The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of immunocytochemical determination with detection in isolated DNA.

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH3)2d(pGpG) (Pt-GG), cis-Pt(NH3)2d(pApG) (Pt-AG) and cis-Pt(NH3)2(dGMP)2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible--at least in part--for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to plantinum-DNA damage may also be involved.

Treatment of age-related memory complaints with Ginkgo biloba extract: a randomized double blind placebo-controlled study.

A growing number of people is subject to age-related cognitive impairment due to the proportional increase of the ageing population. Therefore, there is a growing interest in cognition-enhancing substances. The efficacy of an alcohol/water extract of Ginkgo biloba in elderly individuals with memory- and/or concentration complaints was tested in a randomized, double-blind, placebo-controlled study by using both subjective and objective parameters. After a wash-out period of 4 weeks 241 non-institutionalised patients in the age range 55-86 years were randomly allocated to receive either Ginkgo biloba alcohol/water extract in a high dose (HD), a low dose (LD) or a placebo (PL) for 24 weeks. Patients were assessed using a psychometric testbattery in the following order: Expended Mental Control Test (EMCT) measuring attention and concentration, Benton Test of Visual Retention-Revised (measures short term visual memory), Rey Test part 1 (measures short term memory and learning curve), Beck Depressive Inventory (BDI) measuring the presence and severeness of a depression in order to exclude depressive patients and Rey Test part 2 (measures long term memory: recognition). Furthermore, subjective perception of memory and concentration was measured. 197 patients completed the study (mean MMSE score: 26.29). In the subjective test, the EMCT, the Rey 1 and Rey 2 no significant differences in improvement in time between the groups were observed. In the Benton test increases of 18%, 26% and 11% (expressed as percentage of baseline scores) were observed in the HD, LD and PL respectively (MANOVA; p = 0.0076). No substantial correlation was observed between subjective perception of the severeness of memory complaints and the objective test results. No differences in the number of (gastrointestinal) side effects were observed between placebo and verum groups. These results indicate that the use of Ginkgo extracts in elderly individuals with cognitive impairment might be promising. Further research using both subjective and objective measurements is recommended.

Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells.

The cytotoxicity of cisplatin and cisplatin-DNA adduct formation in vitro and in vivo is clearly enhanced by hyperthermia. We investigated whether cytotoxicity and platinum-DNA adduct formation of two promising new third-generation platinum derivatives, lobaplatin [1,2-diamminomethylcyclobutane platinum(II) lactate] and oxaliplatin [oxalato-1,2-diaminocyclohexane platinum(II)], are also enhanced by hyperthermia. Cisplatin was used for comparison. SW 1573 cells were incubated with cisplatin, lobaplatin or oxaliplatin at different concentrations for 1 h at 37 degrees, 41 degrees and 43 degrees C. The reproductive capacity of cells was determined by cloning experiments. Immunocytochemical detection of platinum-DNA adducts was performed with the rabbit antiserum NKI-A59. At 37 degrees C, cisplatin was the most cytotoxic, followed by oxaliplatin and lobaplatin. Hyperthermia clearly enhanced the cytotoxicity of cisplatin, lobaplatin and oxaliplatin. There was no further increase in cytotoxicity at 43 degrees C compared to 41 degrees C for cisplatin and oxaliplatin. A further increase in cytotoxicity at 43 degrees C was observed for lobaplatin. At 43 degrees C thermal enhancement was higher for lobaplatin than for oxaliplatin, with the reverse pattern at 41 degrees C. For both drugs, thermal enhancement of cytotoxicity was lower than observed for cisplatin. Immunocytochemical detection of platinum-DNA adducts was feasible for all the drugs. Adduct formation was enhanced at 43 degrees C for cisplatin, lobaplatin and oxaliplatin with a relative increase of 410%, 170% and 180%. These results seem to confirm that an increase in platinum-DNA adduct formation is involved in the in vitro thermal enhancement of cytotoxicity. The observed thermal enhancement of cytotoxicity of lobaplatin and oxaliplatin in vitro warrants further in vivo investigations.

In vitro formation of DNA adducts by cisplatin, lobaplatin and oxaliplatin in calf thymus DNA in solution and in cultured human cells.

Two interesting representatives of a new generation of platinum-based cytostatic drugs that are currently being tested in clinical trials are lobaplatin [1,2-diaminomethylcyclobutane platinum(II) lactate] and oxaliplatin [1,2-diaminocyclohexane platinum (II) oxalate]. Since little is known about the DNA adduct formation of these compounds, we studied their formation in DNA in vitro in calf thymus DNA and in cells. The major adducts formed in vitro were the Pt-GG and Pt-AG intrastrand crosslinks. The latter adducts could be detected using a recently developed 32P-postlabelling method. Using both this assay and atomic absorption spectroscopy, it was shown that there is a substantially higher rate of the in vitro adduct formation by cisplatin, compared with lobaplatin and oxaliplatin. Platinum concentrations required to obtain 90% cell kill during a 2 h incubation of A2780 cells were 15 microM for cisplatin and oxaliplatin and 22 microM for lobaplatin. Using an antiserum originally raised against cisplatin-treated DNA, we were also able to detect platinum-DNA adducts induced by lobaplatin and oxaliplatin. Maximal nuclear staining for all three compounds was observed after a 4 h post-incubation period. The nuclear staining level induced by cisplatin was about 10-fold higher than after lobaplatin and oxaliplatin treatment. GG and AG adducts, measured by 32P-postlabelling, also showed maximum levels at about 4 h after treatment. Relative GG peak levels were 4:1:3 for cisplatin, lobaplatin and oxaliplatin, respectively. The ratios of GG over AG intrastrand crosslinks in the A2780 cells were not significantly different for the various compounds. In conclusion, the 32P-postlabelling technique has been shown to be appropriate for adduct analysis, not only for the classical Pt compounds cisplatin and carboplatin but also for novel platinum compounds like lobaplatin and oxaliplatin. Results indicated large differences in reactivity of the latter compounds to DNA in vitro, compared with cisplatin. This difference was smaller in cells, suggesting enhancement of adduct formation by certain cellular mechanisms and/or compounds. From these studies, no conclusions can be drawn with respect to the cytotoxicity of the different Pt-GG and Pt-AG intrastrand crosslinks formed by these compounds.

The formation and persistence of carboplatin-DNA adducts in rats.

The formation and persistence of platinum-DNA adducts were studied with immuno(cyto)chemical methods in male and female Sprague-Dawley rats treated with a single i.p. dose of carboplatin. Linear dose-effect curves were observed for kidney and liver with an immunocytochemical assay using NKI-A59 antiserum that recognizes intrastrand cross-links. With this method, no staining of the nuclei due to platinum-DNA damage could be observed in the spleen, testis, uterus, or ovary after administration of up to 80 mg/kg carboplatin. A homogeneous staining of the nuclei in the liver was observed. The nuclear staining in the kidney was somewhat more intense but less homogeneous, with small groups of intensely stained nuclei occasionally being seen in the outer cortex. An approximately 15 to 20-times lower dose of cisplatin than of carboplatin was needed to reach equal staining levels in the liver and kidney. Plateau staining levels in both tissues were reached at between approximately 8 and 48 h after administration of the carboplatin. This was followed by a significant reduction in the kidney samples, whereas the staining levels in the liver section seemed to be more persistent. No major difference was observed between male and female rats in the formation and removal of DNA damage in these tissues. The levels of the various DNA adducts were measured with a competitive ELISA in liver, kidney, spleen, testis, and combined ovary/uterus samples collected at 8 and 48 h after carboplatin administration. At both 8 and 48 h, the highest platination levels were observed in the kidney, followed--in decreasing order--by the liver, combined uterus and ovary samples, spleen, and testis. At 8 h after administration of carboplatin, the relative occurrence of the bifunctional adducts Pt-GG (34%), Pt-AG (27%), and G-Pt-G (32%), was similar in all tissues. The same held for the monoadducts that amounted to about 7% of the total DNA platination. These data indicate that in the first few hours after carboplatin treatment, no preference for the formation of Pt-GG adducts was observed, which confirms our earlier observations obtained with cultured cells. When the total DNA-platination levels (calculated from the sum of the adducts) seen at 8 and 48 h after treatment were compared, a substantial decrease in DNA platination was observed in the kidney (37%), liver (30%) and ovary/uterus (39%), whereas the repair levels in the testis (9%) and, probably, the spleen (18%) were substantially lower. In all tissues studied, only the relative occurrence of the Pt-GG adducts increased between 8 and 48 h, and as a result, at 48 h, after carboplatin administration the Pt-GG adduct was the major adduct persisting in the DNA samples.

Formation of DNA adducts by the anticancer drug carboplatin: different nucleotide sequence preferences in vitro and in cells.

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)

Detection of platinum-DNA adducts by 32P-postlabelling.

We developed a sensitive 32P-postlabeling method for the detection of bifunctional intrastrand crosslinks d(Pt-GpG) and d(Pt-ApG) in DNA in vitro and in vivo. After enzymatic digestion of DNA the positively charged platinum adducts were purified from unplatinated products, using strong cation exchange chromatography. Subsequently the samples were deplatinated with cyanide, because platinated dinucleotides are very poor substrates for polynucleotide kinase. The excess of cyanide was removed using Sep-pak C18 cartridges, and the resulting dinucleoside monophosphates d(GpG) and d(ApG) were subsequently postlabelled. Analysis of the postlabelling mixture was performed by a combined TLC and HPLC-procedure. Good correlations with existing methods (AAS, immunocytochemistry and ELISA) were found in DNA samples treated in vitro and in vivo with cis- or carboplatin. The detection limit of the assay was 1 adduct/10(7) nucleotides in a 10 micrograms DNA sample.

Selective intra-arterial infusion of high-dose cisplatin in patients with advanced head and neck cancer results in high tumor platinum concentrations and cisplatin-DNA adduct formation.

A group of 23 patients with advanced head and neck cancer were treated with highly selective intra-arterial (IA) cisplatin 150 mg/m2 delivered rapidly through microcatheters. The systemic effects of cisplatin were neutralized by concurrent administration of sodium thiosulfate. Two-to-threefold higher tumor platinum contents were detected in tumor biopsies after selective IA cisplatin administration compared to historical controls (treated with 100 mg/m2 IA). Cisplatin-induced DNA modification in human tumor biopsies was quantitated using the antiserum NKI-A59. High levels of cisplatin DNA adducts were detected which correlated linearly with the tumor platinum content (r2 = 0.62). The addition of radiotherapy to this high dose intensity cisplatin treatment resulted in a 92% complete response (CR) rate (12 of 13 patients achieved a CR). Since no difference in tumor platinum content was detected between patients receiving or not receiving radiotherapy (13 and 10 patients, respectively), but the response rate was substantially different (12 CR and 1 partial response with radiotherapy versus 6 partial and 4 non-responders without radiotherapy), these data suggest that the high platinum levels achieved by selective IA infusion were sufficient to produce enough interaction with radiotherapy to cause a 92% CR rate. Whether this interaction is additive or synergistic is as yet unclear.

Drug-induced DNA modification in buccal cells of cancer patients receiving carboplatin and cisplatin combination chemotherapy, as determined by an immunocytochemical method: interindividual variation and correlation with disease response.

Twenty-six patients with a variety of tumor types were treated according to a phase 1 experimental treatment protocol consisting of repetitive cycles of cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin, 200-480 mg/m2) at day 1 and cis-diamminedichloroplatinum(II) (cisplatin, 50-100 mg/m2) at day 3. Buccal cells were collected in one or two treatment cycles prior to carboplatin, 24 h after carboplatin, just prior to cisplatin, and approximately 24 h after cisplatin administration. Drug-induced DNA modification was visualized at the single cell level by anti-serum NKI-A59 and quantitated by microdensitometry. All (39 of 39) treatments with carboplatin, and almost all (33 of 35) treatments with cisplatin resulted in an increase in nuclear stain. Interindividual variation in drug-induced, adduct-specific nuclear stain amounted to a factor of 5-8 for carboplatin and 5-12 for cisplatin. This drug-induced increase was, however, not related to the dose of either carboplatin or cisplatin, suggesting that large interindividual differences in DNA adduct formation and/or repair obscured the effects of dose variation within the relatively small range used for the drugs (2.4 for carboplatin and 2.0 for cisplatin). This explanation was strengthened by the good reproducibility of the immunocytochemical assay and by the reasonable correlation between carboplatin-induced nuclear stain in cycles 1 and 2 (correlation coefficient, 0.69; P = 0.009). Mean carboplatin-induced nuclear stain was significantly higher in the first cycle than in the second cycle (P = 0.0001) but this difference was no longer significant when drug-induced nuclear stain was corrected for carboplatin dose. Differences in cisplatin-induced nuclear stain between cycle 1 and cycle 2 were small and not significant. Carboplatin-induced nuclear stain was significantly higher in the partial responders than in the nonresponders (P < 0.0001, two cycles combined); the level of statistical significance remained the same after dose correction. Cisplatin-induced nuclear stain did not differ significantly between partial responders and nonresponders; this result might, however, be confounded to some extent by remaining carboplatin-induced nuclear stain at the moment of cisplatin administration. It is concluded that determination of the extent of platinum-induced DNA modification might be helpful in predicting the tumor response in cancer patients.

Effects of ozone, hexachlorobenzene, and bis(tri-n-butyltin)oxide on natural killer activity in the rat lung.

The respiratory tract is a major route of exposure to noxious agents as well as pathogens such as viruses. Natural killer (NK) activity is an important first line of defense to virally infected cells as well as certain neoplasms; therefore, testing the effects of exposure to toxic compounds on this activity is important in understanding the immunotoxic potential of the compound. Lymphoid cell suspensions, obtained after enzymatic dispersion of rat lungs and purification over nylon wool columns, showed in vitro natural killer activity toward YAC lymphoma cells. Validation of the test with well-known NK activity stimulators such as Bacillus Calmette-Guérin (BCG), interleukin-2 (IL-2), interferon (IFN), and inhibitors like anti-asialo-GM1 (ganglio-n-tetrasylceramide) antibody confirmed the reliability of the test as an assay for detecting NK activity in rat lungs. Using this assay, we studied the effects of exposure to ozone (O3), hexachlorobenzene (HCB), and bis(tri-n-butyltin)oxide (TBTO) on NK activity in rat lung. Inhalation exposure to O3 for 7 days at 0.4 and 0.8 mg/m3 resulted in stimulation, and exposure at 1.6 mg O3/m3 resulted in suppression of NK activity. Oral exposure to HCB in concentrations of 150 and 450 mg/kg food for 6 weeks suppressed NK activity in rat lungs in a dose-related manner. This was also true for 6 weeks of oral exposure of rats to 20 and 80 mg TBTO/kg food, but to a lesser extent. In summary, we have developed and validated a method to measure the effects of (toxic) substances on NK activity in rat lung.