Cisplatin-DNA adduct formation in rat spermatozoa and its effect on fetal development.

Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin-DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. The formation of spermatozoa cisplatin-DNA adducts showed dose and time-dependent increases both in vitro, and in vivo up to 168 h (7 days) after dosing. Treatment of rats with 10 mg cisplatin/kg resulted in spermatozoa Pt-GG adduct levels of approximately 1.0 fmol/microg DNA. When cisplatin-treated male rats were bred to untreated females 6-24 h after cisplatin administration, no adverse developmental effects or decreases in body weight were seen in the offspring although there was a trend towards increased early embryo mortality.

Pharmacodynamics of cisplatin in human head and neck cancer: correlation between platinum content, DNA adduct levels and drug sensitivity in vitro and in vivo.

Total platinum contents and cisplatin-DNA adduct levels were determined in vivo in xenografted tumour tissues in mice and in vitro in cultured tumour cells of head and neck squamous cell carcinoma (HNSCC), and correlated with sensitivity to cisplatin. In vivo, a panel of five HNSCC tumour lines growing as xenografts in nude mice was used. In vitro, the panel consisted of five HNSCC cell lines, of which four had an in vivo equivalent. Sensitivity to cisplatin varied three- to sevenfold among cell lines and tumours respectively. However, the ranking of the sensitivities of the tumour lines (in vivo), also after reinjection of the cultured tumour cells, did not coincide with that of the corresponding cell lines, which showed that cell culture systems are not representative for the in vivo situation. Both in vitro and in vivo, however, significant correlations were found between total platinum levels, measured by atomic absorption spectrophotometry (AAS), and tumour response to cisplatin therapy at all time points tested. The levels of the two major cisplatin-DNA adduct types were determined by a recently developed and improved 32P post-labelling assay at various time points after cisplatin treatment. Evidence is presented that the platinum-AG adduct, in which platinum is bound to guanine and an adjacent adenine, may be the cytotoxic lesion because a significant correlation was found between the platinum-AG levels and the sensitivities in our panel of HNSCC, in vitro as well as in vivo. This correlation with the platinum-AG levels was established at 1 h (in vitro) and 3 h (in vivo) after the start of the cisplatin treatment, which emphasizes the importance of early sampling.

Effect of cisplatin exposure on platinum accumulation and growth inhibition in human neoplastic and normal squamous epithelial cells of the mucosa of the upper-aerodigestive tract.

The aim of the present study was to investigate how normal head and neck epithelial cells (NHNEC) respond to cisplatin compared to their neoplastic counterparts with respect to intracellular platinum (Pt) levels and growth inhibition. A colorimetric assay was used to assess growth inhibition after exposure to cisplatin for 72 h. Growth inhibition did not differ between cultures of neoplastic (n = 5) and normal cells (n = 5). Intracellular Pt levels, determined with atomic absorption spectroscopy were about 30-fold higher in the normal epithelial cells. The main finding of this study is that normal epithelial cells from the head and neck region have a much higher tolerance for cisplatin than their neoplastic counterparts. Interestingly, this characteristic is without consequence for growth inhibition.

The potential of plantinum-DNA adduct determination in ex vivo treated tumor fragments for the prediction of sensitivity to cisplatin chemotherapy.

BACKGROUND: Response to cisplatin-therapy is assumed to be related to the formation of platinum (Pt)-DNA adducts. Measurement of these adducts prior to therapy could be of value to improve cisplatin based cancer therapy. MATERIALS AND METHODS: We determined Pt-GG and Pt-AG adduct levels by use of 32P-postlabeling after ex vivo cisplatin treatment of fragments of head and neck squamous cell carcinoma (HNSCC) xenografts (five lines), and of tumor biopsies from patients with HNSCC (n = 8) and testicular cancer (n = 8). RESULTS: Adduct levels in fragments (3 x 3 x 3 mm) exposed to 10 to 80 microM cisplatin for one hour, showed positive correlations with the in vivo response to cisplatin treatment (P < 0.05), as well as with the xenograft adduct levels observed after in vivo cisplatin treatment (P < 0.02). After an additional five-hour drug-free incubation period the correlations were absent. When patient tumor fragments were exposed ex vivo to 80 microM cisplatin for one hour, adduct levels were similar in HNSCC and testicular cancer. Persistence of adducts was observed for testicular cancer in the additional drug-free period. The adduct levels in the samples of two HNSCC patients who received cisplatin chemotherapy were in line with the hypothesis that higher adduct levels are associated with a better response. CONCLUSION: Our preliminary results show that analysis of DNA adducts following ex vivo drug treatment is a feasible approach towards a predictive assay, which warrants further investigation.

Pharmacokinetics of cisplatin with and without amifostine in tumour-bearing nude mice.

Amifostine (Ethyol, WR-2721) is in use in the clinic as a protector against platinum-induced toxicities. We have previously reported that amifostine induced a potentiation of the antitumour activity of carboplatin in human ovarian cancer xenografts. An influence of amifostine on the pharmacokinetics of carboplatin, resulting in higher platinum concentrations in plasma and tissues of the tumour-bearing nude mice, was thought to be the cause of enhancement of the antitumour activity. Therefore, the pharmacokinetics of cisplatin were investigated in tumour-bearing nude mice treated with cisplatin alone or in combination with amifostine. A significant increase in the area under the curve (AUC) of the total platinum concentration in mice treated with amifostine was only observed in the kidney (from 355 to 398 nmol h/g), whereas in the other tissues and plasma no significant changes were measured. The selective protection of normal tissues by amifostine was confirmed by a decrease in the AUC of the cisplatin-DNA adduct levels in normal tissues. The decrease was only significant in the liver (282-240 fmol h/microgram DNA), whereas in tumour tissue a slight increase in the AUC of the cisplatin-DNA adducts could be detected (91.3-110.1 fmol h/microgram DNA). The minor influence of amifostine on the pharmacokinetics of cisplatin may be the reason why amifostine did not potentiate the antitumour activity of cisplatin. The influence of amifostine on cisplatin-DNA adduct levels in normal tissues versus tumour tissues is further evidence for the usefulness of this toxicity modulator in cancer patients.

Influence of amifostine on the pharmacokinetics of cisplatin in cancer patients.

The pharmacokinetics of cisplatin was investigated in 13 patients receiving 18 courses of cisplatin alone or in combination with amifostine to investigate the influence of amifostine (WR 2721; Ethyol) on the pharmacokinetics of cisplatin. Cisplatin was administered as a 1-h i.v. infusion, whereas amifostine was given i.v. over 15 min just before the cisplatin infusion. An increase in the final half-life of ultrafilterable platinum was observed after treatment with cisplatin and amifostine (t1/2, 0.77 +/- 0.10 h; n = 8), compared to cisplatin alone (t1/2, 0.57 +/- 0.15 h; n = 8). This might be caused by an influence of amifostine on the kidney function, because an increase in the serum creatinine levels was also observed 24 h after treatment with cisplatin and amifostine (13.8 +/- 12.6%; n = 9), which was not observed after treatment with cisplatin alone (-0.1 +/- 6.8%; n = 9). Surprisingly, the final half-life of unchanged cisplatin did not increase, but even showed a slight decrease after treatment with amifostine. In vitro data would suggest that this might be due to a chemical interaction between cisplatin and amifostine. Because the AUC values of ultrafilterable platinum and unchanged cisplatin did not change significantly and no change in Pt-DNA adduct (Pt-GG) levels in leukocytes was observed upon addition of amifostine in the treatment schedule, the change in the pharmacokinetics of cisplatin is most probably of minor importance and has no significant impact on the efficacy of cisplatin, as already confirmed by clinical studies.

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.

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines.

Resistance to chemotherapy is a major problem in the treatment of patients with head and neck squamous cell carcinoma (HNSCC). Important factors involved are drug detoxification by glutathione (GSH) and reduced drug accumulation due to active transport out of the cell by so-called 'multidrug resistance-related proteins'. We have studied a panel of eight HNSCC cell lines showing differences in sensitivity to the anti-cancer drug cisplatin. Our previous studies indicated that the IC50 values were inversely correlated with the intracellular accumulation of platinum (Pt). In the present study, cellular GSH levels were found not to be related to the IC50 values. The expression levels of the enzymes glutathione S-transferase (GST) alpha, mu, and pi, the multidrug resistance-related proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP) were determined semiquantitatively by means of immunocytochemistry. The levels of the GSTs, P-gp and LRP were not found to be correlated with the IC50 values of the HNSCC cell lines. Surprisingly, however, an inverse correlation was found between MRP levels and IC50 values. The MRP expression levels were in agreement with the results of the MRP functional assay, based on the transport of calcein across the cell membrane as performed for two of the cell lines. Further studies should prove whether other pump mechanisms or DNA repair are involved in the cisplatin accumulation and the subsequent HNSCC cell growth inhibition.

Cisplatin-DNA adducts and protein-bound platinum in blood of testicular cancer patients.

DNA adducts formed by cisplatin [cis-diamminedichloroplatinum(II)] were measured in blood samples from 48 testicular cancer patients treated in four centers in Europe during four to six cycles with cisplatin infusions on five successive days (total samples, 112). Total protein-bound platinum (Pt) in blood was also measured (total samples, 84). The mean on the main DNA adduct, cis-Pt(NH3)2d(pGpG) (Pt-GG), was 0.75 fmol/microg DNA [standard deviation (SD) = 0.66] on the first day of the first cycle, increased after the infusion at day 5 of the cycle (mean 1.74 fmol/microg DNA, SD = 0.90) and decreased on the following day (mean 1.09 fmol/microg DNA, SD = 0.62). In subsequent cycles, there was a tendency to an increase in the mean Pt-GG levels. The values of protein-bound Pt in blood showed little reduction between day 5 and 6 of each cycle, and a stable increase during the course of the therapy. Strong correlations were seen between day 1, 5 and 6 of the first cycle for both Pt-GG and protein-bound Pt in blood. A strong correlation (r = 0.62, p < 0.001, 69 pairs) was found between the levels of Pt-GG and protein-bound Pt. Only two patients relapsed during the follow-up; therefore, the analysis of the association between Pt-GG levels and response to therapy was not informative. The results of this study suggest that DNA adducts formed by cisplatin at the beginning of chemotherapy are predictive of values found during later days and cycles, and that the value of protein-bound Pt in blood is predictive of the value of DNA adducts.

Improved 32P-postlabelling assay for the quantification of the major platinum-DNA adducts.

For the improvement of chemotherapy with platinum (Pt)-containing drugs a sensitive assay to detect the induced Pt-DNA adducts is needed. Therefore, the 32P-postlabelling assay, described by Blommaert and Saris (Nucleic Acids Res., 1995, 23, 1300-1306), to detect the major adducts Pt-GG and Pt-AG has substantially been improved and compared with ELISA and AAS. For the quantification of the adducts, TpT was added as an internal standard immediately after isolation of the Pt-adducts from digested DNA samples. It was found that 32P-labelling of both GpG and ApG, the dinucleotides obtained after deplatination of the adducts, was equally efficient as that of TpT. To isolate the Pt-adducts on basis of a positive charge, the pH of DNA digests was adjusted to approximately 3 prior to separation by strong cation-exchange chromatography. For the subsequent deplatination a volume of only 12 microl of 0.2 M NaCN was used, which did not interfere with the following labelling step. The quantification of the 32P-labelled dinucleotides was performed by phosphorimaging of spots after separation on TLC as well as by 32P-counting of fractions collected after separation by HPLC. The method was used to determine adduct levels in in vitro cisplatin-treated DNA and in DNA isolated from cisplatin-treated cultured cells, tumor xenografts from cisplatin-treated mice, and from white blood cells and (tumor) tissues from cisplatin-treated patients. The results show a significant correlation with the adduct levels as determined with atomic absorption spectroscopy (high levels) or with specific antibodies (low levels). This assay appears to be useful for the determination of low levels of Pt-adducts in small DNA samples as present in clinical specimens such as blood and tumor tissue, but also in buccal mucosal cells and fine needle aspirates.

Pharmacokinetics of carboplatin with and without amifostine in patients with solid tumors.

We showed previously that amifostine (WR 2721; Ethyol), a protector against carboplatin-induced toxicities, changed the pharmacokinetics of carboplatin in tumor-bearing nude mice. In the present study, the influence of amifostine on the pharmacokinetics of carboplatin was studied in patients when carboplatin was given in combination with three doses of amifostine, administered just before the carboplatin infusion and 2-4 h thereafter. Compared with a control group of patients who received carboplatin alone, the patients receiving the combination had a longer final half-life of ultrafilterable platinum species [5.0 h versus 3.5 h in patients with a normal creatinine clearance (Clcr > 80 ml/min); 5.6 h versus 4.2 h in those with an impaired renal function (50 < Clcr < 80 ml/min)]. This might be caused by an influence of amifostine on the renal clearance of carboplatin as suggested by a transient increase in serum creatinine levels 24 h after treatment in the patients receiving the combination (mean +/- SD: 34.1% +/- 17.2% versus -1.8% +/- 16.5% in patients treated with carboplatin alone). The impact of these changes on the area under the concentration-time curves of the ultrafilterable platinum species was hardly noticeable in patients with a normal renal function but led to a significant increase in patients with an impaired renal function (395 +/- 59 micromol/l.h versus 280 +/- 62 micromol/l.h in patients receiving carboplatin alone). The clinical relevance of this influence is unclear, although theoretically it may result in an increase in the efficacy of carboplatin, as has been observed in tumor-bearing nude mice.

Relationship between the parameters cellular differentiation, doubling time and platinum accumulation and cisplatin sensitivity in a panel of head and neck cancer cell lines.

Patients with head and neck squamous cell carcinoma (HNSCC) treated with cisplatin show a large inter-individual variation in tumor response. Little is known about factors that contribute to this variation. The aim of our study was to correlate the sensitivity to cisplatin with a number of cellular parameters using a panel of 10 human HNSCC cell lines. A 7-fold variation in response after 72 hr of exposure to cisplatin as determined in a colorimetric proliferation assay was observed. The IC50 values did not correlate with the DNA index, the cellular doubling time or the expression of differentiation markers. Intracellular platinum (Pt) concentrations were measured by atomic absorption spectroscopy after exposing the cells to 10 microM cisplatin for 1-72 hr. The intracellular Pt levels increased up to 24 hr. One cell line, derived from the tumor of a patient previously treated with radiotherapy, accumulated much more Pt than the other cell lines. For these other cell lines, a significant positive correlation was found between Pt accumulation and sensitivity. In conclusion, cisplatin-induced growth inhibition in HNSCC in vitro is generally positively correlated with cellular Pt levels. However, the fact that occasionally cancer cells can survive despite high intracellular Pt levels indicates that additional parameters are needed to explain a response unequivocally.

Influence of single and multiple doses of amifostine on the efficacy and the pharmacokinetics of carboplatin in mice.

We have previously reported that amifostine potentiates the anti-tumour activity of carboplatin in mice. The present study was carried out in well-established human ovarian cancer xenografts OVCAR-3, A2780 and FMa grown subcutaneously in the nude mouse. It was found that a single dose of amifostine resulted in a higher increase in the anti-tumour activity of carboplatin than three doses of amifostine. A single dose of amifostine increased the AUC (area under the curve) values of total platinum in plasma ultrafiltrate (30.1 vs 18.2 microM x h), liver (307.7 vs 236.4 nmol g(-1) x h), kidney (500.8 vs 368.3 nmol g(-1) x h) and OVCAR-3 tumour tissue (184.0 vs 146.8 nmol g(-1) x h). Despite this increase in total platinum, a decrease in platinum (Pt)-DNA adduct levels was observed in liver, kidney and bone marrow, which was significant in liver. In tumour tissue an insignificant increase in Pt-DNA adduct levels, specifically the Pt-GG adduct, was observed after treatment with a single dose of amifostine, which may explain the increase in anti-tumour activity. The increase in the AUC of total platinum was probably caused by a reduction in body temperature, which was most severe after three doses of amifostine. The extreme hypothermia may be the reason that three doses of amifostine resulted in less potentiation of the efficacy of carboplatin.

Response of sensitive and resistant IgM immunocytomas to cis-diamminedichloroplatinum (II) does not correlate with the platination level or with the formation or removal of DNA adducts.

An IgM immunocytoma cell line sensitive to cis-diamminedichloroplatinum(II) (CDDP) and a subline with acquired resistance were grown in LOU/M rats. In a previous study with such rats that had been treated with a high dose of CDDP (10 mg/kg) the tumors did not show differences in cellular platinum content or DNA-adduct levels, either immediately after treatment or 24 h later. Recently, this high dose was found to overcome resistance. Therefore, the study was repeated with a 10-fold lower dose (1 mg/kg, i.v.). At 1 and 24 h after treatment, tumor and kidney tissue were assayed for cellular platinum (atomic absorption spectroscopy, AAS) and DNA platination (immunochemical detection of the four CDDP-DNA adducts). The results were compared with previous data. All tissues showed a linear response to dose with regard to platinum uptake as well as adduct formation, with no quantitative difference being seen between the tumors. Also the relative occurrence of the four adducts was very similar. Between 1 and 24 h, in tumors a substantial decrease occurred in both platinum content and adduct level; the kidneys showed little reduction, if any. At the lower CDDP dose a somewhat larger loss of platinum and removal of DNA adducts was observed for the resistant tumor, but these differences could be explained by "dilution", as this tumor continues to grow after low-dose treatment (about 20% within 24 h). Since the strong difference observed between the tumors in sensitivity to CDDP cannot be attributed to differences in CDDP uptake, efficiency of adduct formation, or repair capability, other mechanisms are held responsible.

The interaction of the anti-cancer drug cisplatin with phospholipids is specific for negatively charged phospholipids and takes place at low chloride ion concentration.

The interaction of the anti-cancer drug cis-diamminedichloroplatinum(II) (cisPt) with model membranes was studied, with emphasis on the cisPt and phospholipid species involved. Binding studies using large unilamellar vesicles have revealed that: (i) Interaction involved negatively charged phospholipids only, and (ii) Interaction with negatively charged phospholipids was observed only in buffers with low Cl- concentration, indicating that aquated, positively charged cisPt is involved. Binding to all negatively charged phospholipids tested was highest at pH 6.0. At pH 7.4 a high and specific binding was observed with phosphatidic acid and phosphatidylserine. The consequences of cisPt binding on the organization of lipids was investigated with differential scanning calorimetry studies. These studies have indicated a higher ordering of dispersions of negatively charged phospholipids in the presence of divalent cationic cisPt. Summarizing, the interaction of positively charged cisPt species with negatively charged phospholipids is significant and should be considered in in vivo experiments.

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.

Cisplatin- and carboplatin-DNA adducts: is PT-AG the cytotoxic lesion?

In order to determine the nature of the cytotoxic lesion(s) formed by the antitumour drugs cisplatin and carboplatin, a comparative study was made of bifunctional DNA-adduct formation by these drugs. The kinetics of bifunctional cisplatin adduct formation were studied with DNA in vitro and in cultured Chinese hamster ovary (CHO) cells. Prior to adduct measurements with AAS in in vitro platinated DNA and with ELISA in cellular DNA, the monoadducts were inactivated with thiourea (10 mM; 1 h at 37 degrees C). The data indicated that the conversion of monofunctional to bifunctional adducts, with t1/2 of approximately 2 h (37 degrees C), leads to maximum intrastrand adduct levels after approximately 4-6 h postincubation. This interval coincided with the period during which the cytotoxic effect of cisplatin could be reduced by a 1 h 10 mM thiourea post-incubation of the cells. The formation of interstrand crosslinks continued for approximately 7 h of post-incubation; then these amounted to approximately 2% of the total DNA adducts. When a DNA sample was dialysed against 0.1 M NH4HCO3 (16 h, 37 degrees C) immediately after cisplatin treatment, in order to block mono- to bifunctional adduct conversion, adduct levels were found similar to those after the 4-6 h post-incubation. From this it is clear that the high values reported earlier for bifunctional cisplatin adducts in such DNA samples are not correct. These values apparently represent the amounts of adducts that eventually would have been formed during post-incubation in DNA in vitro but also in cells in the absence of cellular repair. The cisplatin data of CHO cells were compared with those after treatment of the cells with equitoxic doses of carboplatin. The data indicate that after 12 h post-incubation, when all bifunctional adducts are formed, the total amount of the various bifunctional adducts after cisplatin treatment (37.5 +/- 4.5 fmol/micrograms DNA) was in the same range as that after carboplatin (32.8 +/- 6.3 fmol/micrograms DNA). However, because the relative occurrences of the adducts were different, it could also be concluded that if one of the diadducts were exclusively responsible for the cytotoxic effect of these platinum antitumour drugs, Pt-AG is the only likely candidate.

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)

Effects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatin-DNA adducts: consequences for the accurate quantification of adducts in (cellular) DNA.

Cisplatin reacts with DNA by forming mainly bifunctional adducts via reactive monofunctional intermediates. When freshly platinated DNA was postincubated with thiourea (10 mM, at 23 or 37 degrees C) for periods of up to 24 h, followed by determination of mono- and diadducts, a rapid initial decrease was seen in the fraction of diadducts, followed by a much slower decrease. About 40% diadducts were found after 10-min postincubation at 23 degrees C, which dropped to some 14% after 24 h at 37 degrees C; total platination was hardly affected. Postincubation of "aged" platinated DNA (no reactive monoadducts) only showed the slower decrease. The rapid process is likely to represent monoadduct inactivation, preventing the formation of diadducts, whereas the slow reaction must be interpreted as diadduct-to-monoadduct conversion. Similar reactions, but less efficient than with thiourea, occurred during dialysis against NH4HCO3 (0.1-1 M). Pt-containing (di)nucleotides in digested DNA were hardly affected by thiourea. Rapid reduction of the measured level of bifunctional adducts also occurred when cisplatin-treated Chinese hamster ovary cells were postincubated with thiourea, with concomitant increase in survival. It is concluded that quantification of the real levels of mono- and diadducts in freshly platinated DNA requires a posttreatment with thiourea of 30-60 min at 37 degrees C.

Differential formation and enhanced removal of specific cisplatin-DNA adducts in two cisplatin-selected resistant human testicular teratoma sublines.

Mechanisms of cisplatin resistance have been studied in two independently-selected sublines expressing clinically-relevant levels of resistance (3-fold) and established from a primary testicular teratoma obtained from previously untreated patients. Resistance was not associated with any significant modification in cellular uptake of cisplatin, in total glutathione levels or associated enzyme activities. However, immunochemical quantitation of specific platinum-DNA adduct formation and removal revealed that both resistant sublines were more proficient in repairing certain adducts than their generally repair deficient respective parental lines. SUSA/CP+ cells were more efficient in removing the intrastrand adducts in the sequence Pt-AG and the bi-functional Pt-(GMP)2 lesions, as well as DNA-DNA interstrand cross-links, whilst H12.1/DDP cells were highly proficient in removing the major Pt-GG intrastrand adducts.