Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables.

Methotrexate (MTX) steady state concentrations were evaluated in 42 children who had received high-dose infusions (6-8 g/m2) for acute lymphocytic leukemia. Concentrations in serum and cerebrospinal fluid (CSF) measured by immunoassay were found to be highly variable. Reanalysis by a reference high-pressure liquid chromatography method ruled out analytical factors as a source of this variability. The correlation coefficient between the analytical methods was 0.77 for the serum data and 0.88 for the CSF data. The variability of serum and CSF concentrations was higher in younger patients (serum; P = 0.05 and CSF; P = 0.18), and the CSF concentration decreased with decreasing age and in later courses. Body surface area, body mass index, weight, and gender were not significantly related to MTX variability. We conclude that the pronounced pharmacokinetic variability seen during MTX infusions remains largely unexplained.

Pharmacokinetics and metabolism of doxorubicin after short-term infusions in lymphoma patients.

PURPOSE/METHODS: Twenty-four patients (17 males and 7 females with a mean age of 54 years) with malignant lymphoma participated in a study of doxorubicin pharmacokinetics after 50 mg/m(2) as 10-min infusions. In addition to plasma samples, serial leukocyte samples and - in one subject - serial biopsy specimens from lymphoma infiltrates were obtained. The samples were analysed by reversed-phase high-performance liquid chromatography. RESULTS: In contrast to several previous studies, the data suggested that 7-deoxydoxorubicinolone, and not doxorubicinone, is a metabolite of doxorubicin in humans. Doxorubicin, but no metabolites, was present in significant and fairly constant concentrations in circulating leukocytes. These levels may reflect the drug levels in lymphoma infiltrates. The data further suggest that metabolism to 7-deoxydoxorubicinone is subject to large interindividual variation, possibly due to a genetic polymorphism, and that significant levels of a metabolic product which may be a doxorubicin glucuronide can be recovered from plasma of patients treated with doxorubicin.

Sudden, unexpected death in subjects with undiagnosed gliomas.

We report two cases in which a medicolegal autopsy disclosed small and previously undiagnosed gliomas. The first case was a 38-year-old woman who was found dead in bed; her autopsy revealed a 1.3-cm low-grade astrocytoma in the right subthalamic area. The second case involved a 32-year-old man who drowned in shallow water after his canoe capsized. A 0.5-cm oligoden-droglioma of the left temporal lobe and a 0.1-cm ganglionic hamartoma of the hypothalamus were found. In both cases the tumors may, directly or indirectly, have been the underlying cause of death. We emphasize the importance of a thorough neuropathological examination for all cases of sudden unexpected death in which no extracerebral cause of death has been found.

On the prognostic value of systemic methotrexate clearance in childhood acute lymphocytic leukemia.

The prognostic value of systemic methotrexate clearance (ClMTX) during high-dose therapy was evaluated in a cohort of 42 children with acute lymphocytic leukemia (ALL). As part of an extensive chemotherapy protocol, they had received a total of 293 methotrexate (MTX) infusions in the 6-8 g/m2 dose range. At the termination of the study, when they had all been followed up for 3.5 years or more, 26 of these patients were still in continuous complete remission, whereas 16 had suffered relapse. The intrapatient variability in ClMTX during the eight courses was up to six-fold. In 67% of the patients, the maximum level of ClMTX reached at least twice the minimum value. The coefficients of variation for the intra- and interindividual variability in ClMTX were 9-57% and 26-41%, respectively. The cumulative probability of relapse, estimated by the Kaplan-Meier procedure, was increased for patients with a high ClMTX during the initial treatment course, but the difference was not significant on a 5% level. There was no significant relationship between high individual median ClMTX and subsequent relapse of ALL. However, ClMTX during the initial infusion, the time-dependent mean for ClMTX, and the individual patient's median ClMTX, were significant predictors for event-free survival in a Cox proportional hazards regression analysis. The present study demonstrates gross pharmacokinetic variability and unpredictable values of ClMTX in subsequent courses after standardized administration of MTX to paediatric patients with ALL. In spite of the association between ClMTX and prognosis shown by some of the analyses, estimates of ClMTX rates may not necessarily be related to disease outcome in a way that can be exploited to the benefit of the individual patient.

Intrapleurally instilled mitoxantrone in metastatic pleural effusions: a phase II study.

Thirty cases (breast cancer-20 cases, malignant lymphoma-4 cases, different malignancies-6 cases) of histologically/cytologically verified malignant pleural effusion (MPE) in 29 patients were treated with intrapleurally instilled mitoxantrone (30 mg). The therapy was well tolerated. At evaluation, 25 patients had died of progressive disease. The median survival was 3 months (range 0.3-21.3 months). There were 26 responders (12 complete responses (CR), 14 partial responses (PR)), whereas 4 patients relapsed and 3 of these had an early relapse (within 3 months). Patients achieving PR or CR had a low risk (15%) of treatment failure. Five patients were subjected to a pharmacokinetic evaluation. This demonstrated rapidly declining plasma and pleural exudate levels of mitoxantrone within the first 6 hours. At 24 hours after instillation, mitoxantrone was only detected in circulating mononuclear cells. This study shows that mitoxantrone is efficacious in the treatment of MPE, and may represent a cost-effective alternative.

Alterations in methotrexate pharmacokinetics by naproxen in the rat as measured by microdialysis.

Reports of a potentially life-threatening interaction between the antifolate methotrexate (MTX) and drugs belonging to the NSAID class instigated a study of MTX pharmacokinetics by a microdialysis technique in the presence and absence of the NSAID naproxen in anesthetized rats. After pretreatment with naproxen, the animals received either 750 or 1,000 mg/kg MTX as a 6 h continuous intravenous infusion. During infusions, microdialysis effluents were obtained from probes situated intravenously, intrahepatically and intrarenally. In all three compartments, time-concentration AUCs for both MTX and its major extracellular metabolite, 7-hydroxymethotrexate (7-OH-MTX), increased about two-fold in the presence of naproxen. The mechanisms responsible for the MTX-NSAID interaction are briefly discussed. The study demonstrate that the microdialysis technique offers a means to investigate pharmacokinetic drug-drug interactions.

Continuous intratumoral microdialysis during high-dose methotrexate therapy in a patient with malignant fibrous histiocytoma of the femur: a case report.

We used a microdialysis technique to assay intratumoral methotrexate (MTX) levels during high-dose (12 g/m2 given as a 4-h infusion) therapy in a 43-year-old man with a malignant fibrous histiocytoma in the medial femoral condyle. Additional microdialysis probes were implanted in muscle tissue contralateral to the tumor and in an antecubital vein. Microdialysis was attempted during the initial two high-dose courses, but the two latter probes were removed at the start of the second treatment cycle due to leakage. No attempt to correct for microdialysis recovery was made. The intratumorally localized probe gave reproducible data on tumor MTX exposure of 9.3-14% of unbound systemic MTX. There was a close correlation between tumor and systemic levels for both MTX and its major extracellular metabolite 7-hydroxymethotrexate. Although limited to the study of MTX pharmacokinetics in a single subject, the experiment demonstrates that intratumoral microdialysis may provide data on tumor drug exposure, although of an indirect nature and dependent on the probe characteristics, the flow rate, and, possibly, the time after probe implantation. We propose that the application of microdialysis may prove useful for elucidation of the relationship between local drug exposure and the therapeutic response in normally inaccessible compartments during cancer pharmacotherapy.

Intratumoral differences in methotrexate levels within human osteosarcoma xenografts studied by microdialysis.

A central tenet in oncology is the assumed relationship between drug concentration and cytotoxicity. Determinations of drug levels in tumor tissues are, however, generally not undertaken. Microdialysis is a method where continuous drug monitoring may be achieved by sampling of low molecular weight substances from the extracellular space. By employing this technique it is possible to observe variable drug levels within tissues, including tumors, over time. Herein, we present results from a nude rat model where subcutaneous human osteosarcoma xenografts were established prior to the administration of the antifolate methotrexate as an intravenous infusion. Significant differences in drug exposure within single tumors were evident. Generally, peak drug concentrations were lower and drug efflux slower from the center of the tumors as compared to the periphery. The use of microdialysis could be an important tool for optimizing current strategies in anticancer chemotherapy.

Differential effects of the antifolates methotrexate, aminopterin and trimetrexate on murine haemopoietic progenitor cells.

We investigated the effects of the antifolates methotrexate (MTX), aminopterin (AMT) and trimetrexate (TMTX) on murine haemopoietic progenitor cells. Colony formation by late progenitors (CFU-C) was inhibited by the antifolates and the addition of leucovorin (LV) to the cultures on day 0 or day 5 wholly or partially reversed the effects of MTX or AMT. The effect of TMTX was only effectively rescued by hypoxanthine plus thymidine (HT). In the high proliferative potential colony forming cell (HPP-CFU-C) assay the antifolates induced formation of smaller colonies, but not a reduction in absolute colony numbers. This effect was reversed by LV and indicated that the antifolates (except high TMTX concentrations) were not toxic to the HPP-CFU-C but reversibly inhibited proliferation of more mature progenitor cells. The direct effects of the drugs on HPP-CFU-C were investigated in limiting dilution assays performed with fractionated bone marrow cells. Untreated cultures contained almost only large colonies, where the addition of antifolates induced a reversible shift towards smaller colonies. The present study indicates that MTX, AMT and low TMTX concentrations are not toxic to HPP-CFU-C but that the drugs induce a developmental arrest in more mature progenitor cell populations.

Determination of extracellular methotrexate tissue levels by microdialysis in a rat model.

We used a microdialysis technique to determine tissue methotrexate (MTX) levels during steady state in a rodent model. Two different approaches were employed to measure the actual extracellular MTX concentrations in muscle, liver, and kidney tissues of anesthetized Wistar rats. With the reduced-perfusion-rate technique, the flow in the microdialysis perfusate was gradually decreased toward zero to permit calculation of zero-flow intercepts. Using the net change technique, microdialysis probes were perfused with different MTX concentrations to allow an assessment of equilibrium drug levels. For these two methods to be used, drug concentrations in the matrix to be analyzed must remain unchanged during the experimental procedure. In the animal model, steady state was attained after 1.5 h and maintained throughout the rest of the experiments by the administration of MTX as continuous infusions through a venous catheter. In vitro and in vivo, both the reduced-perfusion-rate and net change techniques gave reproducible data that permitted the estimation of extracellular drug concentrations in the dialyzed tissue compartments.The data suggest that the level of unbound MTX in the circulation is fairly similar to the extracellular concentrations in the muscle and liver. In the kidney, MTX levels were measured to be 3-8 times higher than those of unbound, circulating MTX, and a considerable discrepancy between the two methods used for estimations was apparent. These results demonstrate that both the net change and reduced-flow microdialysis techniques can produce reproducible and precise data. The results may constitute a basis for determining recoveries and, thus, true extracellular drug levels during in vivo microdialysis of MTX. This may be of importance in delineation of the relationship between tissue MTX levels and outcome in a variety of normally inaccessible compartments during cancer pharmacotherapy.

Qualitatively different mechanisms of resistance to doxorubicin, both involving altered glutathione pools, in two myeloid cell lines in vitro.

Subclones of the two well-characterized myeloid cell lines HL-60 and KG1a were selected for doxorubicin resistance by systematic exposure to increased concentrations of the drug in vitro. Both subclones demonstrated a threefold increased resistance to the drug as evident from cell growth in liquid culture and clonogenicity in a semisolid matrix. Both resistant subclones displayed a similar degree of reduced total and nuclear doxorubicin levels. The HL-60 and the KG1a cells differed qualitatively and quantitatively with respect to glutathione (GSH) levels during culture, with markedly elevated concentrations in the resistant HL-60 subclone during 1 week of culture. Total GSH pools in resistant and sensitive KG1a cells were similar, but maximum GSH levels were reached earlier in the resistant KG1a clones than in the parental cells. Northern blot analysis suggests that resistance was accompanied by increased mdr1 expression in the KG1a but not in the HL-60 cells, whereas alterations in the glutathione S-transferase P1-1 and topoisomerase II message was evident in the latter. The results demonstrate the complex, multifactorial mechanisms behind the in vitro induction of even moderate resistance in anthracyclines.

A high-pressure liquid chromatographic method for measuring mitotane [1,1-(o,p'-Dichlorodiphenyl)-2,2-dichloroethane] and its metabolite 1,1-(o,p'-Dichlorodiphenyl)-2,2-dichloroethene in plasma.

The adrenolytic agent mitotane [o,p'-DDD or 1,1-(o,p'-dichlorodiphenyl)-2, 2-dichloroethane] has been employed in the nonsurgical treatment of patients with adrenal carcinoma for several decades. Its use is hampered by serious side effects, which may be limited by analytically guided dose modifications in the individual patient. Mitotane analyses have previously been undertaken by gas chromatography with electron capture detection. A sensitive high-pressure liquid chromatographic method for measuring mitotane in plasma is described. After protein precipitation with 1.5 vol of acetone, mitotane and its metabolite 1,1-(o,p'-dichlorodiphenyl)-2,2-dichloroethene (o,p'-DDE) are resolved by isocratic elution from a C18 reversed-phase support and quantified by ultraviolet detection at 230 nm. Recoveries of mitotane and o,p'-DDE after deproteinization were quantitative. Within-run and between-day coefficients of variation were < 4% over the entire therapeutic range. The limit of detection was 0.25 mumol/L and the standard curve was linear in the 1-100 mumol/L range. The method has been evaluated using samples obtained from an adolescent girl who had metastatic adrenocortical carcinoma. Data from this single patient may suggest that systemic absorption of mitotane is adequate, and toxicity possibly decreased, when mitotane is administered by the rectal route.

Effect of methotrexate on murine bone marrow cells in vitro: evidence of a reversible antiproliferative action.

Methotrexate (MTX) acts by inducing cellular depletion of reduced folates, which ultimately leads to an inhibition of DNA synthesis. Like many anticancer drugs, this antimetabolite has little selectivity for tumor cells, and its effectiveness is limited by toxicity to normal tissues, particularly gastrointestinal epithelium and bone marrow. Previous studies have shown that MTX inhibits colony formation of the hematopoietic progenitor cells (CFU-C) in vitro. Whether this effect is due to a cytotoxic or a cytostatic mechanism has not been resolved. The present study was undertaken to eludicate the mechanism by which MTX inhibits CFU-C formation. Bone marrow cells in agarose cultures supplemented with recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) were incubated for 7 days in the presence or absence of MTX. Exposure to 33 nM to 1 microM MTX reduced colony formation by more than 80% when compared to control cultures. When bone marrow suspension cultures supplemented with rmGM-CSF were incubated for 5 days in the presence or absence of MTX, exposure to 10 nM to 1 microM MTX resulted in a 60 to 80% reduction in cell numbers when compared to untreated cultures. Residual CFU-C numbers were determined in the same cultures by replating into agarose. Exposure to 10 nM MTX was found to enhance CFU-C recovery three-fold as compared to controls and cultures exposed to higher MTX concentrations. Addition of 10 microM of the reduced folate leucovorin (LV; 5-formyl-tetrahydrofolate) prevented CFU-C accumulation in the presence of 10 nM MTX. The kinetics of LV rescue of CFU-C, pre-exposed to 100 nM MTX, were investigated in clonogenic assays. The addition of 1 microM LV to semisolid bone marrow cultures preincubated with 100 nM MTX for up to 8 days completely abolished the inhibition of colony formation seen with 100 nM MTX alone. When the dose range of MTX was expanded from 33 nM to 3.3 microM, we found that administration of 10 microM LV on day 5 rescued the hematopoietic progenitors from MTX inhibition in all groups. These observations suggest that MTX is not cytotoxic to hematopoietic progenitors over its entire dose range but that it can induce a reversible block in the proliferation and differentiation of cells in the progenitor compartment.

Quantitation of 6-thioguanine residues in peripheral blood leukocyte DNA obtained from patients receiving 6-mercaptopurine-based maintenance therapy.

The antimetabolite 6-mercaptopurine is widely utilized in maintenance therapy for childhood acute lymphoblastic leukemia. Following p.o. administration, this prodrug undergoes extensive biotransformation, resulting in the generation of a plethora of metabolites including 2'-deoxy-6-thioguanosine triphosphate. Incorporation of 6-thioguanine (6-TG) bases into DNA is generally considered to be central to thiopurine-mediated cytotoxicity. We have developed a novel precolumn derivatization HPLC technique for quantifying 6-TG base accumulation into leukocyte DNA obtained from acute lymphoblastic leukemia patients receiving 6-mercaptopurine maintenance therapy. The method is based on enzymatic degradation of DNA to 2'-deoxyribonucleosides and the derivatization of released 2'-deoxy-6-thioguanosine with a thiol-reactive reagent containing a 7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2'-deoxy-6-thioguanosine-7-amino-4-methylcoumarin-3-acetic acid adduct is resolved by reversed-phase HPLC and quantified fluorometrically. Assay response is linear from 15 pmol to 60 fmol 6-TG bases/microgram DNA with a limit of quantitation corresponding to the incorporation of 1 6-TG residue per 50,000 bases. In a small cohort of acute lymphoblastic leukemia patients receiving p.o. 6-mercaptopurine-based maintenance therapy, significant interindividual variation in the accumulation of 6-TG bases into leukocyte DNA was revealed. The determined levels of drug base incorporation ranged from 95 to 710 fmol 6-TG bases/microgram DNA (6-TG base:nucleotide ratio 1:32,000 to 1:4,000). The assay may provide a novel methodology for pharmacological monitoring of thiopurine therapy either in the routine clinical setting or during studies of alternative routes of drug delivery.

Pharmacokinetics of different doses of methotrexate at steady state by in situ microdialysis in a rat model.

We used a microdialysis technique to monitor extracellular methotrexate (MTX) levels during the steady state in a rodent model. Microdialysis probes were implanted in the muscle, liver, and kidney of anesthetized male Wistar rats. MTX (18.75-500 mg/kg) was given as a continuous infusion through a venous catheter, and blood samples were obtained through a second venous catheter. Heparinized plasma, ultrafiltered plasma, microdialysis effluent from tissues, and tissue samples (obtained at the end of experiments) were analyzed for MTX content by high-performance liquid chromatography (HPLC). Steady state was demonstrated in the blood and tissues from 2 h until the end of the experiments (6 h). Extracellular drug levels in muscle and liver displayed a linear correlation with doses, whereas kidney levels reached a plateau at an MTX dose of 150 mg/kg per 6 h. Microdialysis-fluid endpoint levels for muscle, liver, and kidney were positively correlated to the endpoint total tissue levels (r2 = 0.80, 0.85, and 0.68, respectively). In the kidneys, the maximal relative tissue MTX accumulation was measured at a total dose of 75 mg/kg per 6 h. At higher doses, the relative drug sequestration declined to less than half of the values observed at this dose. This study demonstrates that the microdialysis technique can provide reproducible data on MTX tissue exposure in an animal model and that it offers a means of serial and reproducible monitoring of extracellular-tissue MTX levels at steady state and over a wide dose range. Pending additional studies, microdialysis may be a helpful technique for elucidating the kinetics of drug delivery to both targeted and toxicity-prone tissues during chemotherapy.

Role of the 75-kDa tumor necrosis factor receptor: inhibition of early hematopoiesis.

Biological effects of tumor necrosis factor alpha (TNF-alpha) are mediated through two cell surface receptors, the 55-kDa TNF receptor and the 75-kDa TNF receptor. The present study investigated the relative roles of the two TNF receptors in normal hematopoiesis. Using agonists (antibodies) specific for the 55- and 75-kDa TNF receptors, we demonstrate differential roles of the two TNF receptors in hematopoiesis in that only the 55-kDa TNF receptor mediates antiproliferative effects of TNF-alpha on mature Lin- hematopoietic progenitor cells responding to granulocyte colony-stimulating factor or interleukin 3 alone. In contrast, the 75-kDa TNF receptor is essential in mediating inhibition of primitive Lin-Sca-1+ high-proliferative-potential colony-forming cells and inhibition of the total number of proliferative clones of individually cultured Lin-Sca-1+Rh123lo and Lin-Sca-1+Rh123hi cells.

Evaluation of methotrexate tissue exposure by in situ microdialysis in a rat model.

The feasibility of using a microdialysis technique to obtain pharmacokinetic data on tissue exposure to methotrexate (MTX) was investigated. Microdialysis probes were implanted in the jugular vein, femoral muscle, and liver of anesthetized male Wistar rats. MTX (100 mg/kg) was given as a bolus injection through an indwelling venous catheter, and blood samples were obtained through a second venous access and by microdialysis for a total of 6 h. Heparinized plasma, ultrafiltered plasma, and microdialysis effluent from tissue and venous probes were analyzed by high-performance liquid chromatography. Centrifugal ultrafiltration of rat plasma spiked in vitro with MTX (1-100 microM) revealed a mean binding to plasma proteins of 21%. In vitro microdialysis of this spiked plasma resulted in 23% relative recovery of the unbound fraction. In rats receiving MTX, plasma protein binding was 23% and the relative drug recovery as assessed with venous microdialysis probes was 18%. Plotting of unbound (i.e., ultrafiltrate) MTX concentrations in the blood against venous microdialysis perfusate values in the blood gave a good linear correlation with a coefficient of correlation (r2) of 0.98. There was also a linear correlation between the total MTX concentrations in venous blood and the drug levels in microdialysis samples from muscle and liver (r2 = 0.93 and 0.74, respectively). Area under the curve estimations were consistent with an MTX exposure of 30% and 46% for the muscle and liver as compared with the circulation. The present study demonstrates that the microdialysis technique can provide reproducible data on tissue exposure to MTX in an animal model and indicates that the methodology is adaptable to clinical settings.

Quantitation of cell-associated doxorubicin by high-performance liquid chromatography after enzymatic desequestration.

A method for measuring cellular concentrations of the anthracycline doxorubicin was developed. The assay involves cell lysis and protein degradation by detergent and proteinase K treatment followed by DNA hydrolysis using DNase I. Prior to high-performance liquid chromatography, samples are deproteinized by the addition of ZnSO4 and methanol. The assay is linear with respect to both the cellular drug content and the number of cells assayed over the ranges tested, and drug recovery is close to 100%. The method has a limit of detection of 50 fmol injected doxorubicin. Within run and between-day coefficients of variation have consistently been found to be in the 5% and 10% range, respectively, in different cell lines exposed to doxorubicin in vitro. The method has been evaluated in analyses of doxorubicin levels in mononuclear blood cells of patients. The assay offers several advantages over commonly used organic extraction techniques and may improve cellular drug monitoring during anthracycline therapy in patients.

Pituitary-gonadal dysfunction in male patients with lung cancer. Association with serum inhibin levels.

Male lung cancer patients with poor performance status have an endocrinological dysfunction shown by decreased serum levels of total and free testosterone (AFTC). The intention was to investigate whether or not inhibin plays a role in gonadal dysfunction observed in male patients with malignant pulmonary disease. Twenty-seven patients with locally advanced non-small cell lung cancer were included. Sixteen patients were within ECOG index 1-2 (group A) and 11 patients within ECOG index 3-4 (group B). Gonadal function was monitored by serum LH, FSH, testosterone, SHBG and inhibin levels. Patients with poor performance status displayed significantly lower inhibin (1.6 +/- 0.8 U/I) and AFTC (0.23 +/- 0.07 nmol/l) levels when compared to patients within ECOG index 1-2 (inhibin 2.4 +/- 1.1 U/I; AFTC 0.66 +/- 0.36 nmol/l). Serum inhibin tended to correlate inversely to FSH with a 4.4-fold higher FSH/inhibin ratio in group B compared to A.

A high-performance liquid chromatographic method for the determination of 6-thioguanine residues in DNA using precolumn derivatization and fluorescence detection.

An HPLC method is described for the determination of 6-thioguanine (6-TG) residues in DNA. The assay is based on the release of 2'-deoxy-6-thioguanosine 5'-monophosphate (S6dGMP) by P1-nuclease digestion and its derivatization with the thiol-reactive fluorophore monobromobimane (mBBr). Following treatment with alkaline phosphatase, the resultant 2'-deoxy-6-thioguanosine-mBBr adduct is resolved by isocratic elution from a C18 reversed-phase support and quantified fluorometrically. The chromatographic procedure provides good adduct resolution without interference from reagent peaks or endogenous components present in the DNA. Recoveries of S6dGMP following DNA digestion were quantitative and the assay displayed a linear response from 18 pmol 6-TG bases/microgram DNA down to the lowest concentration tested (0.56 pmol 6-TG bases/microgram DNA). Within-run coefficients of variation were 2.6 and 3.1% for samples containing 18 and 0.9 pmol 6-TG bases/microgram DNA, respectively. Between-day coefficients of variation were 3.1% at 18 pmol and 4.4% at 0.9 pmol 6-TG bases/microgram DNA. In the standard procedure, derivatized sample corresponding to 5 micrograms of DNA (approximately 5 x 10(5) cells) was injected per analysis. This allowed the quantification of < 2.8 pmol adduct and permitted an assessment of 6-TG base incorporation into the DNA of cells exposed to 6-mercaptopurine concentrations as low as 30 nM. This method may be useful in clarifying the relationship between drug metabolite uptake into DNA and the anticancer effect mediated by 6-thiopurines. In addition it may form the basis of improved methods for clinical monitoring during pharmacotherapy with these agents.