Detection of DNA crosslinks in peripheral lymphocytes isolated from patients treated with platinum derivates using modified comet assay.

Platinum-based chemotherapeutic agents induce the formation of crosslinks in DNA, which are accepted as being responsible for the cytotoxicity of these agents. In this study, we used a modification of the alkaline comet assay for detection of the presence of DNA crosslinks in vitro caused by cisplatin, and in peripheral lymphocytes of patients with non-small cell lung carcinoma undergoing chemotherapy with platinum derivatives. The comet technique modified for the detection of DNA crosslinks was calibrated in vitro by treating HeLa cells and human lymphocytes from healthy donors with different concentrations of cisplatin. A cisplatin dose-dependent formation of DNA crosslinks was observed in in vitro measurements using 10-200 µM concentrations of cisplatin. Lymphocytes from cancer patients were also assayed for the formation and repair of DNA crosslinks. Evidence of crosslink formation and repair was observed in peripheral blood lymphocytes of all cancer patients in this study, although some inter-individual differences were observed in the response to chemotherapy and in repair of DNA crosslinks. We propose that monitoring the number of DNA crosslinks in peripheral blood lymphocytes might be a quick and sensitive method for monitoring a patient's sensitivity to this agent. Modification of the method by incubation of analysed cells with styrene oxide before crosslink analysis by comet assay extends the use of the method also to laboratories which have no facilities to use ionizing irradiation for introducing DNA breaks into the cells.

Antioxidants and vitamins in clinical conditions.

Various reactive oxygen species (ROS) may be produced from normal biochemical, essential metabolic processes or from external sources as exposure to a variety of agents presented in the environment. Lipids, proteins, carbohydrates and DNA are all capable of reacting with ROS and can be implicated in etiology of various human disorders (rheumatoid arthritis, reperfusion injury, atherosclerosis, lung diseases etc.). In the organism damage by ROS is counteracted with natural antioxidants (glutathione peroxidases, superoxide dismutases, catalase, glutathione, ubiquinol, uric acid, and essential minerals) and nutritional antioxidants from diet (i.e. vitamins E, C, carotenoids). Possible mechanisms of nutritional depletion and side effects of high intake are in the article described.

Comet assay and its use for evaluating oxidative DNA damage in some pathological states.

The comet assay, or single-cell gel electrophoresis (SCGE), is a sensitive, rapid, relatively simple and inexpensive method for detecting DNA strand breaks in individual cells. It is used in a broad variety of applications and as a tool to investigate DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the DNA incubated with an enzyme, which recognizes a specific kind of DNA damage. This damage induced by oxidative stress plays a pivotal role in many diseases and in aging. This article is a critical review of the possible application of the comet assay in some pathological states in clinical practice. Most of the studies relate to evaluating the response of an organism to chemotherapy or radiotherapy with statistically significant evidence of DNA damage in patients. Other useful applications have been demonstrated for patients with heart or neurodegenerative diseases. Only a few studies have been published on the use of this method in critically ill patients, although its use would be appropriate. There are also other scenarios where the comet assay could prove to be very useful in the future, such as in predicting the likelihood of certain pathological conditions.

Oxidative damage to DNA: do we have a reliable biomarker?

Oxidized bases in DNA can be measured directly by high-performance liquid chromatography (HPLC). 7,8-Dihydro-8-oxo-guanine (8-OHgua), as the most abundant oxidation product, is often regarded as an indicator of oxidative stress. Estimates of endogenous 8-OHgua levels in human lymphocyte DNA are between 2 and 8 for every 10(5) unaltered bases--a high frequency in view of the potential mutagenicity of this base alteration and of the presence of an effective base excision repair pathway in eukaryotic cells. An alternative approach to the measurement of oxidized bases makes use of repair endonucleases with appropriate lesion specificities--endonuclease III, for oxidized pyrimidines and formamidopyrimidine glycosylase for 8-OHgua. These enzymes introduce breaks at sites of damage in DNA. The comet assay (single cell gel electrophoresis) can then be used to detect the DNA breaks. This modified comet assay, like other enzyme-linked DNA breakage assays, gives a value for endogenous oxidative base damage that is more than 10-fold lower than most estimated from HPLC. It is possible that HPLC-based estimates are artificially high because oxidation of guanine occurs during isolation, storage, or hydrolysis of DNA. Using a revised DNA isolation procedure designed to decrease in vitro oxidation, we have obtained results for 8-OHgua concentrations in human lymphocytes that are closure to the figures obtained by the comet assay. It is still an open question whether 8-OHgua, measured by HPLC or by the comet assay, is a valid marker for oxidative damage.

DNA adducts, strand breaks and micronuclei in mice exposed to styrene by inhalation.

Genotoxic and clastogenic effects of styrene were studied in mice. Male NMRI mice were exposed by inhalation to styrene in concentrations of 750 and 1500 mg/m3 for 21, 7, 3 and 1 days (6 h/day, 7 days/week). Followed parameters included styrene in blood, specific styrene oxide (SO) induced DNA adducts, DNA strand breaks and micronuclei. The formation of SO induced 7-SO-guanines and 1-SO-adenines in DNA was analysed from lung tissues by two versions of the 32P-postlabeling technique. In lungs after 21 days of exposure to 1500 mg/m3 the level of 7-SO-guanine was 23.0+/-11.9 adducts/10(8) normal nucleotides, while 1-SO-adenine was detected at the levels of 0.6+/-0.2 adducts/10(8) normal nucleotides. Both 7-SO-guanines and 1-SO-adenines strongly correlated with exposure parameters, particularly with styrene concentration in blood (r=0.875, P=0.0002 and r=0.793, P=0.002, respectively). DNA breaks were measured in peripheral lymphocytes, bone marrow cells and liver cells using comet assay. To discern oxidative damage and abasic sites, endonuclease III was used. In bone marrow of exposed mice slight increase of strand breaks can be detected after 7 days of inhalation. A significant increase was revealed in the endonuclease III-sensitive sites after 21 days of inhalation in bone marrow. In the liver cells inhalation exposure to both concentrations of styrene did not virtually affect either levels of DNA single-strand breaks or endonuclease III-sensitive sites. The inhalation of 1500 mg/m3 of styrene induced significant increase of micronuclei after 7 days of exposure (10.4+/-2.5/1000 cells, i.e. twice higher micronuclei frequency than in controls). After 21 days of inhalation no significant difference between the control group and the two exposed groups was observed. Whether the decrease of micronuclei after 21 days of inhalation was due to the inhibition of cell proliferation caused by styrene or due to the natural elimination of chromatide fragments, remains to be clarified. An interesting link has been found between DNA single-strand breaks in bone marrow and frequencies of micronuclei (r=0.721, P=0.028).

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations.

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 microgram/ml, alkaline elution of DNA at 1 microgram/ml and DNA unwinding at 1-2 micrograms/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations > 1 microgram/ml in VH10 cells (short-term treatment): > 0.2 microgram/ml in V79 cells (long-term treatment) and > 8 micrograms/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2 x 10(-5) M Ara C and 2 x 10(-3) MHU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (< 2 micrograms/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

The comet assay: what can it really tell us?

A range of applications of the alkaline comet assay is covered, from investigations of the physicochemical behaviour of DNA, through studies of cellular responses to DNA damage, to biomonitoring of human populations. The underlying principles of this assay are discussed, and new evidence presented which supports the concept of relaxation of supercoiled loops, rather than alkaline unwinding, as the primary reason for comet tail formation. DNA-damaging agents that do not induce strand breaks directly can be detected when cellular repair processes convert lesions to transient strand breaks; an approach is outlined here which maximises this effect and thus widens the scope of the assay. Purified repair enzymes, applied to DNA during the course of the comet assay procedure, greatly increase the sensitivity and specificity of the assay; recent developments with formamidopyrimidine glycosylase (recognising 8-OH-gua and other damaged purines) and uvrABC (for bulky lesions) are presented. The kinetics of cellular repair after low doses of oxidative damage have been followed with this modified comet assay. Finally, the successful measurement of biomarkers of oxidative damage in human populations establishes the comet assay as a valuable tool in molecular epidemiology.

The influence of DNA-topoisomerase II inhibitors novobiocin and fostriecin on the induction and repair of DNA damage in Chinese hamster ovary (CHO) cells treated with mitoxantrone.

Novobiocin (NB) at the concentration of 2 mmol/l added to the culture medium together with mitoxantrone (MIT) (0.05-0.2 micrograms/ml) reduced the number of MIT-induced single-strand breaks of DNA to approximately one half measured by alkaline DNA unwinding and hydroxyapatite chromatography of DNA and similarly it reduced also the fraction of DNA linked to proteins measured by the K(+) -SDS precipitation method. Neither repair of the induced DNA breaks nor removal of the DNA-protein cross-links were markedly influenced by NB action. The specific inhibitor of topoisomerase II, fostriecin, exerted no effect on the induction of DNA breaks by MIT or their repair. Measurement of intracellular concentration of MIT has revealed that in the presence of NB the uptake of MIT into cells is reduced similarly as the number of induced DNA breaks to approximately one half. The combination of 0.1 mmol araC + 10 mmol HU slightly reduced the number of induced DNA breaks, but did not affect their repair. The present results suggest that (1) MIT induces DNA damage which is not repaired by excision repair, (2) MIT induces protein associated breaks of DNA, (3) topoisomerase II does not probably participate in the formation of DNA breaks induced by MIT, as the specific inhibitor of topoisomerase II, fostriecin exerts no effect on either the induction or repair of these breaks.

The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in CHO Chinese hamster cells.

The inhibition of incorporation of 3H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in CHO Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of 3H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks.

The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in CHO Chinese hamster cells.

The inhibition of incorporation of 3H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in CHO Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of 3H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks.

Induction of DNA single-strand breaks and DNA synthesis inhibition in CHO and AWRF cells after exposure to sterigmatocystin and penicillic acid.

Sterigmatocystin is 12 times more toxic to transformed rat fibroblasts (AWRF) than to Chinese hamster ovary cells (CHO). In contrast, penicillic acid is twice more toxic to CHO cells than to AWRF cells. The ability of sterigmatocystin and penicillic acid to inhibit DNA synthesis correlated well with the differences in cytotoxicity of these mycotoxins in the cell lines used. Sterigmatocystin at a concentration of 10 micrograms/ml inhibited DNA synthesis in AWRF cells during a 3-h exposure to 60% of that found in controls, but did not inhibit DNA synthesis in CHO cells. Within the same time interval penicillic acid inhibited DNA synthesis in AWRF cells at concentrations higher than 5 micrograms/ml and in CHO cells at concentrations over 0.5 microgram/ml. Induction of DNA single-strand breaks (SSB) during a 3-h exposure to sterigmatocystin and penicillic acid was comparable in both cell types. The results suggest that sterigmatocystin is metabolized to reactive metabolites that are responsible for the toxicity and DNA synthesis inhibition at a more rapid rate in AWRF cells than in CHO cells. The observed ability to induce SSB indicates that penicillic acid is potentially carcinogenic.

The effect of factors causing induction of DNA breaks on transfection of chicken cells by the XC DNA, and the kinetics of appearance of virus-producing cells after transfection.

The effect of BUdR and 4NQO treatment of the recipient chicken fibroblast cultures on the efficiency of transfection by the XC DNA was investigated. The efficiency of transfection was 2-fold higher when the recipient cultures were incubated in the presence of 10 micrograms BUdR/ml in medium 199 (48%) than when cultures were not treated (25%). The efficiency was not further increased by exposure of BUdR-treated cultures to visible light for 20 min (31%). Growth of BUdR-treated cultures decreased after light irradiation which indicated that BUdR was incorporated into the host cell DNA. Treatment of the recipient cultures with 4NGO at a concentration of 0.4 micrograms/ml for 2 h before transfection exerted only unfavourable effects on transfection efficiency. In cultures positive in transfection, transformation was first detected 3 days after exposure to DNA by the infectious centre assay, with a frequency of 4.76 +/- 6.46 transformed cells per 10(6) cells.

Induction of DNA single-strand breaks and DNA synthesis inhibition by patulin, ochratoxin A, citrinin, and aflatoxin B1 in cell lines CHO and AWRF.

Four mycotoxins, patulin, ochratoxin A, citrinin, and aflatoxin B1 (AFB1), were compared for their cytotoxicity, DNA synthesis inhibition and DNA damage in CHO and AWRF cells. The concentrations causing a 50% inhibition of colony formation in CHO cells were 0.07, 33, 31, and 10 micrograms/ml, respectively, for patulin, ochratoxin A, citrinin, and AFB1. Values obtained for AWRF cells were 0.011, 6.4, 6.7, and 0.15 micrograms/ml, respectively. Patulin strongly inhibited DNA synthesis in both CHO and AWRF cells, at concentrations over 4 micrograms/ml. Ochratoxin A and citrinin elicited mild inhibition of DNA synthesis (up to 20%) at concentrations greater than 50 micrograms/ml. AFB1 at concentrations over 5 micrograms/ml strongly inhibited DNA synthesis in AWRF cells but stimulated DNA synthesis in CHO cells. In accordance with these findings, AFB1 induced single-strand breaks in the DNA of AWRF cells only. Patulin induced about the same levels of DNA breaks in both types of cells, at concentrations greater than 2 micrograms/ml. Citrinin and ochratoxin A produced DNA breaks in CHO cells, at a very high concentration of 200 micrograms/ml. The number of breaks induced in AWRF cells was not statistically significant. The results suggest that the different effects of AFB1 on CHO and AWRF cells are due to the different ability of these cells to metabolize AFB1 to the ultimate carcinogen AFB1-8,9-oxide by microsomal cytochrome P-450-dependent oxygenases. The effects of the other mycotoxins not requiring the metabolic activation were comparable for both cell types.

Effect of aflatoxin B1 on growth of normal and malignant rat fibroblasts in tissue culture (DNA synthesis).

The effect of aflatoxin B1 on rat embryo fibroblasts WREF, the transformed line of rat fibroblasts AWRF-1 derived from WREF and the XC line of rat fibroblasts transformed by Rous sarcoma virus has been compared. By determining growth inhibition of cultured cells and inhibition of colony formation WREF have been shown to have the sensitivity to AFB1 about 33 times greater than XC cells and 7 times greater than AWRF-1 cells. In accordance with these results, it has been found that the inhibition of DNA synthesis and the binding of 3H-AFB1 to the TCA-insoluble cell fraction proceeds much more rapidly in WREF than in XC and AWRF-1 cells. These results suggest that WREF cells are capable of a more rapid metabolic activation of AFB1 to toxic metabolite AFB1-8, 9-oxide, probably owing to higher levels of cytochrome P-450-dependent microsomal oxidases. The resistance of cells to AFB1 fairly correlates with the ability of cells to grow in semisolid agar medium and the culture time.

Long-term preservation of transfecting activity of DNA isolated from rat virogenic XC cells transformed by Prague strain of Rous sarcoma virus.

The DNA isolated from rat virogenic XC cells transformed by Rous sarcoma virus retains standard transfecting activity for 7 months when the DNA solution in 0.1 X PBS, containing 10% glycerol is stored at -70 degrees C. The value of the sedimentation constant S20w does not significantly change during storage.

[Determination of DNA adducts in persons with occupational exposure to alkenes]. / Stanovení DNA adduktu u osob exponovaných alkenum v pracovním prostredí.

DNA adducts induced by alkenes were studied in in vitro systems (cell cultures) and in experimental animals. Properties of DNA adducts, their relationship with other parameters of genotoxic effect and DNA repair were followed. Based on these results, human biomonitoring studies were conducted in order to elucidate the effect of exposure on enhanced levels of DNA and haemoglobin adducts, single-strand breaks in DNA and HPRT mutant frequencies. Surprisingly, no correlation was found between primary DNA lesions and HPRT mutant frequencies, suggesting that no simple quantitative relationship could be drawn between primary DNA damage and mutagenesis. In order to understand mechanisms of genotoxic effects of xenobiotics, further studies aimed at individual susceptibility, individual repair capacity and the role of specific DNA adducts in mutagenesis, are required.

Transmission of B77 virus double mutant LA334 markers by transfection.

Successful transfection of Brown Leghorn chicken fibroblasts was carried out with DNA isolated from duck cells transformed by the LA334 mutant of avian sarcoma virus B77. Transfection of duck cells was negative. The four viruses recovered after transfection were all temperature-sensitive for transformation. Two were fully temperature-sensitive for replication, as shown by analysis of virus replication, by characterization of virus particles produced at the nonpermissive temperature using density gradient centrifugation, and by electron microscopic examination. The other two viruses were only partially temperature-sensitive for replication. The results suggest that both the src and gag regions of the avian sarcoma virus genome are transferred simultaneously during transfection, probably by a single integral provirus copy.

Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay.

There is a need for a reliable, robust and sensitive assay for DNA repair, suitable for use with human lymphocyte samples in molecular epidemiological investigations. The comet assay (single cell alkaline gel electrophoresis) has been modified to measure the ability of a simple subcellular extract of lymphocytes to carry out the initial step of repair, i.e. incision, on a DNA substrate carrying specific lesions--namely, oxidized bases introduced by visible light in the presence of photosensitizer. The cell extract is free of non-specific nuclease activity, incising DNA only if the DNA has been treated with photosensitizer and light. The activity varies between individuals, but consistency is seen between samples from each individual taken on occasions several months apart. The lack of activity of extract from Ogg1(-) mouse cells (deficient in the glycosylase that excises 8-oxoguanine) in this assay confirms that the activity measured is predominantly excision repair of oxidized bases. This new DNA repair assay is simple, rapid and requires only small quantities of lymphocyte extract (obtainable from 10 ml blood).

7-Alkylguanine adducts of styrene oxide determined by 32P-postlabeling in DNA and human embryonal lung fibroblasts (HEL).

The modified 32P-postlabeling method was used for the detection of N-7-(2-hydroxy-phenylethyl) guanine adducts in DNA and human embryonal lung (HEL) cells treated in vitro with styrene oxide (SO). The total recovery of 7-alkylguanine adducts of styrene oxide in DNA analysed by 32P-postlabeling assay was 4.1 +/- 0.6%. The disappearance of 7-alkyldeoxyguanosine monophosphate adducts from SO-modified DNA at 37 degrees C showed a half-life of 19 h. The levels of 7-alkylguanine DNA adducts and single-strand breaks (SSBs) in DNA were determined in HEL cells treated with SO for 3 and 18 h. In the 3 h treatment there was a concentration-dependent increase of both 7-alkylguanine adducts and SSBs in DNA (r = 0.98, P = 0.012 and r = 0.99, P = 0.003, respectively). We found a significant correlation between 7-alkylguanine DNA adducts and SSBs in DNA (r = 0.98, P = 0.011). In HEL cells approximately 3-fold higher levels of both 7-alkylguanine DNA adducts and SSB in DNA were found after the SO (100 microM) treatment for 3 h than after the treatment for 18 h. A significant concentration-dependent increase was found only for SSB ( r = 0.95, P = 0.024) in the 18 h treatment with SO. There was no significant correlation between 7-alkylguanine adducts and SSBs (r = 0.72, P = 0.15). Our data suggest relatively fast removal of both 7-alkylguanine adducts of SO and DNA SSBs.

The role of various biomarkers in the evaluation of styrene genotoxicity.

We evaluated our data on the occupational exposure to styrene in lamination workers. The battery of parameters included markers of external and internal exposure and biomarkers of biological effects and susceptibility. DNA repair capacities have been determined in both exposed and control groups. Styrene workplace concentration significantly correlated with styrene concentration in blood, exhaled air and urinary mandelic acid. Haemoglobin and O(6)-styrene oxide (SO)-guanine DNA adducts were significantly higher in exposed subjects as compared to controls and correlated with exposure parameters. In styrene-exposed workers 1-SO-adenine DNA adducts were detected (2.6 per 10(9) dNp), while in controls these adducts were below the detection limit. 1-SO-adenine adduct levels were affected by both acute and cumulative exposure (P=0.001, F=86.0 and P=0.017, F=59.0, respectively) and associated with cytochrome P450 2E1 (CYP2E1) polymorphisms (R(2)=0.442). Mutant frequencies (MF) at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus appeared to accumulate with exposure over time and were associated with glutathione S-transferase P1 (GSTP1) polymorphism. DNA repair capacity increased with the exposure, except for the group exposed to the highest styrene concentration. In this particular group, increased DNA repair capacity to remove oxidative DNA damage was found.