Evidence of DNA repair in the guinea pig pancreas, in vivo and in vitro, following exposure to N-methyl-N-nitrosourethane

The nature of DNA damage induced by N-methyl-N-nitrosourethane (NMUT) in the guinea pig pancreas, both in vitro and in vivo, and subsequent repair was investigated by alkaline sucrose density gradient analysis, using a non-radioactive fluorimetric procedure for DNA determination in gradient fractions. In vitro exposure of pancreatic slices to 20 mM NMUT for 30 min damaged DNA to less than 2.24 . 10(6) dalton fragments. However, incubation of NMUT-treated slices for 3 h in a fresh medium resulted in the repair of most of DNA damage, as indicated by the conversion of low molecular weight DNA fragments into heavy DNA of molecular weight comparable to DNA from control slices. Additionally, a single administration of NMUT (30 mg/kg, i.p.) to guinea pigs induced extensive DNA damage, to less than 2.24 . 10(6) dalton fragments in the pancreas within 4 h; similar DNA damage was observed in the liver. However, in the pancreas and liver of guinea pigs sacrificed at increasing intervals after NMUT administration, there was a gradual conversion of shortened DNA fragments to heavy high molecular weight DNA, indicating repair of DNA damage. It appears that most of DNA damage in the pancreas and liver was repaired by 14 and 7 days, respectively, following NMUT administration.     

Metabolism of homovanillamine to homovanillic acid in guinea pig liver slices.

BACKGROUND/AIMS: Homovanillamine is a biogenic amine that it is catalyzed to homovanillyl aldehyde by monoamine oxidase A and B, but the oxidation of its aldehyde to the acid derivative is usually ascribed to aldehyde dehydrogenase and a potential contribution of aldehyde oxidase and xanthine oxidase is usually ignored. METHODS: The present investigation examines the metabolism of homovanillamine to its acid derivative by concurrent incubation with monoamine oxidase and aldehyde oxidase. In addition, the metabolism of homovanillamine in freshly prepared and cryopreserved liver slices is examined and the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activity by using specific inhibitors of each oxidizing enzyme is compared. RESULTS: Homovanillamine was rapidly converted mainly to homovanillic acid when incubated with both momoamine oxidase and aldehyde oxidase. Homovanillic acid was also the main metabolite in the incubations of homovanillamine with freshly prepared or cryopreserved liver slices, via the intermediate homovanillyl aldehyde. The acid formation was 70-75 % inhibited by disulfiram (specific inhibitor of aldehyde dehydrogenase), whereas isovanillin (specific inhibitor of aldehyde oxidase) inhibited acid formation to a lesser extent (50-55 %) and allopurinol (specific inhibitor of xanthine oxidase) had almost no effect. CONCLUSIONS: Homovanillamine is rapidly oxidized to its acid, via homovanillyl aldehyde, by aldehyde dehydrogenase and aldehyde oxidase with little or no contribution from xanthine oxidase.     

Cross-correlation analysis of background neuronal activity in guinea pig neocortical slices in vitro

Interaction between the background activity of adjoining neurons was investigated using simultaneous recording in surviving slices from the guinea pig sensory motor cortex by means of cross-correlation analysis. A numerical connection was found between the timing of successive discharges in sixteen of the twenty six neuronal pairs investigated. Significant discrepancies were observed in correlation tables, mainly in the range of time shifts of ±100 msec from zero point. These emerged as symmetrical or asymmetrical peaks of up to 150 msec in duration and negative shifts measuring up to 200 msec. More complex positive-negative cross-correlation patterns were also encountered. These findings may be compared with those obtained in the cortex of the intact brain. The subject of the contribution made by intrinsic cortical neuronal interaction and that of external afferents to firing correlation is discussed.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 4, pp. 392–399, July–August, 1991.     

Mismatch repair protein MSH2 regulates translesion DNA synthesis following exposure of cells to UV radiation

Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.     

Lipid biosynthesis in liver slices of the foetal guinea pig.

Lipid synthesis as measured by the incorporation of acetate or 3H2O into slices of foetal liver, is much higher than in slices of adult liver and shows a peak at about two-thirds of gestation. At this time the synthesis from glucose was low and reached a peak 10 days later. The changes in the activity of ATP citrate lyase, which mirrored acetate incorporation, and the effect of glucose and pyruvate on acetate corporation into lipid suggests that some of the lipid synthesis occurs via intramitochondrial acetyl-CoA production from acetate. Despite this, lipid synthesis was not inhibited by (-)-hydroxycitrate. The low rate of synthesis from glucose at two-thirds of gestation is ascribed to the low activity of pyruvate carboxylase at this time and a role for a phosphoenolpyruvate carboxykinase in providing oxaloacetate for lipogenesis is proposed. The activity of fatty acid synthetase broadly agreed with the changes in lipid synthesis, whereas the activity of acetyl-CoA carboxylase was barely sufficient to account for the rates of lipid synthesis in vivo. Acetate and short-chain fatty acids are likely to be the major precursors for lipid synthesis in vivo.     

Study of unscheduled DNA synthesis following exposure of human cells to arecoline and extracts of betel nut in vitro.

Aqueous, acetic acid, hydrochloric acid and ethanol extracts of betel nut (Areca catechu L.) have been found to induce unscheduled DNA synthesis in Hep 2 cells obtained from human larynx carcinoma, in vitro. Different concentrations of extracts of betel nut induced dose-dependent unscheduled DNA synthesis in Hep 2 cells. Together with the viability of the Hep 2 cells, our results indicate that the aqueous and acetic acid extracts of betel nut induce relatively more unscheduled DNA synthesis than the hydrochloric acid and ethanol extracts and arecoline. The carcinogenic potency of raw and unprocessed betel nut of North-East India used in this study is discussed.     

Characterization of guinea pig cytomegalovirus DNA.

The genome of guinea pig cytomegalovirus (GPCMV) was analyzed and compared with that of human cytomegalovirus (HCMV). GPCMV and HCMV DNAs were isolated from virions and further purified by CsCl centrifugation. Purified GPCMV DNA sedimented as a single peak in a neutral sucrose gradient and was infectious when transfected into guinea pig embryo fibroblast cells. The cytopathology was characteristic of that seen after infection with GPCMV. Virus DNA purified from virions isolated from infected GPEF or 104C1 cells had a CsCl buoyant density of 1.713 g/cm3, which corresponds to a guanine plus cytosine content of 54.1%. The CsCl buoyant density of GPCMV DNA was slightly less than that of HCMV DNA (1.716 g/cm3), but sufficiently different so that the two virus DNA peaks did not coincide. GPCMV DNA cosedimented with T4 DNA in a neutral sucrose gradient. Restriction endonuclease cleavage of GPCMV or HCMV DNAs with HindIII, XbaI, or EcoRI yielded fragments easily separable by agarose gel electrophoresis and ranging from 1.0 X 10(6) to 25.8 X 10(6) daltons. The number, size, and molarity of GPCMV DNA fragments generated by restriction enzymes were determined. Hybridization of restriction endonuclease-cleaved GPCMV DNA with radioactively labeled HCMV DNA and, conversely, hybridization of restriction endonuclease-cleaved HCMV DNA with radioactively labeled GPCMV DNA indicated sequence homology between the two virus DNAs.     

Escherichia coli DNA synthesis in vitro: insensitivity of ATP-dependent DNA repair to inhibition by novobiocin.

Novobiocin, an effective inhibitor of DNA replicaion in Escherichia coli, is shown to have no effect on the ATP-dependent DNA repair carried out by toluenized cells after ultraviolet irradiation. Therefore novobiocin can be considered a selective inhibitor of replicative DNA synthesis in vitro.     

Lactate-induced inhibition of glucose catabolism in guinea pig cortical brain slices.

Extracellular lactate concentration rises following ischaemic stroke in both the infarcted area and in the surrounding ischaemic penumbra. We investigated the effect of lactate accumulation on glucose metabolism in cortical slices from guinea pigs initially by varying superfusion medium to tissue volumes. Stable intracellular K+ concentrations indicated that a decrease in media/ tissue volume did not impair viability of the tissue, but 13C NMR demonstrated that lactate accumulation in the superfusion medium reduced glucose oxidation with inhibition of glial metabolism via pyruvate carboxylase. The concentration of lactate which had accumulated when significant inhibition was observed was approximately 0.85 mM. In independent experiments we found that superfusion of brain slices with lactate at this concentration (even using a 'high-volume' of superfusion fluid) decreased oxygen consumption by 40 +/- 3%. K(-)-induced depolarisation partially reversed this effect. These results suggest that even low extracellular lactate concentrations may depress metabolic rates in inactive and poorly perfused brain tissue in vivo through inhibition of glial metabolism of glucose.     

Short-term exposure to cigarette smoke induces endothelial dysfunction in small intrapulmonary arteries: analysis using guinea pig precision cut lung slices.

The pathogenesis of cigarette smoke-induced pulmonary hypertension is not understood. We have previously shown that smoke rapidly and persistently, but discoordinately, upregulates gene expression of mediators that control vasoconstriction, vasoproliferation, and vasorelaxation in small intrapulmonary arteries. To investigate the possibility that smoke also induces endothelial dysfunction, a finding common to other forms of pulmonary hypertension, we exposed guinea pigs to smoke or air (control) daily for 2 wk and then prepared precision-cut lung slices. After exposure to endothelin-1, a vasoconstrictor, intra-acinar arteries in lung slices derived from smoke-exposed animals constricted more rapidly (greater constriction at a given concentration of endothelin) than did vessels from air-exposed animals. To examine relaxation responses, arteries were constricted with the vasoconstrictor U-46619 and then relaxed with progressively increasing doses of acetylcholine. Vessels from smokers had a delayed response to acetylcholine compared with vessels from controls. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester reduced relaxation in both control and smoke-exposed arteries, whereas the NO donor sodium nitroprusside increased relaxation of the smoke-exposed arteries, confirming that endothelial dysfunction with decreased effective NO production is present. These findings show that precision cut lung slices can be used to examine the physiological effects of cigarette smoke on intra-acinar pulmonary arteries and indicate that even relatively short-term exposure to smoke produces endothelial dysfunction with a resulting tendency to earlier constriction and later relaxation in cigarette smokers. These changes may be important in the development of pulmonary hypertension.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro.

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

Neuronal firing in guinea pig neocortical slices surviving in vitro during adenosine-induced blockade of synaptic transmission

Background firing activity was recorded in guinea pig neocortical slices maintained using extracellular techniques. Between 30 and 40% of neurons continued to generate action potentials, although at a reduced rate, when synaptic disruption had been induced by adenosine or adenosine 5-monophosphate action. These cells were classed as endogenously active. No connection could be shown between neuronal firing pattern and capacity for autonomous generation of action potentials. The remaining neurons tested remained inactive after synaptic disruption, but regained their capacity for spontaneous firing following washout. The activity of these cells was classified as exogenous (or the result of synaptic excitation induced by other neurons in the same slice). The majority of cells with a highly regular discharge pattern initially stopped discharging during synaptic blockade and resumed their activity following washout. This would suggest that a miniature excitatory circuit with 30–140 msec cycles operates in these slices.Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 816–824, November–December, 1987.     

Induction of guinea pig antibody responses in vitro.

Guinea pig spleen cells cultured together with peritoneal exudate lymphocytes (PEL) were found to generate large numbers of antibody-forming cells (AFC) in vitro in response to hapten-protein antigens. Neither cell type cultured alone yielded appreciable responses. Strain 13 or F1 (Strain 2 X Strain 13) lymphocytes, but not those from strain 2 animals, are able to respond to the genetically controlled antigen, DNP-guinea pig albumin (DNP-GPA). Antisera directed against responder (strain 13) parent Ia antigens selectively blocked the generation of AFC by F1 (strain 2 X strain 13) spleen-PEL mixtures in response to DNP-GPA. Both allogeneic (strain 2) and syngeneic macrophages functioned equally well in presentation of DNP-GPA to strain 13 lymphocytes.