxylated the substrate at a rate of 17.6 nmoles/min/mg protein at 37°. A Type I difference spectrum was observed with phenobarbital induced microsomes that also displayed a substrate binding constant (K_s of 4 × 10^?5 M.     

Hepatic cytochrome P450 2B induction by ethyl/ phenyl-substituted congeners of phenobarbital in the B6C3F1 mouse

The abilities of structural congeners of phenobarbital to induce immunoreactive hepatic cytochrome P450 2B (CYP2B) protein and associated catalytic activity (benzyloxyresorufin O-dealkylation) in the male B6C3F1 mouse were examined. Interspecies differences in inducing ability were examined through comparison of the results with induction data obtained previously with the male F344/NCr rat. The congeners were administered in the diet for 2 weeks at concentrations equimolar to 500 ppm of the prototype CYP2B inducer, phenobarbital. Of the series of compounds tested, phenobarbital was the most effective inducer of benzyloxyresorufin O,-dealkylation and immunoreactive CYP2B protein, with 2-ethyl-2-phenylsuccinimide, 5-ethyl-5-phenylhydantoin, primidone, and glutethimide being only 19–42% as effective. 5-Ethyl-5-phenyloxazolidinedione and the ring-opened and decarboxylated congeners, N-(2-ethyl-2-phenylacetyl)urea and 2-ethyl-2-phenyl-malonamide, displayed minimal induction of these catalytic activities. Dose-response experiments performed with 5-ethyl-5-phenylhydantoin indicated that the intrinsic CYP2B-inducing activity of this congener was as great as that of phenobarbital in the mouse, although a fourfold greater dietary concentration of this hydantoin (2000 ppm) was required to elicit a response equivalent to that caused by 500 ppm phenobarbital. When extent of induction was related to serum total xenobiotic concentration rather than to administered dietary concentration, the potencies of the two congeners were determined to be more similar (58 vs. ≥78 μ M for phenobarbital and 5-ethyl-5-phenylhydantoin, respectively).     

Deacylation of carcinogenic 5-nitrofuran derivatives by mammalian tissues

The deacylations of N-[4-(5-nitro-2-furyl)-2-thiazolyl] fonnamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl] acetarnide (NFTA) and formic acid 2-[4-(5-nitro-2-furyl)-2-thiazolyl] hydrazide (FNT) by liver, kidney, small intestines and stomach of mouse, rat, hamster and guinea pig were investigated. FANFT was deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT). FANFT formamidase activity was higher in the liver and small intestines of mouse, hamster and guinea pig, and small intestines and stomach of rat. There was no detectable FANFT formamidase activity in the stomach of the mouse and hamster. Neither NFTA nor FNT was deacylated by the rodent tissue homogenates studied. It is suggested that (1)4 ANFT is a metabolite of FANFT but not NFTA; (2) 2-hydrazino-4-(5-nitro-2-furyl)thiazole (HNFT) may not be a metabolite of FNT; and (3) the induction of tumors by FANFT, NFTA and FNT may not be due to a common carcinogenic metabolite, although these chemicals demonstrate similar organ specificities in some of these rodents.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.     

Synthesis,Molecular Conformation and Activity Against Herpes Simplex Virus of (E)-5-(2-Bromovinyl)-2′-Deoxycytidine Analogs

Analogs of (E)-5-(2-bromovinyl)-2 ′-deoxycytidine (BrVdCyd) (1) by substitution at N⁴ were synthesized to impart resistance against deamination. The anti-HSV-1 activity and solution conformation of these analogs were determined. N⁴-Acetyl-BrVdCyd (2) was a potent inhibitor of HSV-1 replication whereas N⁴-propanoyl-BrVdCyd (3) had good activity and N⁴-Butanoyl-BrVdCyd (4) had only low activity against HSV-1 replication. N⁴-Methyl-BrVdCyd (5) was devoid of activity against HSV-1.     

Increased activity of rat liver N2-guanine tRNA methyltransferase II in response to liver damage

Alterations in rat liver transfer RNA (tRNA) methyltransferase activities have been observed after liver damage by various chemicals or by partial hepatectomy. The qualitative and quantitative nature of these activity changes and the time course for their induction have been studied. Since homologous tRNAs are essentially fully modified in vivo, E. coli tRNAs were used as in vitro substrates for the rat liver enzymes in these studies. Each of the liver-damaging agents tested rapidly caused increases in activities of the enzyme(s) catalyzing methyl group transfer to tRNAs that have an unmodified guanine at position 26 from the 5′ end of the molecule. This group of tRNAs includes E. coli tRNA^Nfmet, tRNA^Ala₁, tRNA^Leu₁, or ^Leu₂, and tRNA^Ser₃ (Group 1). In each case N²-methylguanine and N²,N²-dimethylguanine represented 90% or more of the products of these in vitro methylations. The product and substrate specificity observed are characteristic of N²-guanine methyltransferase II ($\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine:tRNA}$ (guanine-2)-methyltransferase, EC 2.1.1.32). In crude and partially purified preparations derived from livers of both control and treated animals this enzyme activity was not diminished significantly by exposure to 50°C for 10 min. The same liver-damaging agents induced little or no change in the activities of enzymes that catalyze methyl group transfer to various other E. coli tRNAs that do not have guanine at position 26 (Group 2). The results of mixing experiments appear to rule out the likelihood that the observed enzyme activity changes are due to stimulatory or inhibitory materials present in the enzyme preperations from control or treated animals. Thus, our experiments indicate that liver damage by each of several different methods, including surgery or administration of chemicals that are strong carcinogens, hepatotoxins, or cancer-promoting substances, all produce changes in liver tRNA methyltransferase activity that represent a selective increase in activity of N²-guanine tRNA methyltransferase II. It is proposed that the specificity of this change is not fortuitous, but is the manifestation of an as yet unidentified regulatory process.     

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.     

Rat liver glutathione S-transferase B: the functional mRNAs specific for the Ya Yc subunits are induced differentially by phenobarbital

Liver poly(A⁺)-RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-free system in order to examine the kinetics of induction of the translatable mRNAs encoding each subunit of glutathione S-transferase B. Translatable glutathione S-transferase B mRNA levels were maximally elevated at 16 to 24 h after a single injection of phenobarbital. Interestingly, the functional mRNA specific for the low-molecular-weight subunit was elevated markedly by phenobarbital administration whereas the mRNA specific for the high-molecular-weight subunit was only increased slightly. Our data suggest that different mRNAs direct the synthesis of the two subunits of glutathione S-transferase B and that these two mRNAs are under independent regulation.     

Induction of cytochrome P-450-dependent hydroxylases in primary monolayer cultures of rat hepatocytes

Hydroxylation of androstenedione was studied in rat hepatocytes in primary monolayer culture following induction with phenobarbital. Six days after addition of phenobarbital and seven days after isolation of cells from liver, a maximal induction of total androstenedione hydroxylation of 5–6 times was seen at a phenobarbital concentration of 1·10⁻⁴ M. The 6β-, 7α- and 16α-hydroxylase activities showed different responses towards phenobarbital in agreement with the contension that different forms of cytochrome P-450 with different sensitivity towards phenobarbital participate in hepatic steroid hydroxylation. These results were obtained with cells supplemented with 1% (v/v) rat serum. The present cell culture system should be suitable for in vitro studies on mechanisms of induction of cytochrome P-450-dependent enzymes in normal liver cells.     

Assessment of membrane damage in continuous cultures of mammalian cells

The present studies were aimed at evaluating procedures for assessing the effect of chemicals on the integrity of the plasma membrane in continuous cell cultures. The degree of membrane damage was monitored by determining the ‘leakage’ of α-[³H]aminoisobutyric acid ([³H]AIB) and [¹⁴C]deoxy-2-fluoro-D-glucose ([¹⁴C]FdG) from the prelabelled cells. These parameters were compared to the loss of lactate dehydrogenase (LDH) from the cells and the decrease in the intracellular level of K⁺. Triton X-100, sodium dodecylsulfate (SDS), phospholipase C and nystatin which are known to affect membranes by different mechanisms served as test agents. In parallel, we monitored the effects of the chemicals on the viability of the cells. The following results were obtained:(1) The two radioactive markers [³H]AIB and [¹⁴C]FdG were found to be suitable to probe for damages of the plasma membrane in a variety of continuous cell lines which differ widely in their phenotype, rate of growth and degree of differentiation. (2) The leakage of the two markers could conveniently be monitored by double labelling techniques. (3) The loss from the cells of the 3 markers of smaller molecular size, K⁺, [³H]AIB, [¹⁴C] FdG, differed considerably depending on the test agent used. (4) Intracellular K⁺ level and [³H]AIB leakage generally appeared to follow a similar pattern, whereas [¹⁴C]FdG leakage may have shown a distinctly different response. (5) The leakage of LDH was an insensitive indicator for membrane damage. (6) No clear relationship was detectable between a particular leakage pattern of the markers and the loss of cellular viability.     

Induction of mixed function oxidase (MFO) system in two species of antarctic fish from Terra Nova Bay (Ross Sea)

Summary The aim of this study was to investigate the detoxicant Mixed Function Oxidase system in two species of Antarctic fish, C. hamatus and P. bernacchii, collected during the Antarctic summer of 1989–1990 at the Italian Scientific Station at Terra Nova Bay (Ross Sea). Several specimens were induced by injection of phenobarbital (PB), 3-methylcholanthrene (3-MC) and PCBs in the caudal vein. The results show significant differences between the two species. In C. hamatus, basal activity of benzo(a)pyrene monooxygenase (BPMO) was among the lowest measured even in fish of temperate seas, and in P. bernacchii it was 20 times lower. The values of regenerating activities (NADPH-cytochrome c reductase (NADPH-CYTCRED), NADH-cytochrome c reductase (NADH-CYTCRED) and NADH-ferricyanide reductase (NADH-FERRIRED) suggest that the two species use different electron donor molecules. Injection of chemicals in the caudal vein did not provoke induction of MFO activity in P. bernacchii. In C. hamatus, phenobarbital and PB-type inducers did not cause induction but there was a statistically significant response to 3-MC.     

Manipulation of ginsenoside heterogeneity of <Emphasis Type="Italic">Panax notoginseng</Emphasis> cells in flask and bioreactor cultivations with addition of phenobarbital

Structure-similar ginsenosides have different or even totally opposite biological activities, and manipulation of ginsenoside heterogeneity is interesting and significant to biotechnological application. In this work, addition of 1 mM phenobarbital to cell cultures of Panax notoginseng at a relatively high inoculation size of 7.6 g dry cell weight (DW)/L enhanced the production of protopanaxatriol-type (Rg₁ + Re) ginsenosides in both shake flask and airlift bioreactor (ALR, 1 L working volume). The content of Rg₁ + Re in the ALR was increased from 42.5 ± 4.0 mg per gram DW in untreated cell cultures (control) to 56.4 ± 4.6 mg per gram DW with addition of 1.0 mM phenobarbital. The maximum productivity of Rg₁ + Re in the ALR reached 5.66 ± 0.38 mg L⁻¹ d⁻¹, which was almost 3.3-fold that of control. The maximum ratio of the detectable ginsenosides protopanaxatriol:protopanaxadiol (Rb₁) was 7.6, which was about twofold that of control. The response of protopanaxadiol 6-hydroxylase (P6H) activity to phenobarbital addition coincided with the above-mentioned change of ginsenoside heterogeneity (distribution). Phenobarbital addition is considered as a useful strategy for manipulating the ginsenoside heterogeneity in bioreactor with enhanced biosynthesis of protopanaxatriol by P. notoginseng cells.     

Repair replication of opossum lymphocyte DNA: effect of compounds that bind to DNA

Opossum lymphocytes were used for studies of DNA repair. Several compounds were assessed for their capacity to induce repair. Specially interesting was the fact that some intercalators (proflavin, ICR-170, quinacrine and acridine orange) did induce repair, as determined by [³H]thymidine incorporation in the presence of hydroxyurea, CsCl density gradient centrifugation of bromodeoxyuridine-containing DNA and autoradiographically detected unscheduled DNA synthesis.A comparison of the inhibitory effect of several chemicals on DNA replication and DNA repair was also carried out. In this study, repair synthesis was induced by UV irradiation. For most of the compounds, the concentration necessary to inhibit 50% of DNA replication or DNA repair was similar. The most notable exception was cycloheximide which inhibited replication much more effectively than repair. None of the compounds used in this study was found to specifically inhibit repair synthesis.Inhibition of DNA replication and DNA repair was a general effect exhibited by the compounds which bind to DNA. However, only some of these compounds were able to induce repair. As most of these compounds were mutagens it was concluded that the inhibitory effect could be more relevant to mutagenesis that the repair-induction effect.     

Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbital and iron on hepatic uroporphyrinogen decarboxylase. Implications for the pathogenesis of porphyria.

Treatment of cultured chick embryo hepatocytes with phenobarbital, polychlorinated biphenyl compounds and 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in increased delta-aminolaevulinate synthase and decreased uroporphyrinogen decarboxylase activities and porphyrin accumulation; uroporphyrin and heptacarboxyporphyrin predominated. Iron had no effect on these changes. Simultaneous treatment of cultures with dioxin and phenobarbital produced a synergistic response in delta-aminolaevulinate synthase induction, uroporphyrinogen decarboxylase inhibition and porphyrin accumulation. These data suggest that an inhibitor of uroporphyrinogen decarboxylase may be generated in the liver from polychlorinated biphenyl compounds or dioxin by metabolic activation. Additionally these findings bear on the postulated role of these and related chemicals in determining the low levels of uroporphyrinogen decarboxylase activity in porphyria cutanea tarda patients.