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Topotecan- or mitoxantrone-selected cell lines (T8 and MX3, respectively), derived from the human IGROV1 ovarian cancer cell line, were resistant to the topoisomerase I inhibitors topotecan, SN-38 (the active metabolite of irinotecan), and 9-aminocamptothecin, as well as to the topoisomerase II drug mitoxantrone. In both resistant cell lines, decreased accumulation of topotecan and mitoxantrone was observed, caused by enhanced energy-dependent efflux of the drugs involved. In both cell lines, we found that the breast cancer resistance protein/mitoxantrone resistance/placenta-specific ATP binding cassette (BCRP/MXR/ABCP) gene was overexpressed. Furthermore, BCRP/MXR/ABCP expression levels in various partially revertant T8 cells correlated with the levels of resistance to topotecan, SN-38, and mitoxantrone, strongly suggesting BCRP/MXR/ABCP to be the transporter responsible for the enhanced efflux. Pharmacodynamic analysis demonstrated that BCRP/MXR/ABCP is a very efficient transporter of topotecan; in vitro, 70% of the intracellular topotecan pool was transported out of the T8 or MX3 cells within 30 s. In conclusion, we report for the first time that BCRP/MXR/ABCP can also be up-regulated upon exposure of tumor cells to the clinically important drug topotecan, and that BCRP-mediated efflux of topotecan is very efficient. This highly efficient efflux of topotecan by BCRP/MXR/ABCP may have clinical relevance for patients being treated with topotecan.

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Binding of the cytostatic drug carboplatin to DNA was studied in solution, in RIF-1 and CHO cell lines and in human buccal cells after in vitro or in situ drug exposure. Results were compared with DNA adduction by cisplatin. The rate of binding in solution, determined by atomic absorption spectroscopy, was 35 times lower for carboplatin than for cisplatin. Adduct formation in cells in vitro was determined in a quantitative immunostaining assay. Staining intensities after carboplatin treatment were at least 29 times lower than after an equimolar dose of cisplatin. For RIF-1 and CHO cells, maximum levels of carboplatin-induced DNA modification were obtained 24 h after treatment; these levels correlated with cell killing. Adduct-specific staining in buccal cells from two carboplatin-treated patients increased 5-7 fold between 0 and 14 h after infusion, reaching a maximum at 10-14 h. This strongly contrasts with buccal cells from a cisplatin-treated patient, in which the adduct-specific staining signal increased by only 23% between 0 and 6 h after infusion, and then declined. This difference in the rate of adduct formation in vivo is consistent with the in vitro data.

The relationship between cell killing and the binding of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) to DNA was studied in six mammalian cell lines. Two of the human cell lines (COV413B) were of the same origin, comprising one sensitive to cis-DDP and the other with induced resistance to the drug. The four other lines, two rodent (RIF-1, Chinese hamster ovary) and two human (A2780, A1847), were unrelated. The cell lines differed in their sensitivity to cis-DDP, as tested in a clonogenic assay. cis-DDP-DNA binding was determined by quantitative immunocytochemistry using an antiserum against cis-DDP-modified DNA. The resistance factors relative to RIF-1, calculated from full survival curves for cis-DDP, were 3.8 +/- 0.4 for Chinese hamster ovary cells and 8.8 +/- 0.7 for both A2780 and A1847 lines. Using quantitative immunocytochemistry, the levels of the adduct-specific nuclear staining density compared with RIF-1 cells were 4.8 +/- 0.2 for Chinese hamster ovary cells, 9.1 +/- 0.2 for A2780, and 10.0 +/- 0.1 for A1847 cells, i.e., in good agreement with the resistance factors. In studies with the COV413B cells and their cis-DDP-resistant counterpart COV413B-PtR, immunologically detected adduct levels again correlated closely with resistance factors (correlation coefficient = 0.97). The kinetics of cis-DDP-DNA adduct formation and loss was investigated in RIF-1, A2780, and A1847 cells by the immunocytochemistry technique. Adduct levels after a 1-h incubation with approximately equitoxic doses of cis-DDP increased by 18 to 32% (average, 27%) between 0 and 6.5 h after treatment and then declined. Adduct half-lives in this latter phase did not correlate with the sensitivities of the cells for cis-DDP. These results indicate that the initial level of cis-DDP-DNA binding measured by quantitative immunocytochemistry may be a reasonable predictor of sensitivity to this chemotherapeutic drug.

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.

The formation and stability of interaction products between the anti-cancer drug cis-diamminedichloroplatinum(II) (cis-DDP) and DNA were studied in buccal epithelial and urinary cells from ten cancer patients who received cis-DDP-based therapy. Buccal cells were collected 1 h before and 1-2 h after i.v. infusions with cis-DDP. The interaction products were visualized in an immunocytochemical peroxidase assay, using an antiserum against cis-DDP-modified calf thymus DNA. The nuclear staining density was measured by microdensitometry. Nuclear staining densities in buccal cells after infusions of greater than or equal to 20 mg/m2 cis-DDP were always higher than pretreatment values. Repeated sampling from individual patients treated for 2-5 consecutive days with daily doses of 20-70 mg/m2 cis-DDP indicated that cis-DDP-DNA binding in buccal cells increased in proportion to the cumulative total dose of cis-DDP. The variation in dose-density response between patients was 17%. Apparent adduct loss in buccal cells from four patients, as measured 8-17 days after the last infusion, amounted to 67-86%. Platinum-induced DNA modifications could also be detected in buccal cells from two cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)-treated patients. In vitro experiments with human buccal cells and lymphocytes indicated linear relationships between DNA modification and either cis-DDP concentration or incubation time. Nuclear staining densities in pretreatment buccal cells from ten cancer patients treated in vitro with 33 microM cis-DDP for 1 h revealed that interpatient variation in in vitro DNA modification by cis-DDP was low. No quantitative correlation was found between in situ and in vitro DNA modification.

Calf thymus DNA was modified in vitro by cis-diamminedichloroplatinum(II) (cisDDP), complexed with methylated bovine serum albumin and used to immunize rabbits. The anti-cisDDP-DNA antiserum obtained was applied in a double peroxidase-antiperoxidase staining procedure to localize cisDDP-DNA and cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA)-DNA interaction products in cryostat tissue sections of mice and rats. Rats received cisDDP (0-10 mg/kg) and were killed after 24 h. Mice received cisDDP (0-15 mg/kg) or CBDCA (200 mg/kg), and were killed after 2 h-162 days. For each time-dose combination two mice or one rat were used; agents were given i.p. Specific nuclear staining was observed in all tissues examined from cisDDP- or CBDCA-treated animals. No significant nuclear staining could be observed in tissue sections from control rats and mice. The extent of staining after cisDDP was dose and time dependent. The lowest dose of cisDDP after which specific nuclear staining could be detected varied from tissue to tissue [e.g., 0.1 mg/kg, pancreas (mouse); 0.5 mg/kg, liver, kidney (mouse, rat)]. The longest time interval after a single dose of 6 mg/kg cisDDP in which adducts could be visualized also depended on the tissue and varied between 9 days (spleen, testis) and 162 days (kidney). The staining intensity in liver and kidney, measured microdensitometrically, decreased relatively fast in the first days after treatment, but much slower thereafter. In the kidney, cisDDP-induced DNA modification showed regional variation: inner cortex greater than outer cortex greater than medulla (rat) and cortex greater than medulla (mouse). In the mouse kidney, a small subpopulation of tubular cells in close association with the renal corpuscles showed a remarkably high staining intensity after both cisDDP and CBDCA administration. Tissues that showed clear cisDDP-induced histological alterations (kidney, pancreas, testis, and duodenum) also showed moderate to high levels of cisDDP-DNA interaction products. A correlation between cell damage (measured histologically) and cisDDP-DNA binding within one tissue type was demonstrated in the rat inner renal cortex, the murine renal cortex, and in duodenal epithelial cells of both mice and rats.