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.

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)

A 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA.

Within the group of DNA alkylation products, phosphotriesters (PTE) are among the most stable lesions. Hence, alkyl PTE are attractive biomarkers for DNA alkylation monitoring purposes. We have developed a 32P-postlabelling method for the analysis of both methyl and ethyl PTE in DNA. Since PTE bonds are not cleaved by any known DNA degrading enzyme, they are easily obtainable as PTE dinucleoside monophospates. A purification step, separating the PTE dinucleoside monophosphates from interfering compounds, such as mono- or oligonucleotides resulting from incomplete digestion of DNA, was developed using Waters C18 Sep-Pak cartridges. Phosphotriester dinucleoside monophosphates themselves are not a substrate for phosphorylation by polynucleotide kinase. Polynucleotide kinase probably requires a negative charge on the phosphate closest to the 5'-end. Therefore, prior to the post-labelling step they have to be converted into either phosphodiester dinucleoside monophosphates or 3'-phosphate alkylated mononucleotides by treatment with alkali. For analysis of the labelled compounds we developed a two-step procedure, combining TLC and HPLC, that gave very straightforward information on the composition of the rather complex mixture. The detection limit is approximately fmol PTE.

Polycyclic aromatic hydrocarbon-DNA adducts in white blood cell DNA and 1-hydroxypyrene in the urine from aluminum workers: relation with job category and synergistic effect of smoking.

We examined a group of 105 workers from a primary aluminum plant for the presence of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in their WBC and 1-hydroxypyrene in their urine. Workers were recruited from five job categories with different PAH exposure: the anode factory; the bake oven; and the electrolysis and the pot-relining departments. Unexposed workers from the foundry department served as the control group. The exposure to PAH was measured by personal monitoring, and the average PAH concentrations in the work atmosphere ranged from 0.4 micrograms/m3 in the foundry to 150 micrograms/m3 in the pot-relining department. The average exposure to benzo(a)pyrene was under the Swedish exposure limit of 5 micrograms/m3. The internal dose of pyrene was measured utilizing the 1-hydroxypyrene concentration in pre- and postshift urine samples. Higher exposure to PAH in the work atmosphere was associated with increased concentrations of 1-hydroxypyrene in the urine. The average increase in concentration of 1-hydroxypyrene ranged from 0.2 mumol/mol creatinine in the control group to 5.9 mumol/mol creatinine in the pot-relining department; an accumulation of 1-hydroxypyrene over a 5-day working period was observed. A good correlation was found between PAH exposure and the concentration of 1-hydroxypyrene in the urine on a group level (rs = 0.90; P = 0.02). PAH-DNA adducts were determined by 32P-postlabeling analysis (nuclease P1 enrichment procedure).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The present paper reports about an immunocytochemical inventory of the cell types involved in the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to a DNA methylating metabolite. The formation and distribution of the methylated DNA bases O6-methylguanine (O6-meGua) and 7-methylguanine (7-MeGua) were studied in respiratory tissues, oesophagus, liver, kidneys, pancreas, small intestine, colon and prostate of rat, mouse and hamster 6 h after treatment with a single dose of 30 mg NNK/kg. The tissue- and cell-specific distribution of O6-meGua- and 7-meGua-specific nuclear staining showed the same patterns and were remarkably similar in rat, mouse and hamster in spite of the diverging spectra of NNK-induced tumours in these species. In nasal tissue, a target for NNK-induced tumourigenesis in rat and hamster, but not in mouse, adduct-specific nuclear staining was observed in all three species in sustentacular cells, Bowman glands, respiratory epithelial cells and serous glands. Both methylated DNA bases were also observed in basal cells of the olfactory epithelium of rat and (occasionally) hamster, but not in those of the mouse. In the trachea, a target for NNK-induced tumourigenesis in hamster only, substantial adduct-specific nuclear staining was found in basal epithelial and glandular cells of the hamster; in the same cells of rat and mouse only a weak nuclear staining was found. In the lung, a common target for NNK-induced tumourigenesis, the formation of O6-meGua and 7-meGua was restricted predominantly to bronchial and proximal bronchiolar epithelium. Nuclear staining in the rat was occasionally found in alveolar cells and was also observed in hepatocytes. In the three species investigated, O6-meGua- and 7-MeGua-specific nuclear staining was found in target and non-target tissues. Apparently, and in analogy with results obtained in other studies, the species-specific organotropy for tumour formation of NNK is not exclusively determined by DNA methylation. Expanding methylation data with literature data on factors considered to be involved in tumour formation, namely proliferation, toxicity and DNA repair among others, still did not lead to a satisfactory explanation for the species-specific organotropy observed. Additional factors (yet to be identified), need to be taken into account in order to explain (and predict) tumourigenic effects induced by monofunctional methylating agents.

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 temperature on the interaction of cisplatin and carboplatin with cellular DNA.

Increased levels of cisplatin (cDDP)- and carboplatin (CBDCA)-DNA adducts were detected in cDDP (10 microM)- and CBDCA (6 mM)-treated CC531 cells when the temperature was raised from 37 degrees to 43 degrees. In the case of cDDP, increased DNA adduct formation was already detectable at 38.5 degrees; additional temperature steps led to further increases in DNA modification. Increased CBDCA-DNA adduct formation was observed only at temperatures higher than 40 degrees. In vitro studies on the interaction of CDDP and CBDCA with isolated salmon sperm DNA, however, demonstrated no significant differences in the DNA binding rate between 37 degrees and 43 degrees for cDDP and a minor effect for CBDCA only at 43 degrees, almost totally excluding a direct temperature effect on DNA platination in this temperature range. Furthermore, neither the stability of the formed platinum-DNA adducts nor the rate of adduct loss in CC531 cells was changed at higher temperatures. The observed difference in cellular adduct formation, however, could be related to increased uptake of cDDP and CBDCA into CC531 cells at higher temperatures. In the case of cDDP, a temperature shift from 37 degrees to 38.5 degrees resulted in a significantly higher intracellular platinum concentration (0.03 +/- 0.01 vs 0.071 +/- 0.021 micrograms platinum/10(6) cells, respectively); for CBDCA, temperatures > or = 41.5 degrees were needed to increase the platinum concentration significantly above 37 degree values (0.3 +/- 0.1 vs 0.6 +/- 0.1 micrograms platinum/10(6) cells, respectively). In addition, the increase in DNA adduct formation of cDDP and CBDCA at elevated temperatures was comparable with the increase in cDDP-DNA adducts after a cDDP concentration escalation at 37 degrees, indicating a concentration-dependent increase in cDDP-DNA adducts. It seems that heat affects primarily the cellular uptake of cDDP and CBDCA and not their covalent binding to DNA.

DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons.

Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell(WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk than PAH in the work atmosphere, or the amount of a metabolite in the urine, because adduct levels reflect that part of the dose that escapes detoxification and binds to DNA. We analyzed WBC DNA from coke-oven workers and from workers in an aluminum production plant and demonstrated the presence of PAH-DNA adducts. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their WBC compared with 27% of the controls (p < 0.05), measured with ELISA. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. In the aluminum workers, no PAH-DNA adducts were detected by ELISA, although the benzo[a]pyrene concentrations in the work atmosphere were comparable to those of the coke-oven workers. The more sensitive 32P-postlabeling assay showed the presence of PAH-DNA adducts in 91% of the aluminum workers. There was no correlation of WBC adduct levels with the concentration of PAH in the work atmosphere. Recently we showed that total PAH-DNA adduct levels in WBC from lung cancer patients were much higher than those generally found in healthy smokers.(ABSTRACT TRUNCATED AT 250 WORDS)

32P-postlabelling analysis of alkylphosphotriesters in DNA.

We have developed a highly sensitive 32P-postlabelling assay for the detection of alkylphosphotriesters in DNA using selective enzymatic DNA hydrolysis, sample preparation by C18 Sep-Pak solid phase extraction and product analysis by HPLC. The assay provides information on the quantity of the alkylphosphotriesters and gives some clues with respect to their identity.

Accumulation of O6- and 7-methylguanine in DNA of N-nitroso-N-methylbenzylamine-treated rats is restricted to non-target organs for N-nitroso-N-methylbenzylamine-induced carcinogenesis.

The distribution and accumulation of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) was investigated immunocytochemically in target and non-target tissues of rats injected twice weekly with 0.5 mg N-nitroso-N-methylbenzylamine (NMBzA)/kg for 16.5 weeks. Seventy two hours after every two or three doses, two NMBzA-treated rats and one control rat were killed. Tissue-specific cell proliferation was investigated after two injections of bromodeoxyuridine (BrdU) to rats unexposed to NMBzA. Neither O6-meGua- nor 7-meGua-specific immunostaining could be observed in the target tissues for tumor induction, i.e. esophagus, tongue and forestomach. Accumulation of both O6-meGua and 7-meGua was found, however, in nasal, tracheal and bronchiolar epithelia and glands--tissues for which tumor induction by NMBzA has not been reported. An explanation for this phenomenon might be the relatively low levels of cellular proliferation we observed in the latter epithelia. The present results support the hypothesis that the tumorigenic organotropism of NMBzA is determined both by the level of DNA methylation and the proliferative capacity of the methylated cells.

Polycyclic aromatic hydrocarbon--DNA adducts in white blood cells from lung cancer patients: no correlation with adduct levels in lung.

Smokers of cigarettes are exposed to a number of carcinogens, including polycyclic aromatic hydrocarbons (PAHs), and are at a high risk for lung cancer. PAHs exert their carcinogenic activity after metabolic activation to reactive intermediates that can damage DNA through adduct formation. Measuring DNA adducts in peripheral white blood cells (WBC) could serve as a means of monitoring human exposure to genotoxic agents and subsequently risk assessment. In this study, DNA from WBC obtained from 39 lung cancer patients was examined for PAH-DNA adducts both in an ELISA using a polyclonal antibody against benzo[a]pyrene 7,8-diol-9,10-epoxide (BPDE)-DNA and the 32P-post-labeling technique. The ELISA results showed BPDE-DNA antigenicity in WBC DNA from 12/38 (32%) patients and adduct levels ranged from 1.5 to greater than 150 adducts in 10(8) nucleotides. The autoradiographs of chromatograms of 32P-post-labeled digests of WBC DNA from the 38 patients showed a variety of adduct spots; relative adduct labeling (RAL) values ranged from 0.3 to 407 adducts in 10(8) nucleotides. In 18 of the 38 (47%) persons an adduct spot was detected that co-chromatographed with the major BPDE-DNA adduct (BPDE-dG); RAL values ranged from 0.03 to 382 adducts in 10(8) nucleotides. Correlations were not significant between the adduct levels in WBC and smoking habits, age or sex. From 20 patients of the same group lung tissue was collected at surgery and examined for PAH-DNA adducts by ELISA and 32P-post-labeling assay. No significant correlation was found between DNA adduct levels in blood and lung. This finding stresses the limitations of the use of WBC as a surrogate for adduct levels in the target organ.

A rationale for carboplatin treatment and abdominal hyperthermia in cancers restricted to the peritoneal cavity.

The purpose of this study was to optimize the treatment of cancers restricted to the peritoneal cavity by combining i.p. chemotherapy with abdominal hyperthermia. In vitro experiments demonstrated that the uptake of carboplatin into CC531 tumor cells was increased at temperatures higher than 41.5 degrees C at dose levels of 5 and 50% cell kill. Carboplatin-DNA adduct formation and cytotoxicity, however, were already increased at temperatures of about 40 degrees C, indicating that carboplatin-DNA adduct formation and consequently cytotoxicity could be enhanced by mild hyperthermia (temperatures in the range of 39-41.5 degrees C). CC531 tumor bearing rats were treated i.v. and i.p. with carboplatin (6.15 mg/kg) in combination with regional hyperthermia of the abdomen (41.5 degrees C for 1 h). The mean temperature was 41.5 +/- 0.3 degrees C (SD) in the peritoneal cavity and 40.5 +/- 0.3 degrees C in the esophagus. Enhanced platinum concentrations were found in peritoneal tumors (factor 3) and in kidney, liver, spleen, and lung (a factor 2 average), after the combined i.v. or i.p. carboplatin-hyperthermia treatment. Pharmacokinetic data of i.p. CBDCA combined with hyperthermia demonstrated an increased tumor exposure for total and ultrafiltered platinum in plasma. The areas under the concentration x time curve for total platinum at 37 degrees C and 41.5 degrees C were 69 and 210 microM/h, respectively; for ultrafiltered platinum these values were 47 and 173 microM/h. This may have been due to a slower elimination of platinum from the blood at the higher temperature (t1/2 beta for total platinum 99 and 156 min at 37 and 41.5 degrees C, respectively). The direct exposure of the tumor via the peritoneal fluid appeared to diminish, since the area under the curve for total platinum was lower at 41.5 degrees C than at 37 degrees C (576 microM/h versus 1255 microM/h, respectively). Our results indicate that the advantage of adding hyperthermia is caused by an increased drug exposure of the tumor via the circulation. This was supported by the fact that platinum concentrations in peritoneal tumors after carboplatin treatment at elevated temperatures were similar for the i.p. and i.v. routes.

Immunocytochemical localization of DNA adducts induced by a single dose of N-nitroso-N-methylbenzylamine in target and non-target tissues of tumor formation in the rat.

The formation and short-term persistence of O6-methylguanine (O6-meGua) and 7-methylguanine (7-meGua) in individual cells of various target and non-target tissues for tumor induction in rats were examined after a single dose of N-nitroso-N-methylbenzylamine (NMBzA). In the principal target organ, the esophagus, both adducts were observed at 6 h after 0.5, 1.0 and 2.5 mg NMBzA/kg in a dose-dependent manner in nuclei of epithelial cells only. Nuclear staining in this organ had apparently declined by 72 h and modified nuclei were found in the more differentiated cells located closer to the lumen. In epithelial cells of the tongue, another target organ of NMBzA, methylation at 6 h was also dose dependent. At 72 h nuclear staining was lower and again largely located in differentiated cells. In the liver, a non-target organ, O6-meGua was not detectable and 7-meGua-specific staining was weak, being only observed at 6 h after the highest dose. Dose-dependent DNA methylation was seen, both at 6 and 72 h, in other non-target organs such as lung (bronchiolar epithelial cells), trachea (epithelial and glandular cells) and nasal cavity (respiratory epithelial cells, ductal cells of the respiratory lamina propria and cells of Bowman glands of the olfactory lamina propria); the nuclei of the glandular cells were highly methylated. Visual inspection of lung, trachea and nasal cavity indicated no or only minor losses of O6-meGua and 7-meGua between 6 and 72 h. Microdensitometric determination of the nuclear staining at 6 and 72 h indicated that the promutagenic O6-meGua was partially lost from cells of the tongue epithelium but did persist in esophageal epithelial cells; 7-meGua was lost to a substantial extent from both tongue and esophagus. The present results imply that the organotropism of NMBzA is not uniquely determined either by the initial level or the short-term persistence of DNA methylation.

Immunocytochemical visualization of DNA adducts in mouse tissues and human white blood cells following treatment with benzo[a]pyrene or its diol epoxide. A quantitative approach.

The formation and stability of benzo[a]pyrene DNA adducts were studied in tissues of BALB/c mice exposed to benzo[a]pyrene (B[a]P). The DNA adducts were visualized with an immunocytochemical peroxidase staining technique using an antiserum specific for the major B[a]P-derived adduct in DNA [(+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-N2-dG)]. The nuclear staining density was measured by microdensitometry. When mice were treated with an increasing dose of B[a]P the nuclear staining increased in the tissues studied (lung, heart and kidney). A linear relationship was found between the immunocytochemical nuclear staining signal and the actual DNA adduct level in the lung as measured by 32P-postlabeling. Maximum adduct formation was found 5 days after a single i.p. injection of B[a]P. Adduct levels decreased gradually after 7 days, but even after 61 days a slight specific staining was still present, suggesting that not all adducts had disappeared at that time. As judged from the disappearance of [3H]thymidine from prelabeled DNA the loss of adducts from the lung was not a result of DNA repair but one of cell turnover. In human white blood cells B[a]P-derived adducts could be detected after in vitro incubation with the reactive metabolite of B[a]P (BPDE). Dose-response studies demonstrated a positive relationship between BPDE-DNA adduct formation, the immunocytochemical staining signal and the BPDE concentration in the culture medium.

Formation of interaction products of carboplatin with DNA in vitro and in cancer patients.

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.

Dose-escalation study of carboplatin (day 1) and cisplatin (day 3): tolerance and relation to leukocyte and buccal cell platinum--DNA adducts.

Carboplatin and cisplatin have similar antitumor activities but different toxicities. Combining these two analogs may be expected to balance the toxicities and allow higher doses of platinum compounds to be administered with tolerable toxicity. To test this concept, a Phase I trial of carboplatin in combination with cisplatin was performed. Thirty-three eligible patients received carboplatin doses ranging from 200-480 mg/m2 on day 1 and cisplatin doses ranging from 50-100 mg/m2 on day 3 of the 28 day cycle. A 2-day interval ensured no interference in renal excretion of carboplatin by cisplatin. Myelosuppression was the dose limiting toxicity. With the usual full dose of carboplatin, 480 mg/m2, patients tolerated 50 mg/m2 of cisplatin, without apparent additional toxicity. At 100 mg/m2 of cisplatin, non-hematologic as well as hematologic toxicities frequently precluded administration of more than 300 mg/m2 of carboplatin. Platinum-DNA adduct quantitation was done in leukocytes and buccal cells during cycle 1 in most patients. The adduct-specific immunosignal in buccal cells was always increased after carboplatin and in all but one after cisplatin. The level of adducts in buccal cells increased with increasing doses of carboplatin and cisplatin. In leukocytes, measurable levels of adducts were formed after carboplatin with further contribution made by cisplatin but not obviously in a dose dependent fashion. We conclude from the toxicities observed, that combinations of carboplatin with cisplatin may have advantages over either drug alone in certain clinical situations; and that further study of platinum-DNA adducts may shed light on platinum dose-response relationships.

Antibodies against cisplatin-modified DNA and cisplatin-modified (di)nucleotides.

Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding of cis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore raised and characterized four rabbit antisera: one against cis-DDP-modified DNA (antiserum NKI-A59) and three others against the cis-DDP-modified (di)nucleotides cis-Pt(NH3)2d(pApG) (NKI-A68), cis-Pt(NH3)2d(GMP)2 (NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with either cis-DDP or CBDCA [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity to cis-DDP-modified poly[d(G-C)].poly[d(G-C)], poly(dC), and poly(dG). No affinity was found for trans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digested cis-DDP-DNA, or cis-DDP-DNA, or cis-DDP-modified poly(dA).poly(dT), oligo(dA)15.oligo(dT)15, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding to cis-DDP-DNA decreased with decreasing DNA modification levels. Although other cis-DDP-DNA- and cis-DDP-(di)nucleotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-hapten cis-DDP-DNA adducts. Their affinity for digested cis-DDP-modified DNA was up to 30 times that for intact cis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining of cis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.

Localization of DNA adducts formed in the nasal cavity of the rat by the tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK).

The tissue localization of the DNA adducts O6- and 7-methylguanine induced in the nasal cavity by the nicotine-derived carcinogen 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK, 30 mg/kg intraperitoneally) has been investigated immunocytochemically in male Sprague-Dawley rats. Adduct-specific nuclear staining, indicative of the metabolic activation of NNK to a methylating compound, was observed in both respiratory and olfactory mucosa. In the respiratory epithelium, strong staining was generally observed in areas devoid of goblet cells. Less intense staining was observed both in the serous gland cells and their efferent ducts in the respiratory submucosa, whereas the mucous gland cells were unstained. In the olfactory mucosa, the sustentacular and basal cells of the olfactory epithelium were moderately stained; staining varied substantially from site to site. No DNA adduct was detected in the olfactory cells. Strong nuclear staining, similar to that in the respiratory mucosa, was observed in the cells of the Bowman glands of the olfactory submucosa. A similar distribution of methylated DNA bases in nasal tissues has been observed in rats after exposure to other N-nitrosamines and in Syrian hamsters after exposure to NNK. This finding may indicate that in man the same cell types undergo DNA adduct formation after exposure to NNK and other N-nitrosamines.