Collagenase perfusion of rat liver induces DNA damage and DNA repair in hepatocytes

Evidence is presented that the collagenase perfusion of adult rat liver results in significant damage to nuclear DNA as evaluated by the alkaline elution technique. The extent of the damage is related to the perfusion time as well as to the clostridial enzyme preparation used. The DNA structure of isolated cells is almost completely repaired within 12 h of their culture in chemically defined medium.     

Induction of DNA repair in human and rat hepatocytes by 1,6-dinitropyrene

1,6-Dinitropyrene (DNP) was found to be an extremely potent genotoxicant in metabolically competent primary cultures of human and rat hepatocytes. Dose-dependent increases in DNA repair as measured by unscheduled DNA synthesis (UDS) were observed in the range from 0.05 to 5 microM 1,6-DNP for both species, indicating that the rat-hepatocyte assay is an appropriate model for assessing genotoxic potential in human hepatocytes for this class of compound. Unlike some nitroaromatic compounds, 1,6-DNP did not require gut flora for metabolic activation. No DNA repair was observed in hepatocytes isolated from rats treated with 50 mg/kg 1,6-DNP in corn oil by gavage 2, 12 or 24 h previously. The reason for the lack of a response in vivo is not known, but may relate to detoxification or distribution of the compound in the animal.     

Characterization of gene-specific DNA repair by primary cultures of rat hepatocytes

At present, almost all the information on gene-specific DNA repair in mammals comes from studies with transformed cell lines and proliferating primary cells obtained from rodents and humans. In the present study, we measured the repair of specific DNA regions in primary cultures of nondividing rat hepatocytes (parenchymal cells). DNA damage was induced by irradiating the primary cultures of hepatocytes with ultraviolet (UV) light, and the presence of cyclobutane pyrimidine dimers (CPDs) was measured by using T4 endonuclease V in the following: a 21-kb BamHI fragment containing the albumin gene, a 14-kb BamHI fragment containing the H-ras gene, and the genome overall. The frequency of CPDs in the two BamHI fragments and the genome overall were similar and ranged from 0.5 to 1.3 CPDs per 10 kb for UV doses of 5–30 J/m². However, the removal of CPDs from the DNA fragment containing the albumin gene was significantly higher than from that of the genome overall and the DNA fragment containing the H-ras gene. Within 24 hr, approximately 67% of the CPDs was removed from the DNA fragment containing the albumin gene versus less than 40% for the genome overall and the DNA fragment containing the H-ras gene. The lower repair observed for the 14-kb fragment containing the H-ras gene is probably indicative of repair of the nontranscribed region of this fragment because the H-ras gene makes up only 2.4 kb of the 14-kb fragment. Primary cultures of hepatocytes removed CPDs from the transcribed strand of albumin fragment more efficiently than from the nontranscribed strand; however, no differences were observed in the repair of the two strands of the fragment containing the H-ras gene. These results demonstrate that primary cultures of nondividing rat hepatocytes show differential repair of UV-induced DNA damage that is comparable to what has been reported for transformed, proliferating mammalian cell lines. J. Cell. Physiol. 176:314–322, 1998. © 1998 Wiley-Liss, Inc.     

Kinetics of induction and decay of error-prone DNA repair activity in Escherichia coli after treatment with nalidixic acid

Inducible error-prone DNA repair activity was detected by infecting nalidixic acid-pretreated E. coli cells with UV-irradiated phage phi X174. Induction and decay kinetics of reactivation very much resembled that of mutagenesis of the UV-damaged phage. Repair as well as mutagenic activity increased for about 30 min. The maximal error-prone repair capacity, which was induced in the cell during the 30 min nalidixic acid treatment, rapidly died out during subsequent cell growth in absence of nalidixic acid. Induction of this repair mode was not observed in a recA- mutant. In the presence of nalidixic acid plus rifampicin both repair and mutagenic effects were abolished.     

Tumor-promoting barbiturates act on DNA repair of cultured hepatocytes

We have observed that two promoters of liver carcinogenesis, i.e. phenobarbital and barbital, markedly increase DNA-repair synthesis of cultured hepatocytes following treatment with the ultimate carcinogens methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, N-acetoxy-2-acetylaminofluorene, and UV light of 254 nm. Phenobarbital also increased the incorporation rates of deoxynucleoside triphosphates into nuclear DNA of permeabilized hepatocytes following carcinogen treatment. The action of these barbiturates apparently correlates with their potential to promote hepatocarcinogenesis in vivo, since the non-promoting agent barbituric acid did not modify carcinogen-induced repair synthesis. Moreover, the mechanisms of action of tumor-promoting barbiturates is different from the known enhancing action on repair synthesis of inhibitors of nuclear poly(ADP-ribose) biosynthesis.     

The effects of putative DNA repair inhibitors on DNA adduct levels and unscheduled DNA synthesis in rat hepatocytes exposed to 2-acetylaminofluorene

The enzymology of DNA repair is currently under active investigation. The purpose of the present study was to examine the involvement of a number of enzymes (DNA polymerase alpha and beta, DNA topoisomerase II and ribonucleotide reductase) in the repair of chemically induced DNA damage in a mammalian cell system. This was done by studying the effects of inhibitors of these enzymes on the levels of 2-acetylaminofluorene (2-AAF)-DNA adducts and on the induction of UDS in primary cultures of rat hepatocytes exposed to the carcinogen in vitro. The results obtained with aphidicolin (an inhibitor of DNA polymerase alpha) show that the binding of 2-AAF to cellular DNA was significantly higher in samples exposed to this compound. Moreover, induction of UDS by 2-AAF was completely blocked in the presence of this compound. Dideoxythymidine, a DNA polymerase beta inhibitor, led to complex results. It produced a reduced DNA-specific activity due to [3H]2-AAF adduct formation as well as a diminished but still detectable UDS response in the presence of 2-AAF. Inhibitors of DNA topoisomerase II (nalidixic acid) and ribonucleotide reductase (hydroxyurea) did not cause any statistically significant change in the accumulation of 2-AAF adducts nor did they affect the induction of UDS. The data clearly suggest that DNA polymerase alpha participates in the repair of 2-AAF adducts in hepatocytes. In addition, neither DNA topoisomerase II activity, nor limitations in the precursor nucleotide pools appear to be critical factors in this process.     

Effect of the bile acid taurolithocholate on cell calcium in saponin-treated rat hepatocytes

Neomycin was used to assess the involvement of Ins (1,4,5)P3 in the Ca2+ release from the endoplasmic reticulum induced by the bile acid taurolithocholate. In saponin-permeabilized rat hepatocytes, neomycin via its ability to bind Ins (1,4,5)P3 abolished the release of Ca2+ induced by added Ins (1,4,5)P3. In contrast, it did not alter the Ca2+ release initiated by the bile acid. In intact cells, neomycin had no effect on the [Ca2+]i rises promoted by taurolithocholate and vasopressin. It is suggested that the effect of taurolithocholate in liver is not mediated by Ins (1,4,5)P3 but results from a primary action on endoplasmic reticulum.     

Effect of methylamalonic acid on gluconeogenesis in isolated rat and guinea-pig hepatocytes.

Gluconeogenesis from pyruvate, alanine, lactate and propionate was inhibited by methylmalonate in both rat and guinea-pig hepatocytes. The effect was dose-dependent. Gluconeogenesis from glycerol and xylitol was not affected.     

Effect of the bacterial DNA gyrase inhibitors, novobiocin, nalidixic acid, and oxolinic acid, on oxidative phosphorylation

When incubated with isolated intact rat liver mitochondria, novobiocin and nalidixic acid act as uncouplers of oxidative phosphorylation; they stimulate oxygen uptake and inhibit ATP synthesis. Novobiocin is about as powerful an uncoupler as is 2,4-dinitrophenol, nalidixic acid is somewhat less powerful, and oxolinic acid exerts no inhibition whatsoever at the concentrations used. The three inhibitors are without effect on oxidative phosphorylation in Escherichia coli nor does novobiocin affect this process in a novobiocin-permeable mutant of yeast. While it would appear that oxolinic acid may be a relatively specific tool for the manipulation of the superhelicity of DNA in complex systems such as mammalian mitochondria and intact mammalian cells, the specificity of each of these inhibitors may depend upon the particular conditions and species used and such experiments require adequate controls on oxidative phosphorylation.     

Influence of the culture time on DNA damage and repair in isolated rat hepatocytes exposed to nitrochlorobenzene derivatives

The induction of DNA damage on 1.5- and 24-h cultured hepatocytes was tested after a 3-h exposure to 5 and 50 microM mono-, di-, and trinitrochlorobenzene (100-00-5; 97-00-7; 88-88-0). DNA-repair synthesis, elicited by nitrochlorobenzene treatment, was also estimated 24 and 48 h after the withdrawal of the nitro-aryl halides. DNA damage and repair were evaluated by determining the DNA elution rate in alkali. A dose-related rate of DNA damage was obtained by exposure of 1.5-h-cultured hepatocytes to 5 and 50 microM nitrochlorobenzenes . DNA of 24-h-cultured cells was not affected by nitrochlorobenzene treatment. The data obtained by exposure to 5 microM methyl methanesulfonate (66-27-3) and nitrosodimethylamine (62-75-9), direct and indirect methylating agents, suggest that 24-h-cultured liver cells are still able to transform nitrosodimethylamine but not nitrochlorobenzenes . Isolated hepatocytes maintain their capability of repairing the induced DNA damage when cultured for 24 and 48 h in fresh medium. The system offers an interesting model to investigate the perturbations related to the metabolism of xenobiotics.     

Effect of nalidixic acid on sporulation of catabolite-resistant mutants ofBacillus subtilis

A number ofBacillus subtilis mutants that are able to sporulate in the presence of relatively high concentrations of carbon sources have been isolated in our laboratory. The present study shows that some of the mutants are also able to sporulate in the presence of nalidixic acid (Nal) under the condition where sporulation of wild-type strains is inhibited. Furthermore, it has been found that a Nal-resistant mutant is unable to sporulate normally when Nal (40 μg/ml) or glucose (55 mM) is present. Since the adverse effect of Nal on inducible enzymes is eliminated when bacterial strains carry a Nal-resistance marker, the above result suggests that the effect of this drug on sporulation might be mediated by a unique mechanism.     

Effect of nalidixic acid on Euglena gracilis: induced loss of chloroplast deoxyribonucleic acid

Optimum conditions for bleaching of Euglena gracilis by nalidixic acid were 50 μg/ml at pH 3.8. The inhibitor did not affect cell division; however, the bleaching efficiency approached 100% after 2 generations. Within 1.5 generations of exposure to nalidixic acid the quantity of chloroplast DNA decreased at least 10-fold and was no longer detectable by equilibrium density-gradient ultracentrifugation. Replication of chloroplast deoxyribonucleic acid was completely inhibited by nalidixic acid.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Bile acid synthesis in isolated rat hepatocytes

Normal adult rat hepatocytes were incubated for 48h and the concentration of total and individual bile acids in homogenized samples of the culture was measured at intervals during the incubation, using radiogas chromatography and isotope derivative assay. The net increase in bile acids over the value observed at the start of the culture was taken as synthesis. The results showed that bile acid synthesis was linear up to 24h of incubation, at a rate of 20nmol/g hepatocytes per hour, and that 85% of the newly synthesized bile acid was cholic acid. The bile acid synthesized was mainly conjugated with taurine. These results suggest that isolated hepatocytes cultured in the way described could be a useful in vitro model for the study of bile acid synthesis.