Evidence for ethylation of rat liver deoxyribonucleic acid after administration of ethionine

1. Administration of a large dose (500mg/kg body wt.) of (3)H-labelled l-ethionine to rats resulted in the incorporation of a small amount of radioactivity into the liver DNA. Considerable evidence that this radioactivity was not due to contamination of the isolated DNA with labelled protein, RNA, S-adenosyl-l-ethionine or l-ethionine was obtained. 2. After acidic hydrolysis of the DNA isolated from the livers of rats treated with labelled l-ethionine, virtually all of the radioactivity present in the DNA was found in a fraction with similar chromatographic properties to 7-ethylguanine. 3. Treatment of rats with comparable doses of l-methionine did not lead to the formation of 7-methylguanine in the liver DNA. 4. These results are discussed in relation to the induction of liver tumours by ethionine.     

Postnatal growth inhibition in the rat retina after actinomycin D adminstration]

The effects of actinomycin D on the development of the rats retina were observed. At the day of birth the inner neurons and the inner cells of the bipolar layer are vulnerable. The pale degeneration of these neurons accompanied by a dilatation of the endoplasmatic reticulum and the dark degeneration accompanied by a pycnosis and a shrinkage of the cytoplasm persist during the first 11 days after birth. The same alterations are to be seen in bipolar cells on day 11 after birth. The transient disorganisation of the inner layers could effect the ramification because the stratum reticulare internum is smaller as in untreated animals.     

Evidence for peroxisomal hydroxylase activity in rat liver

This study presents evidence for the first time that rat liver peroxisomes contain a hydroxylase capable of converting 3 alpha, 7 alpha, 12 alpha,- trihydroxy-5 beta-cholestane to a cholestanetetrol. Furthermore, this hydroxylase differs from both the mitochondrial and microsomal enzymes in its response to various co-factors. Highly purified peroxisomal, mitochondrial, and microsomal fractions from cholestryamine-treated rats were incubated with [22(23)-3H]3 alpha,7 alpha,12 alpha,-trihydroxy-5 beta-cholestane under a variety of conditions. The products were acidified, extracted, and subjected to thin-layer chromatography to determine the amount of cholestanetetrol produced. The identification of the 25- and 26-hydroxylated products from the incubations with the microsomes was confirmed by gas chromatography-mass spectrometry. Peroxisomal fractions incubated with a NADPH-generating system, Mg2+, and ATP showed a rate of 40 pmol/min/mg conversion of 3 alpha,7 alpha,12 alpha,-trihydroxy-5 beta-cholestane to a cholestanetetrol. Co-factor studies indicated that both the peroxisomal and mitochondrial hydroxylase activities were dependent on NADPH, Mg2+, and ATP (with different concentration requirements) whereas the microsomal hydroxylase(s) required only NADPH. An abstract of this work has been published (1).     

Evidence for gliotoxin-glutathione conjugate adducts

The equilibrium constant for the gliotoxin/glutathione pair was found to be 1200+/-100 M(-1) at pH 7.0 at 25 degrees C. Under conditions where the reaction was quenched rapidly with the addition of acid, gliotoxin-glutathione conjugate adducts were detected.     

DNA adducts in humans after exposure to methylating agents

Human exposure to methylating agents appears to be widespread, as indicated by the frequent occurrence of methylated DNA adducts in human DNA. The high incidence of methylated DNA adducts even in humans thought not to have suffered extensive exposure to environmental methylating agents implies that chemicals of endogenous origin, probably N-nitroso compounds such as the strongly carcinogenic N-nitrosodimethylamine (NDMA), may be primarily responsible for their formation and raises the question of the carcinogenic risks associated with such exposure. In addition to accumulation of DNA damage, other factors (such as induced cell proliferation) appear to be important in determining the probability of induction of mutation or cancer by NDMA, implying that high to low dose risk extrapolations should not be based on the assumption of dose- or even adduct-linearity. Comparative studies of the accumulation and repair of methylated adducts in humans and animals treated with methylating cytostatic drugs do not reveal significant species differences. Based on this and the dosimetry of adduct accumulation in rats chronically exposed to very low doses of NDMA, it is suggested that the exposure needed to account for the levels of adducts found in human DNA may be of the order of hundreds of micrograms NDMA (or equivalent) per day, a level of exposure which may well represent a significant carcinogenic hazard for man.     

Quantification of aristolochic acid-derived DNA adducts in rat kidney and liver by using liquid chromatography-electrospray ionization mass spectrometry

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts. The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA. In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS) was used to identify and quantify the AA-DNA adducts isolated from the kidney and liver tissues of the AA-dosed rats. The deoxycytidine adduct of AA (dC-AA) and the deoxyadenosine-AA adduct (dA-AA) were detected and quantified in the tissues of rats with one single oral dose (5mg or 30mg AA/kg body weight). The deoxyguanosine adduct (dG-AA), however, was detected only in the kidney of rats that were dosed at 30mg AA/kg body weight for three consecutive days. The amount of AA-DNA adducts found in the rats correlated well with the dosage.     

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.     

Evidence for nucleoside channeling in vivo: deoxythymidine incorporation into rat liver dTTP and nuclear matrix DNA

Previous studies in prokaryotes and in eukaryotic cell lines have indicated the possible existence of more than one dTTP pool accessible to DNA synthesis. To investigate this possibility in eukaryotes in vivo, the incorporation of [3H] deoxythymidine into nuclear matrix-attached DNA and intracellular dTTP was examined in regenerating rat liver. The labeling of matrix DNA reached a maximum after a 5 min pulse and then began to rapidly decrease. Conversely, [3H] deoxythymidine incorporation into dTTP began to increase after 5 min and peaked 10 min after injection. Since the peak specific activity for [3H] deoxythymidine incorporation into matrix DNA precedes that into dTTP, there seems to be channeling of exogenous thymidine directly to sites of DNA replication, bypassing existing nucleotide pools.     

DNA adducts formation and induction of apoptosis in rat liver epithelial 'stem-like' cells exposed to carcinogenic polycyclic aromatic hydrocarbons

The bipotent liver progenitor cells, so called oval cells, may participate at the early stages of hepatocarcinogenesis induced by chemical carcinogens. Unlike in mature parenchymal cells, little is known about formation of DNA adducts and other genotoxic events in oval cells. In the present study, we employed spontaneously immortalized rat liver WB-F344 cell line, which is an established in vitro model of oval cells, in order to study genotoxic effects of selected carcinogenic polycyclic aromatic hydrocarbons (PAHs). With exception of dibenzo[a,l]pyrene, and partly also benzo[g]chrysene and benz[a]anthracene, all other PAHs under the study induced high levels of CYP1A1 and CYP1B1 mRNA. In contrast, we observed distinct genotoxic and cytotoxic potencies of PAHs. Dibenzo[a,l]pyrene, and to a lesser extent also benzo[a]pyrene, benzo[g]chrysene and dibenzo[a,e]pyrene, formed high levels of DNA adducts. This was accompanied with accumulation of Ser-15 phosphorylated form of p53 protein and induction of apoptosis. Contrary to that, benz[a]anthracene, chrysene, benzo[b]fluoranthene and dibenzo[a,h]anthracene induced only low amounts of DNA adducts formation and minimal apoptosis, without exerting significant effects on p53 phosphorylation. Finally, we studied effects of 2,4,3',5'-tetramethoxystilbene and fluoranthene, inhibitors of CYP1B1 activity, which plays a central role in metabolic activation of dibenzo[a,l]pyrene. In a dose-dependent manner, both compounds inhibited apoptosis induced by dibenzo[a,l]pyrene, suggesting that it interferes with the metabolic activation of the latter one. The present data show that in model cell line sharing phenotypic properties with oval cells, PAHs can be efficiently metabolized to form ultimate genotoxic metabolites. Liver progenitor cells could be thus susceptible to this type of genotoxic insult, which makes WB-F344 cell line a useful tool for studies of genotoxic effects of organic contaminants in liver cells. Our results also suggest that, unlike in mature hepatocytes, CYP1B1 might be a primary enzyme responsible for formation of DNA adducts in liver progenitor cells.     

Evidence for a new activator of rat liver phosphofructokinase

A low molecular weight compound that activates purified rat liver phosphofructokinase has been isolated and partially purified from rat hepatocyte extracts. It can be separated from both fructose bisphosphate and AMP on DEAE-Sephadex. Incubation of rat hepatocytes with glucagon lowers the level of this activator, and this accounts for the inhibition of phosphofructokinase that was observed in hepatocyte extracts (S. Pilkis, et al. (1979) Biochem. Biophys. Res. Commun. $\text{88}$, 960–967). Other characteristics of this activator are described which suggest that it is not any of the known effectors of rat liver phosphofructokinase.     

Evidence for glucose-6-phosphate transport in rat liver microsomes

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.