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LacZ transgenic mice are suitable for short-term mutagenicity studies in vivo. Mutagenicity in these mice is determined in the lacZ transgene. Since the lacZ gene is of bacterial origin the question has been raised whether DNA-adduct formation and repair in the transgene are comparable to those in total genomic DNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) and killed at several time points following treatment. Some mice were pretreated with O6-benzylguanine to inactivate the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). O6-ethylguanine (O6-EtG) was determined in lacZ in liver and brain by means of a monoclonal antibody-based immunoaffinity assay. In addition, O6-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomic DNA of liver and brain with an immunoslotblot procedure. In liver, the initial O6-EtG level in total genomic DNA was 1.6 times that in lacZ. The extent of repair of O6-EtG during the first 1.5 h after treatment was 2.1 times that in lacZ. At later time points, O6-EtG repair was the same. N7-EtG repair in genomic DNA was evident. In contrast to the liver, little repair of O6-EtG in total genomic and lacZ DNA occurred in the brain while N7-EtG was repaired. No initial difference in O6-EtG levels were found in lacZ and genomic brain DNA. These findings indicate that in the liver, total genomic DNA is more accessible than lacZ to ENU and/or the AGT protein, during the first 1.5 h following treatment. Because the difference in O6-EtG levels in the transgene and genomic DNA in the liver is restricted to the first 1.5 h after treatment, while the fixation of mutations occurs at later time points, O6-EtG-induced mutagenesis most likely is also very similar in both types of DNA.

We have studied the formation of adducts upon carboplatin treatment of isolated DNA and in cells. The major adduct formed in vitro, determined with atomic absorption spectroscopy and enzyme-linked immunosorbent assay, was the intrastrand cross-link cis-Pt(NH3)2d(pGpG)(Pt-GG) (58%). cis-Pt-(NH3)2d(pApG) (Pt-AG) (11%), cis-Pt(NH3)2d(GMP)2 (G-Pt-G) (9%), and monofunctionally bound platinum (cis-Pt(NH3)3dGMP (Pt-G), 22%) were formed in smaller amounts. These relative occurrences of the adducts, average values found between 1 and 16 h of incubation, are comparable with those after incubation with cisplatin. The formation of carboplatin-DNA adducts was slow, and about 230-fold more carboplatin than cisplatin (molar dose) was required to obtain equal levels of platination after 4 h of incubation. However, less than 20 times more carboplatin was needed to obtain equal levels of cytotoxicity after 1 h of exposure of CHO cells. The percentages of the carboplatin-DNA adducts after 7-12 h postincubation of the cells (determined with ELISA), Pt-GG (30%), Pt-AG (16%), G-Pt-G (40%), and Pt-G (14%), were different from those of the in vitro data. After 12 h postincubation, the number of interstrand cross-links (determined by alkaline elution) amounted to about 10% of the G-Pt-G adducts and 3-4% of the total amount of adducts. The immunocytochemical detection (with antiserum NKI-A59) of the platinum-DNA modifications showed a pattern similar to that found for the various bifunctional adducts: the initially low levels slowly increased to maximum values within 7-12 h and then slowly decreased. In conclusion, carboplatin forms the same bifunctional adducts as cisplatin.(ABSTRACT TRUNCATED AT 250 WORDS)

During chemotherapy with a cisplatin-containing combination of drugs, 217 blood samples from 30 cancer patients were analyzed for the presence of the main cisplatin-DNA adduct cis-Pt(NH3)2d(pGpG) (Pt-GG). Cisplatin was administered during 3-h infusions on each of 5 consecutive days, resulting in increasing adduct levels which, on the average, were about twice as high after the fifth as after the first infusion. Higher levels were found in blood samples of patients who received the same total amount of cisplatin in one single 3-h infusion. No significant differences in adduct levels were found during first and repeated courses. The nonlinear dependence of adduct levels on total dose can be attributed to removal of adducts. At 21 h after a very first cisplatin infusion 76% of the adducts were removed. Lower percentages of removal were observed over the 21-h periods following the fourth and fifth infusions of 5-day courses (49 and 53%, respectively). After the initial 21 h the removal of adducts continued, albeit at a slower rate. Substantial interindividual variation was found in the adduct levels, which did correlate with the levels obtained after in vitro cisplatin treatment of blood samples from the same patients but not with their age or gender. Testicular cancer patients with complete tumor response showed higher adduct levels in their blood than those with partial response or progressive disease. When blood samples from 8 healthy volunteers were treated with cisplatin in vitro, the person-to-person variation in adduct levels and the intraindividual variation observed over a 2-year period were found to be in the same range, which was narrower than that observed with samples from treated patients. In vitro studies with human blood showed that the formation of the Pt-GG adduct is proportional to cisplatin concentration and complete after about 1 hour. In some of the in vivo and in vitro cisplatin-treated blood samples, all 4 known platinum-DNA adducts were determined. In all cases Pt-GG was by far the major adduct, and no significant differences were observed with respect to the relative amounts of the 4 adducts. Similar adduct ratios were found in DNA from a testicular tumor obtained from a patient who underwent orchidectomy; the Pt-GG adduct level was about 10-fold higher than that in his blood cells.

As a result of permanent exposure to low levels of various endogenous and exogenous genotoxic agents, large numbers of lesions are continuously induced in the DNA of cells of living organisms. Such lesions could lead to dysfunction of cells and tissues, and they might well be the underlying cause of the age-related reduction of homeostatic capacity and the increased incidence of cancer and other diseases of old age. The rate of damage induction as well as the persistence of the lesions depends on the activity, efficiency and reliability of a wide variety of molecular defense systems. However, a certain degree of imperfection seems to be a general characteristic of most of these defense systems and this could lead to a gradual accumulation of DNA alterations during aging. Even when the original lesions are quickly removed, they can still lead to secondary changes in the DNA, such as DNA-sequence changes and changes in gene expression. This process would be accelerated in case of the occurrence of an age-related decline in the efficiency of these molecular defense systems. This review deals with the present knowledge on the occurrence of 'spontaneous' DNA damage in aging organisms, its potential sources, the influence of preventive and processive cellular defense mechanisms and its consequences in terms of DNA-sequence changes, DNA conformational and configurational changes and changes in gene expression. In general, it can be concluded from the data discussed here that, in spite of a number of discrepancies and conflicting results, an age-related accumulation of DNA alterations occurs at all levels, e.g., chemical structure, DNA-sequence organization and gene expression.

The formation and repair of cisplatin [cis-PtCl2(NH3)2] adducts in the DNA of cultured normal and repair-deficient human fibroblasts are presented in relation to cell survival after cisplatin treatment. Directly after treatment with cisplatin, in normal (MB), Fanconi's anemia (FA), and xeroderma pigmentosum (XP) fibroblasts four platinated products are found. The major adduct is cisplatin bound to two neighboring guanines, Pt-GG (62-75%). A less abundant product is cisplatin bound to an AG sequence (Pt-AG). Binding to two guanines separated by one or more bases or to two guanines in opposite DNA strands (together measured as G-Pt-G) and cisplatin bound monofunctionally to guanine (Pt-G) are also found in small amounts. The distribution of the four products is similar to that found previously, in in vitro systems as well as in living cells. Directly after cisplatin treatment, the removal of cisplatin-DNA adducts is fast in normal and FA fibroblasts, whereas in XP fibroblasts adduct removal proceeds slowly throughout the repair period studied. Both FA and XP fibroblasts are extremely sensitive to cisplatin with regard to cell killing. For FA fibroblasts this sensitivity may be attributed to the fact that in these cells initially more DNA-adducts are formed than in normal fibroblasts, and/or to their known deficiency in the repair of DNA interstrand cross-links. For XP fibroblasts this sensitivity may be caused by their deficiency in the fast repair process, known as excision repair.

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.

Two different enzyme-linked immunosorbent assays (ELISAs) are used for the sensitive determination of cisplatin-DNA adducts in, for example, blood cells of cisplatin-treated cancer patients. Poirier et al. determined the adducts in native DNA with an antiserum raised against highly modified cisplatin-DNA. Fichtinger-Schepman et al. assayed the various adducts after chromatography of enzymatically digested DNA samples, with antibodies raised against synthetic haptens mimicking the Pt-containing digestion products. In identical human samples analysed by both methods, 14- to 300-fold higher adduct levels were found with the Fichtinger-Schepman method. Adduct levels in organ samples of cisplatin-treated rats and mice allowed comparison of both ELISAs with independent Pt assays (atomic absorption spectroscopy, AAS). AAS data confirmed the results of the Fichtinger-Schepman assay, whereas ELISAs according to Poirier showed differences up to a factor of 1000. Analysis of cisplatin adducts in in vitro modified DNA revealed that the Poirier antibodies yield correct estimates with highly modified DNA, but fall short at low platination levels: no adducts could be detected in samples modified to a level similar to that found in blood cells from cisplatin-treated patients. The positive responses obtained with samples of such patients, who had been treated repeatedly, might be explained if cisplatin adducts are preferentially formed around persistent lesions from the previous treatments.

Cisplatinum [cis-diamminedichloroplatinum(II)] is one of the most active antitumour agents and its effect is mainly due to the formation of cisplatinum-DNA crosslinks. Formation of cisplatinum-DNA intrastrand crosslinks in nucleated white blood cells of patients was measured, both in (pretreatment) samples exposed in vitro and in cells collected immediately after in vivo exposure to cisplatinum. Large interpatient variations were found. The in vitro results showed a linear correlation with the in vivo data (cc = 0.91). The in vitro measurements of crosslinks may allow prediction of response and avoidance of toxicity in individual patients.

A commercial sample of the tuberculostatic drug isoniazid (INH) was found to have a weak mutagenic activity towards Salmonella typhimurium strains TA100 and TA1535. The addition of a rat or mouse liver homogenate to the test system decreased the mutagenic effect of INH. Hydrazine, an impurity of the INH sample, was also weakly mutagenic in strains TA100 and TA1535, but not in the extent that could account for the mutagenicity of the INH sample. An inhibition of DNA synthesis in human fibroblasts was observed for INH, this effect being potentiated by the addition of manganese to the test system. There was no induction of unscheduled DNA synthesis in human cells by INH except in the presence of manganese.

The same spectrum of cisplatin adducts was detected in DNA isolated from white blood cells of a cisplatin-treated cancer patient as had been found in cisplatin-treated DNA in vitro. The adducts were quantified in femtomole amounts by competitive enzyme-linked immunosorbent assay (ELISA) with three antisera raised against synthetic cisplatin-containing (oligo)nucleotides. For this assay, DNA samples digested with nucleases were fractionated by ion-exchange chromatography; the fractions were used as inhibitors of antibody binding. Determinations of the main adduct formed, cis-Pt(NH3)2d(pGpG), in patients immediately after a first treatment with equal doses of cisplatin showed interindividual differences in the platination levels of the white blood cells. These differences were found to correlate with those found after in-vitro exposure to cisplatin of blood samples taken from patients before treatment. In vivo, about 75% of the adducts formed after the first treatment were removed within 24 h. During a five-day course, the amounts of the main adduct increased after the first three administrations; no increase was seen on day 4 or 5. By day 6, considerable removal of adducts had occurred. Analysis of the formation and repair of the cis-Pt(NH3)2d(pGpG) adducts in cultured cells, i.e., human fibroblasts with different DNA repair capacities and one bladder and two testicular human cancer cell lines, indicated that both the amounts of adducts formed and the ability of the cells to repair the adducts can differ. These differences appear to determine the susceptibility of the cells for the cytotoxic action of cisplatin.

A comparative study was performed with a variety of human cell lines on the effects of treatments with cis-diamminedichloroplatinum(II) (cisplatin) on cell survival and the induction of unscheduled DNA synthesis. In addition to control fibroblasts (Han, MB), cell lines defective in DNA repair were used [xeroderma pigmentosum, XP(A) and XP(F), and Fanconi's anemia (FA)], as well as cells deficient in arylsulfatase A (mucolipidosis II, ML1 and ML2). Ultraviolet light and mitomycin C were included in this study as model DNA-damaging agents. Furthermore, induction of DNA interstrand cross-links by cisplatin and their repair were studied. As for survival, only XP cells were abnormally sensitive to ultraviolet light, and only FA cells were abnormally sensitive to mitomycin C. To cisplatin, however, all mutants tested were more sensitive (2 to 5 times) than were normal cells. Unscheduled DNA synthesis induction by ultraviolet light was strong in all but the XP cells; the other two agents did not induce unscheduled DNA synthesis. Induction of DNA interstrand cross-links by cisplatin was linear with dose. Formation continued for up to 18 to 24 h after treatment. During this period, all cells but the ML mutants responded similarly. In ML cells, much fewer cross-links were induced, which were repaired rapidly. In FA cells, accumulation continued for at least 96 h; in the other cells, most of the cross-links had been removed after that period. In the discussion, the cisplatin-induced DNA interstrand cross-links are proposed as an important potentially lethal lesion, in view of their persistence in the highly sensitive FA cells. Furthermore, the possible involvement of certain steps of the long-patch excision repair pathway in the removal of this lesion is considered. The sensitivity of ML cells to cisplatin is attributed to cytoplasmic effects, rather than to chromosomal damage.

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (ssb) or alkali-labile sites were measured by centrifugation in alkaline sucrose gradients after lysis in alkali. 4 agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate (EMS) and diethyl sulphonate (DES). The compounds differed greatly in their potency to induce the lesions measured when compared on a molar basis, but comparison at equicytotoxic doses showed relatively small differences. Upon prolonged incubation of the DNA in alkali, the number of ssb increased considerably. DNA from untreated cells showed biphasic kinetics: slow ssb formation for about 10 h, then the rate increased and remained constant for up to 40 h. Treated cells showed an accelerated, dose-dependent linear generation of ssb for 10 h, followed by a short plateau; then ssb were formed again at a constant rate, somewhat higher than that in controls. Ssb formed in the initial phase are ascribed to phosphotriester hydrolysis, those after the plateau to unidentified causes. Zero intercepts appeared to be a measure of apurinic sites generated intracellularly. A 24-h repair period preceding lysis reduced the ENNG intercept, but not that of DES. Rapid degradation of DES during the 1-h treatment occurred, so most "apurinic-site lesions" were induced in the beginning of exposure and possibly were already repaired at the end. The types of lesion distinguished (reparable and non-reparable apurinic sites, phosphotriesters) appeared of little consequence for cell survival.