The effect of aflatoxin B1 on normal and cortisol-stimulated rat liver ribonucleic acid synthesis

1. Aflatoxin B(1), administered in vivo, inhibits the incorporation of [(14)C]orotic acid in vivo into rat liver nuclei, and also inhibits both Mg(2+)- and Mn(2+)-dependent RNA polymerase activities in nuclei assayed in vitro. 2. Aflatoxin B(1) inhibits the cortisol-induced increase in incorporation of [(14)C]leucine in vivo, but does not affect the control value of this activity. 3. Aflatoxin B(1) administered in vivo inhibits the increase in nuclear Mg(2+)-dependent RNA polymerase activity, assayed in vitro, which results from the treatment with cortisol. 4. Adrenalectomy causes a decrease in Mg(2+)-dependent RNA polymerase activity. The effect on this enzymic activity of adrenalectomy plus treatment with aflatoxin B(1) is no greater than that of treatment with aflatoxin B(1) alone. 5. These results suggest that the inhibition of cortisol-stimulated biochemical pathways by aflatoxin B(1) is due to an inhibition of cortisol-stimulated RNA synthesis. 6. The cytoplasmic action of aflatoxin is thought to be due to a competition for receptor sites on the endoplasmic reticulum between steroid hormones and aflatoxin B(1). No evidence was obtained for a similar competition for nuclear receptor sites between [(3)H]cortisol and aflatoxin B(1). 7. No differences were observed between the activities of RNA polymerase preparations solubilized from control or aflatoxin-inhibited nuclei. 8. No differences in ;melting' profiles were observed between DNA and chromatin preparations isolated from control nuclei or from aflatoxin-inhibited nuclei. 9. It is suggested that aflatoxin B(1) exerts its effect on RNA polymerase by decreasing the template capacity of the chromatin and that the aflatoxin ;target' area of the chromatin includes that region which is stimulated by cortisol. This process, however, does not involve inhibiting the movement of cortisol from the outside of the hepatic cell to the nuclear chromatin.     

Actinomycin D-binding in vivo: active chromatin preferred

Escherichia coli RNA polymerase and the endogenous engaged RNA polymerase I were used as specific probes to monitor the physiologically inactive and active nucleolar chromatin template function, respectively. Actinomycin D bound preferentially to the physiologically active regions of rat liver nucleolar chromatin in vivo.     

Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin.

Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.     

Inhibition of hepatic deoxyribonucleic acid-dependent ribonucleic acid polymerases by the exotoxin of Bacillus thuringiensis in comparison with the effects of α-amanitin and cordycepin

The action of Bacillus thuringiensis exotoxin, a structural analogue of ATP, on mouse liver DNA-dependent RNA polymerases was studied and its effects were compared with those of alpha-amanitin and cordycepin. (1) Administration of exotoxin in vivo caused a marked decrease in RNA polymerase activity of isolated nuclei at various concentrations of Mg(2+), Mn(2+) and (NH(4))(2)SO(4). A similar action was recorded after addition of exotoxin to isolated nuclei from control or exotoxin-treated mice. (2) Chromatographic separation of nuclear RNA polymerases from mice treated in vivo with exotoxin showed a drastic decrease of the peak of nucleoplasmic RNA polymerase, whereas the peak of nucleolar RNA polymerase remained unaltered. The same effect was observed after administration of alpha-amanitin in vivo, but cordycepin did not alter the relative amounts of the two main RNA polymerase peaks. (3) Administration of exotoxin in vivo did not alter the template activity of isolated DNA or chromatin tested with different fractions of RNA polymerase from control or exotoxin-treated mice. (4) Addition of exotoxin to isolated liver RNA polymerases inhibited both enzyme fractions. However, the alpha-amanitin-sensitive RNA polymerase was also 50-100-fold more sensitive to exotoxin inhibition than was the alpha-amanitin-insensitive RNA polymerase. Kinetic analysis indicated the exotoxin produces a competitive inhibition with ATP on the nucleolar enzyme, but a mixed type of inhibition with nucleoplasmic enzyme. The results obtained indicate that the B. thuringiensis exotoxin inhibits liver RNA synthesis by affecting nuclear RNA polymerases, showing a preferential inhibition of the nucleoplasmic alpha-amanitin-sensitive RNA polymerase.     

Some studies of the effects of aflatoxin B1 in vivo and in vitro on nucleic acid synthesis in rat and mouse.

The mouse compared with the rat, is more resistent to the acute toxic action of aflatoxin B1 and is refractory to its hepatocarcinogenic properties. Aflatoxin B1 inhibits DNA synthesis more strongly than RNA synthesis in the rat, and both nucleic acid syntheses more strongly in rat than in the mouse. Mouse hepatic microsomes, like those of the rat, are capable of metabolizing aflatoxin B1 in vitro in the presence of NADPH, to an active form which binds to DNA both in solution and in intact nuclei and also inhibits nuclear RNA synthesis. Non NADPH-dependent binding of aflatoxin B1 to nuclei is not effective in inhibiting RNA polymerase and is largely removed by washing with lipid solvents. Mouse nuclear RNA polymerases particularly Mn 2+ (NH4)2SO4 primed acitivity are more resistant to inhibition in vitro by activated aflatoxin B1 than are the corresponding enzyme activities in rat liver nuclei. This would appear to be due to the bound aflatoxin B1 being less efficient in the case of the mouse nucleus, in inhibiting RNA synthesis. Mouse liver slices exhibit a much lesser degree of inhibition of RNA synthesis by aflatoxin B1 than do rat liver slices. Accompanying this is a lower level of binding of aflatoxin B1 to subcellular particulate fractions in the mouse liver slice compared to the rat, this disparity being most marked in the case of the nuclear fraction. The suggestion is made that the resistance of RNA synthesis in the mouse liver, to aflatoxin B1, and perhaps also resistance to its toxicity, is dependent, not on a lower capacity to activate the toxin, but (a) on a less efficient inhibition of RNA synthesis by nuclear bound toxin, and (b) a detoxifying mechanism at least partially situated in the cytosol fraction.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.     

Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Androgenic regulation of elongation of polyribonucleotide chains on rat ventral-prostate chromatin.

The kinetics of polyribonucleotide-chain elongation by rat ventral-prostate RNA polymerase B with homologous chromatin as a template were investigated. Chain elongation was measured under conditions wherein all initiation had occurred, no reinitiation took place and the reaction rate was constant. The kinetic behaviour of prostate RNA polymerase B was consistent with a mathematical model formulated for the multisubstrate enzyme. The addition of each nucleoside triphosphate was independent of the other three. The overall rate of chain elongation was lower when prostate chromatin from castrated rats was used than with prostate chromatin from normal rats. The inclusion of dihydrotestosterone-receptor complexes stimulated the rate of elongation. Androgenic effects did not appear to be directed towards the addition of individual nucleoside triphosphates, but probably towards one of the other major events in RNA-chain elongation, i.e., unwinding of DNA or movement of the enzyme along the template.     

The effects of O-acetylsterigmatocystin and related compounds on rat liver and cultured chicken embryonal liver cells.

Fine structural nucleolar changes induced in rat liver and primary tissue culture cells from 10-day-old chicken embryonal liver by O-acetylsterigmatocystin (AcO-stg), related compounds and aflatoxin B1 were compared. (1) Male Wistar rats were given a single i.p. injection of sterigmatocystin (stg), AcO-stg, and aflatoxin B1. 3 days after the injection of 15 mg/kg of stg, sporadic single cell necrosis was observed in rat liver, whereas rats treated with 8 mg/kg AcO-stg or more, and 3 mg/kg of aflatoxin B1 showed massive liver necrosis. Acetylation resulted in a marked increase in solubility in polar organic solvents. This increased solubility could play an important role in determining toxicity. (II) Treatment with the compounds with an unsaturateddelta1,2-furobenzofuranring system, such as AcO-stg, demethyl-diacetyl-stg (deMe-diAc-stg), and aflatoxin B1, resulted in nucleolar segregation and fragmentation of primary culture cells. Both parenchymal and mesenchymal cells in culture were susceptible to AcO-stg and deMe-diAc-stg, while the mesenchymal cells were more resistant to aflatoxin B1 than the hepatocytes. The inhibition of RNA synthesis in both cell types as determined in radioautography was in accordance with the electron-microscopic observations. Acetyldihydrosterigmatocystin (AcO-dihyd-stg), a saturated delta1,2-furobenzofuranring compound, was less toxic to primary tissue culture cells.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.