Increase In DNA Strand Breaks Research Articles

Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide.

Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro. This mechanism is not well understood. The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma. Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h. Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma. The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM. By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan. Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide. Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma. ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD. All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h. Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD. Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT. Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma. The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity. Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma. Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro. Intracellular glutathione has been shown to protect cells against oxidative damage by various agents. Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively. These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth. Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.

DNA lesions and DNA degradation in mouse lymphoma L5178Y cells after photodynamic treatment sensitized by chloroaluminum phthalocyanine.

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabeled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.

Using the DNA alkaline unwinding assay to detect DNA damage in laboratory and environmentally exposed cells and tissues

The DNA alkaline unwinding assay is being evaluated for use in the detection of DNA damage in marine animals exposed to environmental pollutants. In preliminary work, DNA unwinding methods were used with in vitro cell systems to demonstrate DNA strand breaks. Cultured mammalian fibroblasts and sperm from marine fish and invertebrates ( Pseudopleuronectes, Arbacia and Mytilus) showed concentration-dependent increases in DNA strand breaks after brief exposures to alkylating agents. DNA unwinding methods were also used on DNA extracted from marine animals injected with genotoxicants and from animals exposed in situ at an estuarine site. Gills from blue mussels caged at the New Bedford Harbor Superfund Site (MA, USA) highly contaminated with many organic (e.g. PCBs) and inorganic contaminants, were also examined. A significant increase in DNA strand breaks was seen in gill tissues of animals held in the contaminated site for as little as 3 days. Although not as severe, an increase in strand breaks was also seen in animals held at the control site for 28 days. This apparent damage may have resulted from low-level contamination and/or from generally poor tissue condition. These results suggest that DNA alkaline unwinding results from controlled experiments using cells briefly exposed to toxicants can be used to identify DNA strand breaking agents. While whole-animal exposures to toxicants may affect DNA integrity, unwinding results may not specifically reflect genotoxicity.

Enzyme activities of leukemic cells and biochemical changes induced by deoxycoformycin in vitro—Lack of correlation with clinical response

Deoxycoformycin (DCF) is a specific inhibitor of adenosine deaminase (ADA) and has been shown to be active in lymphoid neoplasms. Cytotoxicity is thought to be mediated by the accumulation of deoxyadenosine (AdR) and deoxyadenosine triphosphate (dATP) which inhibits ribonucleotide reductase and DNA synthesis in rapidly proliferating cells. Others suggested mechanisms leading to cell death particularly in non-dividing cells include depletion of ATP and NAD pools, inhibition of S-adenosylhomocysteine (SAH) hydrolase and induction of DNA strand breaks. In patients with high leukemic counts who were subsequently treated with DCF, we have studied (a) the levels of ADA, ecto-5′-nucleotidase (5NT), deoxyadenosine kinase (AdR-kinase) and SAH-hydrolase in the leukemic cells; (b) the in-vitro effects of DCF on dATP, ATP, NAD, SAH-hydrolase levels and on DNA strand breaks; and (c) the correlation between these parameters with clinical response to DCF. No significant difference in ADA, 5NT, AdR-kinase and SAH-hydrolase activities could be found between responders and non-responders. Incubation of the leukemic cells in vitro with DCF caused an inhibition of ADA, an accumulation of dATP, a moderate reduction in ATP and NAD levels, a suppression of SAH-hydrolase activity and an increase in DNA strand breaks in practically all the leukemic samples, irrespective of clinical response. Our results show that neither measurement of these enzymes nor studies of these biochemical sequelae of ADA inhibition in vitro predicts clinical responsiveness to DCF therapy.

Radiation-induced DNA-strand breakage and repair in the marine polychaete, Neanthes arenaceodentata

We used an alkaline unwinding assay, which provides an index of DNA-strand breakage and repair, to study the genotoxic effects of gamma radiation in adult Neanthes arenaceodentata. To determine radiation-induced DNA-strand damage, we exposed the worms to radiation levels from 3·13 to 100 Gy. Significant DNA-strand damage was found at exposures ≥3·13 Gy. To determine the repair of radiation-induced DNA-strand damage, we irradiated worms at 100 Gy and then harvested and analyzed their DNA on days 0, 2, 5 and 14 post-irradiation. A significant increase in DNA-strand breaks was found to exist between control and radiation-exposed groups on days 0–5. By day 14 post-irradiation, however, no significant relationship was found between control and radiation-exposed groups.

Alterations in levels of 5′-adenyl dinucleotides following DNA damage in normal human fibroblasts and fibroblasts derived from patients with xeroderma pigmentosum

Levels of 5'-adenyl dinucleotides, measured as diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A), were found to accumulate in cultured human fibroblasts following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the radiomimetic drug bleomycin, and nitroquinoline-1-oxide (NQO) or UV-irradiation in the presence of cytosine arabinofuranoside (araC). In contrast, cells derived from patients with xeroderma pigmentosum complementation group A (XP-A) did not demonstrate an increase in DNA-strand breaks following UV irradiation or NQO in the presence of araC nor an increase in Ap4A levels. Ap4A accumulation did occur in XP-A cells following treatment with MNNG. Cells derived from patients characterized as XP variants, which are incision repair-proficient, accumulated 5'-dinucleotides following bleomycin, MNNG and UV or NQO in the presence of araC. Taken together, these data suggest that Ap4A accumulates as a response to DNA-strand breaks.

Programmed cell death and adenine deoxynucleotide metabolism in human lymphocytes

Agents that cause the accumulation of DNA strand breaks are directly cytotoxic to non-dividing normal human peripheral blood lymphocytes, and to chronic lymphocytic leukemia (CLL) cells. Activation of poly(ADP-ribose) polymerase (ADPRP), and the resultant consumption of NAD, play an essential role in mediating the toxicity of these agents. Human peripheral blood lymphocytes contain a substantial number of alkali-sensitive DNA sites, reflecting ongoing DNA strand breakage and repair. However, resting lymphocytes have a limited capacity to synthesize NAD. Pulse-chase experiments indicate that approximately 75% of their NAD turnover is due to ADPRP activity. Exposure of the cells in vitro to deoxyadenosine, or to 2-chlorodeoxyadenosine (CdA, an adenosine deaminase resistant deoxyadenosine congener), caused an increase in DNA strand breaks, rapid NAD consumption, ATP depletion and cell death. Supplementation of the medium with inhibitors of poly(ADP-ribose) polymerase blocks the fall in cellular NAD and ATP, and protects the lymphocytes from the toxicity of DNA damaging agents. Slowly dividing malignant lymphocytes from patients with CLL are also susceptible to lethal NAD depletion following DNA damage. 2-chlorodeoxyadenosine (CdA) induced massive DNA strand break formation in CLL cells in vitro and a fall in NAD and ATP pools. In an initial clinical trial, several CLL patients, and two patients with hairy cell leukemia, have responded to treatment with CdA, with minimal toxicity. Thus, the suicidal activation of ADPRP in response to DNA damage has been rationally exploited in the treatment of chronic lymphoid malignancies.

Cytotoxicity of 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) on Mer+, Mer+Rem- and Mer- cell lines: differential potentiation by 3-acetamidobenzamide.

Mechanisms of resistance to the active metabolite 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) of the drug 5-(3,3-dimethyl-1-triázeno)imidazole-4-carboxamide (DTIC) were studied in three human cell lines with differing amounts of the repair enzyme O6-alkylguanine-DNA alkyltransferase (O6AT). The lines were HT29 (Mer+Rem+), A549 (Mer+Rem-) and VA13 (Mer-). The ability to repair O6 methyl-guanine was directly related to resistance to MTIC (HT29 ID50 650 mumol l-1, A549 ID50 210 mumol l-1, VA13 ID50 15 mumol l-1. MTIC produced DNA single strand breaks over the range of one log of cell kill, but depletion of cellular NAD levels could not be detected until there was greater than 95% cell kill. Inhibitors of the repair enzyme adenosine diphosphoribosyl transferase (ADPRT) potentiated killing by 2-fold in the Mer+ cell lines but not the Mer- line. The enhancement was directly proportional to an increase in DNA strand breaks but not a change in their half-life. Therefore resistance to the clinically used methylating agent MTIC can be partly overcome by inhibiting ADPRT but a role for ADPRT as a suicide mechanism in response to alkylating agent damage is unlikely.

Elevated levels of the c-myc protein in Bloom's syndrome and induction of c-myc by DNA strand breakage.

We demonstrate elevated levels of the c-myc protein in lymphoblastoid cell lines derived from Bloom’s syndrome patients and, furthermore, show that treatment of normal lymphocytes with DNA damaging agents results in induction of c-myc protein. We present a model whereby increased DNA strand breakage in Bloom’s syndrome, as a consequence of the reduced activity of DNA ligase I, results in the constitutive induction of c-myc. Increased c-myc protein may thus contribute towards the predisposition to cancer observed in Bloom’s syndrome.

Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators.

This study was undertaken to investigate the hypothesis linking peroxisome proliferation with the production of reactive oxygen species and subsequent DNA damage. Hepatic peroxisomal proliferation was induced in male Wistar-derived rats by the administration of clofibrate, methyl clofenapate, di(2-ethylhexyl)phthalate, or its metabolite mono (2-ethylhexyl)phthalate (MEHP) for periods of up to 28 days. Genotoxicity was monitored using an alkaline elution technique to assay for DNA strandbreaks and cytotoxicity was monitored by measuring lipid peroxidation. Both parameters might be expected to be elevated if peroxisome proliferation is accompanied by an elevated level of oxygen free radicals within the cell. Enzyme measurements made on the livers of the treated rats showed that peroxisomal palmitoyl CoA oxidase activity was markedly increased over control whereas peroxisomal catalase activity was not. In addition, both the cytosolic glutathione peroxidase and superoxide dismutase activities were found to be lowered in the treated animals by up to 50 and 20% respectively. Despite such changes in enzyme activity, no evidence for increases in DNA strandbreaks or lipid peroxidation was obtained with any of the chemicals at any of the time points examined. DNA strandbreaks were also assayed on hepatocytes treated in culture with MEHP (0.5 mM) for 3 days and then exposed to inhibitors of DNA repair for 2 h immediately before assay. Again, no significant increase over controls was observed. Our data suggest that any increase in radical production in the livers of rats exposed to peroxisome proliferators is not large enough to give rise to a biologically significant degree of DNA damage and that the mechanism whereby such chemicals produce liver tumours in certain rodent species may be one other than simply DNA damage due to increased production of radical species.

Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP-ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP-ribose) DNA strand break DNA repair

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10–200 μM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1,3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinotropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5–20 μM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine.

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m2). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10(8) daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2-9.4 breaks/10(8) daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2-14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10(8) daltons, respectively). The same level of repair inhibition was observed in confluent XP-variants receiving ara-C/HU, but was reduced by 62-68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.

Gossypol induces DNA strand breaks in human fibroblasts and sister chromatid exchanges in human lymphocytes in vitro.

The male contraceptive agent gossypol was found to induce a dose related increase of DNA strand breaks in human fibroblasts in vitro at concentrations of 5 to 40 micrograms/ml. The effect was reduced in the presence of 2% fetal calf serum. A weak but reproducible increase in the SCE frequency was found in human lymphocytes treated for 1 hour in serum-free medium with 0.04 to 4 micrograms/ml of gossypol.

The genotoxicity of aromatic amines in primary hepatocytes isolated from C57BL/6 and DBA/2 mice.

The capacity of the chemical carcinogen 2-acetylaminofluorene (AAF) and its derivates to cause DNA damage in primary mouse hepatocytes from aryl-hydrocarbon responsive C57BL/6 and non-responsive DBA/2 mice was studied using the alkaline elution technique. Low levels of DNA damage were observed after exposure of hepatocytes to either AAF or 2-aminofluorene (AF) (50-100 microM). Quantitation of metabolites produced from AAF in hepatocytes from untreated C57BL/6 and DBA/2 mice using h.p.l.c. showed a similar metabolic profile with respect to C- and N-hydroxylations. After in vivo pretreatment with the potent monooxygenase inducer TCDD (50 micrograms/kg), N-hydroxylation in the C57BL/6- and DBA/2-derived hepatocytes increased 25- and 5-fold, respectively. However, the C-hydroxylation pathways were still responsible for approximately 90% of the metabolism in cells from both strains. This may explain why only a slight increase in the DNA damage was observed in C57BL/6 mouse hepatocytes after incubation with AF or AAF and no increase in DNA damage was seen in the DBA/2 hepatocytes isolated from TCDD treated animals. Both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene (N- OAc -AAF) caused clear dose-dependent increases in DNA strand breaks (5-100 microM), suggesting that N-hydroxylation was the rate limiting step in the activation process of AAF leading to the DNA damage. Treatment of hepatocytes with paraoxon, an inhibitor of microsomal deacetylase activity, prior to exposure to either N-OH-AAF or N- OAc -AAF completely inhibited the damage caused by N-OH-AAF, while the damage caused by N- OAc -AAF was only partially inhibited. This suggests that these compounds are causing genotoxic effects after deacetylation. In accordance with this, N-hydroxy-2-aminofluorene (N-OH-AF), the deacetylated metabolite of N-OH-AAF, was an effective genotoxic agent, causing DNA strand breaks at low doses. Depletion of cellular glutathione by pretreatment with diethyl maleate, increased the sensitivity of the cells to the damage induced by N-OH-AF. These data indicate that glutathione may play an important role in the detoxification of N-OH-AF in mouse hepatocytes.

DNA repair replication, DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10 −3 moles/1. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10 −6 moles/1). Fibroblasts exposed to AM at 10 −4 moles/1 in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes. A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (<10 −7 moles/1). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G 0 or G 1. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles. These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.

Determination of DNA superhelicity and extremely low levels of DNA strand breaks in low numbers of nonradiolabeled cells by DNA-4′,6-diamidino-2-phenylindole fluorescence in nucleoid gradients

Nonradiolabeled cells derived from animals or cell cultures can be examined in nucleoid gradients labeled with 4′,6-diamidino-2-phenylindole to quantitate extremely low levels of DNA strand breaks and DNA superhelicity. In vitro: 4′,6-Diamidino-2-phenylindole detection of DNA in nucleoid gradients requires only 15,000 nonradiolabeled human dermal fibroblasts per gradient. Quantization of increased DNA strand breaks, relative to controls, occurring at frequencies as low as one break per 2.2 × 10 10 daltons DNA is possible. This technique was employed to examine low-passage (“young”) and high-passage (“old”) normal human dermal fibroblasts. DNA from old cells contains approximately one more strand break per 5 × 10 9 daltons that does the DNA from young cells, and this difference is unobservable using conventional radiolabeled alkaline-sucrose gradients. Decreased DNA negative superhelicity in the old cells was also observed. In vivo: This technique was used to examine nucleoids derived from female Fisher rat livers treated with aflatoxin B 1. Treated cells exhibit decreased DNA negative superhelicity. Since this technique cannot quantitate DNA strand breaks occurring at the higher levels required by alkaline techniques, it is proposed that this technique is a necessary complement to alkaline techniques when low levels of DNA strand breaks must be quantitated.

Visceral Transposition as a Mendelian Recessive

The male contraceptive agent gossypol was found to induce a dose related increase of DNA strand breaks in human fibroblasts in vitro at concentrations of 5 to 40 micrograms/ml. The effect was reduced in the presence of 2% fetal calf serum. A weak but reproducible increase in the SCE frequency was found in human lymphocytes treated for 1 hour in serum-free medium with 0.04 to 4 micrograms/ml of gossypol.