DNA damage and cell death induced by 1,2-dibromo-3-chloropropane (DBCP) and structural analogs in monolayer culture of rat hepatocytes: 3-aminobenzamide inhibits the toxicity of DBCP

1,2-Dibromo-3-chloropropane (DBCP) and a number of halogenated propane analogs induced DNA damage in rat hepatocytes in vitro measured by an automated alkaline elution method. Short-term (2 hrs) cytotoxic effects of DBCP were not observed until the DBCP concentration exceeded 1 mM. The short-term cytotoxicity of all the DBCP analogs occurred in the same concentration range. Significant membrane damage, measured as cell detachment, was observed after extended exposure to lower concentrations of DBCP (100 M) for 20 hrs. The relative, delayed cytotoxic effect of DBCP and analogs correlated with their ability to cause DNA damage. In general, the halogenated propanes with more bromines relative to chlorines were the more potent compounds. Propane analogs lacking the third halogen had little cytotoxic activity. The addition of the proposed specific poly(ADP-ribosyl)transferase inhibitor 3-aminobenzamide (3-ABA) protected against DBCP-induced cytotoxic effects and NAD⁺ depletion. However, 3-ABA also reduced DBCP-induced DNA damage, DBCP metabolic loss, and the formation of water soluble and covalently bound DBCP metabolites. Thus, 3-ABA may block DBCP-induced cell death by decreasing the formation of reactive DBCP-metabolites.Abbreviations 3-ABA 3-aminobenzamide - 3-AB acid 3-aminobenzoic acid - Asc ascorbate - BSA bovine serum albumin - DBCP, 1,2-diB-3-CP 1,2-dibromo-3-chloropropane - DMSO dimethylsulfoxide - DPPD N,N-diphenyl-p-phenylenediamine - GSH glutathione - Hoechst 33258 [2(2-(4-hydroxy-phenyl)-6-benzimidazole-6-(1-methyl-4-piperazyl)-benzimidazole trihydrochloride)] - 1,2,3-triBP 1,2,3-tribromopropane - 1,3-diB-2-CP 1,3-dibromo-2-chloropropane - 1,3-diC-2-BP 1,3-dichloro-2-bromopropane - 1,2,3-triCP 1,2,3-trichloropropane - 1,2-diBP 1,2-dibromopropane     

Induction of proteinase K-sensitive sites and M. luteus endonuclease-sensitive sites in DNA of barley embryos by sodium azide

DNA damage induced in germinating barley embryos by mutagenic and sublethal doses (0.1–2 mM, 2 h) of sodium azide, applied at pH 3, was measured by alkaline elution. Isolated nuclei were lysed at a high pH with either 2% SDS or 2 M NaCl on polyvinyl chloride filters and digested with proteinase K or with Micrococcus luteus endonuclease prior to elution. The azide treatments resulted in a dose-dependent increase of proteinase K-sensitive sites and an appearance of Micrococcus luteus endonuclease-sensitive sites. These sites were detected as DNA single-strand breaks after digestion of the DNA with either one or both of the enzymes. The two types of lesion were additive and occurred in a ratio of about 1:1. The additive effect suggested independent origin for the two types of lesion. Breaks independent of proteinase K digestion appeared only when DNA was analysed 24 h after the action of azide. The nature and significance of these DNA lesions are discussed.     

A technique for the measurement of breakage and repair of DNA alkylated in vivo.

The alkaline elution technique has been adapted for use in the assessment of DNA damage induced in the livers and lungs of mice after administration of an alkylating agent, methylemthanesulfonat (MMS). At 4 h after administration of MMS, damage ot DNA was readily demonstrable; the damage was repaired in liver by 24 h. The lung, particularly of the A/J mouse, exhibited an increased alkaline elution rate when compared to C57BL/6J, and repair was not entirely complete (as judged from the rate of alkaline elution of DNA) by 24 h. The rate of elution was dependent upon temperature. It is believed that this adaptation should have great utility in examining DNA repair in vivo.     

Metabolism of selectively methylated and deuterated analogs of 1,2-dibromo-3-chloropropane: role in organ toxicity and mutagenicity

In vitro bromide release and in vivo glutathione (GSH) depletion in rat liver, kidney and testis by 1,2-dibromo-3-chloropropane (DBCP) and selectively methylated and deuterated DBCP analogs were studied. With liver microsomes from phenobarbital-pretreated rats the bromide release from the C1-C3-D4- and the perdeuterated DBCP analogs were 54% and 26% of that of DBCP, respectively. Inhibitors of P-450 reduced the bromide release to 10-20% of that without additions. This correlated with the effects of deuterium substitution and additions of P-450 inhibitors on DBCP-induced bacterial mutagenicity as reported elsewhere by this laboratory. To study the importance of GSH-dependent metabolism in DBCP toxicity, bromide release was assayed in cytosolic preparations using methylated analogs of DBCP. With the C1-methyl-derivative, bromide release was markedly reduced compared to that with DBCP in cytosols from liver, kidney and testis. A similar reduction in in vivo nephrotoxicity and testicular damage has recently been reported. The obtained correlation between in vitro GSH-dependent metabolism of methylated DBCP analogs and their in vivo organ damaging potential, points to an involvement of GSH-dependent metabolism in DBCP-induced in vivo toxicity. Both DBCP and the methylated analogs (360 mumol/kg i.p.) depleted the GSH levels in liver after 1 and 3 h and in kidney after 1 h, whereas in the testis no significant depletion of GSH was obtained. As kidney and testis are reported to be the primary target organs for DBCP, there was an apparent lack of correlation between tissue depletion of GSH and organ toxicity.     

Measurement of radiation-induced DNA damage using gel electrophoresis or neutral filter elution shows an increased frequency of DNA strand breaks after exposure to pH 9.6

The filter elution technique using nondenaturing conditions is widely used to assay DNA double-strand break (DSB) induction and repair. It has been reported that in the measurement of strand breaks higher rates of elution and of initial rejoining are obtained at pH 9.6 compared to pH 7.2. In the present experiments neutral elution at pH 7.2 and 9.6 were compared in the assay of damage to DNA induced by X rays, 125I decay, and restriction enzyme digestion, in an effort to explain this discrepancy and to determine whether the higher rate of elution observed at pH 9.6 corresponds to a greater number of DSBs. X-ray damage to cellular DNA resulted in significantly different elution profiles at the two pH values. In contrast the elution profiles of the DSB induced by intragenomic 125I decays or restriction endonuclease were independent of the pH of the elution buffer. When gamma-irradiated SV40 DNA was exposed to pH 7.2 or 9.6 elution buffer prior to analysis by gel electrophoresis, a significantly greater number of DNA DSBs were detected in the DNA exposed to pH 9.6. We conclude that X and gamma radiation produce lesions (pH 9.6-labile lesions), in proportion to dose, that have the potential of becoming measurable DSBs following incubation under the mildly alkaline condition of pH 9.6. The data suggest that these lesions may result from single-hit events.     

Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells

Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05–1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.     

DNA cross-links in human leucocytes treated with vinyl acetate and acetaldehyde in vitro

Human leucocytes were incubated in the presence of vinyl acetate or acetaldehyde (10-20 mM) for 4 h at 37 degrees C in vitro. DNA damage was analysed by alkaline elution. None of the compounds induced a detectable increase in the frequency of DNA strand breaks. Cells exposed to 5 Gy of X-ray immediately after treatment and before alkaline elution showed a clear, dose-dependent retardation of the elution rate in comparison with X-irradiated control cells. These results demonstrate that both vinyl acetate and acetaldehyde induce DNA cross-links in human cells.     

Alkaline phosphatase activity does not mediate phosphate transport in the renal-cortical brush-border membrane.

We studied (1) the effect of primary modulators of phosphate transport, namely the hypophosphataemic mouse mutant (Hyp) and low-phosphorus diet, on alkaline phosphatase activity in mouse renal-cortex brush-border membrane vesicles and (2) the effect of several primary inhibitors of alkaline phosphatase on phosphate transport. Brush-border membrane vesicles from Hyp-mouse kidney had 50% loss of Na+-dependent phosphate transport, but only 18% decrease in alkaline phosphatase activity. The low-phosphorus diet effectively stimulated Na+/phosphate co-transport in brush-border membrane vesicles (+ 118%), but increased alkaline phosphatase activity only slightly (+13%). Levamisole (0.1 mM) and EDTA (1.0 mM) inhibited brush-border membrane-vesicle alkaline phosphatase activity of 82% and 93% respectively, but had no significant effect on Na+/phosphate co-transport. We conclude that alkaline phosphatase does not play a direct role in phosphate transport across the brush-border membrane of mouse kidney.     

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 7g/ml to 20 7g/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

Alkaline DNA fragmentation,DNA disentanglement evaluated viscosimetrically and sister chromatid exchanges,after treatment in vivo with nitrofurantoin

Nitrofurantoin was not positive as a carcinogen in long term assays. In vitro it was positive in some short term tests and negative in others. We have examined Nitrofurantoin for its capability of inducing DNA damage in vivo. With the alkaline elution technique, Nitrofurantoin appeared clearly positive in all the tissues examined (liver, kidney, lung, spleen and bone marrow). In the liver we also observed some cross-linking effect. In bone marrow cells Nitrofurantoin was also clearly positive in terms of sister chromatid exchanges (SCEs) induction. DNA damage in vivo was also examined with a viscosimetric method, more sensitive than alkaline elution. With this method the results were essentially negative, suggesting that the two methods detect different types of damage. In view of its positivity in many organs and in two short term tests in vivo, the carcinogenic potential of Nitrofurantoin should be reconsidered.     

Induced DNA Damage Measured by the Comet Assay in 10 Weed Species

For most plant species growing in polluted areas no mutagenicity assays are available. We have studied the possibility of using the alkaline protocol of the Comet assay as a method for detecting induced DNA damage in wildly growing weeds. The monofuctional alkylating agent ethyl methanesulphonate (EMS) was applied on leaves of 10 weed species (ordered according to the diameter of the nuclei): Arabidopsis thaliana, Convolvulus arvensis, Bellis perennis, Urtica dioica, Lamium album, Chenopodium rubrum, Plantago media, Poa annua, Taraxacum officinale, and Agropyron repens. With increasing concentrations of EMS (2 to 10 mM) the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all weeds studied. Using the Head Extent parameter of the Komet version 3.1, we have measured the diameter size of the nuclei of the 10 weed species either immediately after the isolation of the nuclei or after 20 or 45 min of treatment with alkaline buffer (pH > 13). According to the increase of the diameter of the nuclei (including the formed halo) resulting from the to alkaline buffer treatment, electrophoretic conditions (unwinding and electrophoresis time) for the Comet assay can be selected for the individual weed species.     

DNA damage induced by some bile acids, evaluated with alkaline elution technique

Bile acids are promoting agents in colon carcinogenesis. In this work we have tried to characterize the DNA alteration induced by bile acids in Sprague-Dawley male rats. Confirming previous findings, a clear increase in elution rate was observed at alkaline pH. No effect could be observed when the nuclei were washed before the elution, in condition totally unsuitable for the repair of the type of DNA damage induced by typical genotoxic agents. We advanced the hypothesis that the increased alkaline elution rate observed with bile acids could be independent of DNA fragmentation and related to changes in chromatin structure.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

Fractionation of DNA from marine invertebrate (Maja crispata, Mytilus galloprovincialis) haemolymph by alkaline elution.

1. An alkaline elution procedure for the detection of DNA damage in marine invertebrate haemolymph has been developed. 2. Provided that three criteria are optimized, such as buffer composition, small filter pores (0.22 microns GVWP 025 00, Millipore), and optimal amounts of haemolymph applied, flow rates may be changed within the range of 0.2 ml/min to 0.05 ml/min without adverse back-pressure on the filter and without blocking filter pores. 3. Under optimal conditions, 70% of mussel haemolymph DNA, and 80% of crab haemolymph DNA will be retained on the filter after 6 hr of elution, indicating shorter DNA in mussel haemolymph. 4. The technique is applicable for testing the in vivo effects of different compounds on DNA in marine invertebrates, and to measurements of DNA damage in naturally exposed mussels. 5. This argues an important case for the use of alkaline elution technique for assessment of environmental genotoxicity, and especially for investigation of DNA damage in different marine organisms which cover a broad range in their DNA molecular weights.     

Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography

In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5-8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris-HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.     

The use of alkaline elution procedures to measure DNA damage in spermiogenic stages of mice exposed to methyl methanesulfonate

The ability of methyl methanesulfonate (MMS) to induce DNA breakage in spermiogenic stages of the mouse was studied using an alkaline elution technique. At daily intervals over a 3-week period following i.p. injection of 50 mg MMS/kg, mature spermatozoa were recovered from treated (3H-labeled) and control (14C-labeled) animals, lysed together on polycarbonate filters, and eluted with a high pH (12.2) buffer. Elution of germ-cell DNA from MMS-treated animals was found to increase in stages in which genetic damage from MMS is greatest. In general, the pattern of DNA elution from treated, spermiogenic stages paralleled the pattern of sensitivity to dominant lethals, specific-locus mutations and heritable translocations found by other investigators. It also paralleled the pattern of sperm-head methylation and protamine methylation measured in an earlier study (Sega and Owens, 1983). At 9 days post treatment (sperm sampled were in mid-to late-spermatid stages at the time of MMS exposure) the elution of sperm DNA did not change significantly over a pH range of 11.6-12.8, suggesting that, at the time of assay, DNA breaks were already present in the sperm. Because of the parallelism found between increased sperm DNA elution and increased genetic damage after mutagen treatment, alkaline elution may prove useful in monitoring potential genetic damage in human sperm.     

Biochemical characterization of alkaline phosphatase in guinea pig thymus.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.     

Effect of magnesium on ATP labelling by kidney brush border membrane

1. Brush border membranes purified from rat kidney cortex were incubated in the presence of ATP and analysed by SDS polyacrylamide gel electrophoresis. 2. Quantitative analysis of phosphorylation was performed with a calibration curve obtained by autoradiography. 3. The presence of magnesium was required for the phosphorylation of membrane proteins. 4. EDTA completely inhibited the labelling of all bands, except for the alkaline phosphatase band. 5. In contrast, alkaline phosphatase was inhibited by 52, 65 and 85% in the presence of 1 mM bromotetramisole, 10 mM NaF and 10 mM Na arsenate respectively. 6. However these inhibitors had only minor effects on the labelling of other proteins. 7. High concentrations of magnesium caused a pronounced inhibition on the labelling of the alkaline phosphatase band but had no effect on the phosphorylation of other proteins.     

Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.