Accelerator mass spectrometry allows for cellular quantification of doxorubicin at femtomolar concentrations.

Accelerator mass spectrometry (AMS) is a highly sensitive analytical methodology used to quantify the content of radioisotopes, such as (14)C, in a sample. The primary goals of this work were to demonstrate the utility of AMS in determining total cellular [(14)C]anthracycline concentrations following administration of doxorubicin (DOX) and to develop a sensitive assay that is superior to high performance liquid chromatography (HPLC) for the quantification of [(14)C]anthracycline at the tumor level. In order to validate the sensitivity of AMS versus HPLC with fluorescence detection, we performed three studies comparing the cellular accumulation of DOX: one in vitro cell line study, and two in vivo xenograft mouse studies. Using AMS, we quantified cellular [(14)C]anthracycline content up to 4 h following in vitro exposure at concentrations ranging from 0.2 pg/ml (345 fM) to 2 microg/ml (3.45 microM) [(14)C]DOX. The results of this study show that, compared to standard fluorescence-based HPLC, the AMS method was over five orders of magnitude more sensitive. Two in vivo studies compared the sensitivity of AMS to HPLC using a nude mouse xenograft model in which breast cancer cells were implanted subcutaneously. After sufficiently large tumors formed, [(14)C]DOX was administered intravenously at two dose levels. Additionally, we tested the AMS method in a nude mouse xenograft model of multidrug resistance (MDR) in which each mouse was implanted with both wild type and MDR+ cells on opposite flanks. The results of the second and third studies showed that [(14)C]anthracycline concentrations were significantly higher in the wild type tumors compared to the MDR+ tumors, consistent with the MDR model. Although this method does not discriminate between parent drug and metabolites, the extreme sensitivity of AMS should facilitate similar studies in humans to establish target site drug delivery and to potentially determine the optimal treatment dose and regimen.

Hyperthermic potentiation of cis-diamminedichloroplatinum(II) cytotoxicity in Chinese hamster ovary cells resistant to the drug.

The cytotoxic and pharmacological properties of hyperthermia and cis-diamminedichloroplatinum(II) (DDP) were studied in DDP-sensitive and DDP-resistant Chinese hamster ovary cells in vitro. Cytotoxicity was measured by cell survival using colony formation assay and cellular platinum levels were determined by atomic absorption spectrophotometry. Hyperthermia potentiated DDP cytotoxicity in both DDP-sensitive and DDP-resistant Chinese hamster ovary cells. Dose enhancement ratios increased from 1.4 to 6.5 over the temperature range of 39-43 degrees C. Cellular accumulation of platinum at 37 degrees C in the sensitive cells was 2.3- to 3.3-fold greater than that in the drug-resistant cells. Cellular accumulation of DDP was increased by factors of 1.5 and 2.2 at elevated temperature. DDP resistance did not confer cross-resistance to heat alone. The results suggest that hyperthermia could be used to circumvent DDP resistance.

Differential expression of ganglioside GD3 by human leukocytes and leukemia cells.

Gangliosides from normal leukocytes and the cells of 25 patients with acute and chronic leukemia were tested for the presence of the disialoganglioside II3-alpha-N-acetylneuraminosyl-alpha 2----8-N-acetylneuraminosyllactosylceramide (GD3). GD3 was detected by immunostaining thin-layer chromatographs with an anti-GD3 monoclonal antibody (AbR24). Among the myeloid cells tested, acute leukemia cells were positive for GD3, whereas chronic leukemia cells and normal neutrophils did not have detectable GD3. A range of GD3 reactivity was apparent within the acute myeloid leukemia cells; gangliosides from pure myeloid leukemia cells stained more intensely than those from leukemia cells with monocytic characteristics. All lymphocytic leukemia cells (chronic and acute) contained GD3, but this ganglioside could not be detected in extracts from normal lymphocytes. A ganglioside extract from the cells of a patient with hairy cell leukemia was also positive for GD3 immunostaining. These results demonstrate that normal leukocytes and chronic myelogenous leukemia cells are distinguished from other lymphoid and nonlymphoid leukemia cells on the basis of GD3 ganglioside expression.

Toremifene: pharmacologic and pharmacokinetic basis of reversing multidrug resistance.

Triphenylethylene compounds, such as tamoxifen, have shown chemosensitizing activity independent of estrogen receptor status in doxorubicin-resistant cells. We examined the chemosensitizing activity of a new triphenylethylene, toremifene, and its major metabolites in a doxorubicin-resistant human breast cell line, MCF-7/DOX. In addition, we examined the chemosensitizing activity of unbound plasma toremifene and its metabolites isolated from patients treated with toremifene doses of 20 to 400 mg/d. MCF-7/DOX cells were exposed to ultrafiltrate plasma specimens in the absence and presence of doxorubicin. These latter studies were single-blinded. Toremifene and its major metabolites were capable of sensitizing multidrug-resistant cells to doxorubicin. The degree of chemosensitizing activity in vitro correlated with the plasma concentrations of toremifene and its metabolites (P less than .05). Plasma samples isolated from patients receiving high-dose toremifene (400 mg/d) had the greatest chemosensitizing activity. We present evidence that toremifene and its metabolites can sensitize resistant MCF-7/DOX cells to doxorubicin, that this effect is concentration-dependent, and that sensitizing activity can be detected at clinically achieved concentrations.

Identification of estrogenic tamoxifen metabolite(s) in tamoxifen-resistant human breast tumors.

PURPOSE: We have shown previously that acquired tamoxifen resistance in an in vivo experimental model is associated with reduced tamoxifen accumulation, isomerization of trans-4-hydroxytamoxifen, and tamoxifen-stimulated tumor growth. The purpose of this study is to isolate and verify the presence of estrogenic tamoxifen metabolites in human breast tumors using high-performance liquid chromatography (HPLC) and mass-spectrometry (MS) techniques. PATIENTS AND METHODS: In the present study, we used HPLC and MS to identify the presence of estrogenic metabolites in tumor samples excised from athymic nude mice and in human breast tumors isolated from patients receiving adjuvant tamoxifen therapy. RESULTS: We identified the presence of metabolite E, a known estrogenic metabolite of tamoxifen, in tamoxifen-resistant MCF-7 human breast tumors implanted in athymic nude mice, as well as in tumors from patients with clinical resistance. Additionally, we separated another estrogenic metabolite, bisphenol, by HPLC, and this was also tentatively confirmed by MS analysis. CONCLUSION: These data suggest that cellular tamoxifen metabolism to estrogenic metabolites may in part contribute to stimulating the growth of hormone-responsive breast tumors following prolonged exposure to tamoxifen. Further evaluation of the relationship between cellular metabolism and acquired tamoxifen resistance is warranted.

Tamoxifen and the isomers of 4-hydroxytamoxifen in tamoxifen-resistant tumors from breast cancer patients.

PURPOSE: The antiestrogen tamoxifen is effective in therapy for breast cancer. However, its use is limited by the eventual development of acquired tamoxifen resistance in many patients. The mechanisms responsible for tamoxifen resistance remain unknown; loss of estrogen receptor (ER), selection of hormone-independent breast cancer clones, or alterations in serum tamoxifen levels after long-term use do not explain acquired resistance in most patients. Using an experimental model in which human breast cancer cells develop resistance in athymic mice treated with tamoxifen, we have recently shown that acquired resistance is associated with markedly reduced cellular concentrations of tamoxifen and by isomerization of the trans-4-hydroxy metabolite to the less potent cis isomer. MATERIALS AND METHODS: Using a sensitive high-performance liquid chromatography (HPLC) assay, we have now measured levels of tamoxifen and its major metabolites in a series of 14 tumors from patients treated with tamoxifen. The duration of therapy ranged from 1 month to 6 years. RESULTS: Tumor tamoxifen levels varied over a wide range. Low concentrations were observed in tumors from eight patients, all demonstrating progressive disease at the time of biopsy after a minimum duration of treatment of 6 months. Six tumors had moderate to high tamoxifen levels, two from patients responding to tamoxifen, one from a patient with stable disease, and three from patients with disease progression. Both the cis and trans isomers of the potent antiestrogenic metabolite 4-hydroxy-tamoxifen were detected in 11 tumors. Six tumors had high ratios of the cis to trans isomer (1.10:2.06), all from patients not responding to tamoxifen. The five tumors with low cis:trans ratios included the two tumors from responding patients and three from patients with progression. All but one of the 11 nonresponding patients had either a low tumor tamoxifen level, a high cis:trans ratio, or both. CONCLUSION: This study clearly demonstrates a wide range of tumor tamoxifen levels and accumulation of the less antiestrogenic cis isomer of 4-hydroxytamoxifen in some patients on tamoxifen therapy. Additional study is necessary to determine if these metabolic profiles are related to the development of tamoxifen resistance.

High-dose cisplatin in hypertonic saline: reduced toxicity of a modified dose schedule and correlation with plasma pharmacokinetics. A Northern California Oncology Group Pilot Study in non-small-cell lung cancer.

Although increased efficacy has been described with a five-day schedule of high-dose cisplatin (CDDP) in hypertonic saline, severe myelosuppression and cumulative neurotoxicity have limited the usefulness of this therapy. In order to evaluate a possible dose-response relationship in non-small-cell lung cancer (NSCLC), 17 patients with metastatic disease were treated with a modified dose schedule delivering the same total dose (200 mg/m2) in a divided day 1 and 8 schedule. During a pilot study, a total of 47 cycles of therapy were administered, with a median of three cycles per patient and a median total cumulative dose of 600 mg/m2. Nine of 17 patients received at least 600 mg/m2. While nephrotoxicity was similar to previous reports of the five-day schedule, the incidence and severity of myelosuppression and peripheral neuropathy were markedly reduced. Using this modified schedule, severe myelosuppression did not occur. Clinically severe peripheral neuropathy developed in only one patient (6%). The overall response rate was 47% (eight of 17 patients). Plasma platinum pharmacokinetics during five cycles of the modified day 1 and 8 schedule were compared with pharmacokinetics of the five-day schedule. Accumulation of plasma ultrafiltrate platinum occurred in the five-day schedule, but not in the day 1 and 8 schedule. This difference in pharmacokinetics is one possible explanation for the reduced toxicity of this modified schedule. Although the degree of activity seen in this pilot study is encouraging, the efficacy of high-dose CDDP in NSCLC remains to be defined. In view of reduced myelosuppression and neurotoxicity, further trials with this modified schedule are indicated.

Tamoxifen resistance in breast cancer.

Tamoxifen (TAM) resistance is the underlying cause of treatment failure in many breast cancer patients receiving TAM. The mechanism(s) involved in TAM resistance are poorly understood. A variety of mechanisms have been proposed but only limited evidence exists to substantiate them. Studies have now shown that in many patients TAM resistance is not related to the down regulation or loss of estrogen receptors (ER). Variant ER have been identified, but their significance clinically remains to be proven. Since breast cancer cells secrete several estrogen-regulated growth factors and growth inhibitors that may have autocrine or paracrine activity, altered growth factor production is another possible mechanism for TAM resistance. Tissue-specific transcription activating factors that may alter how the signal induced by TAM binding to the receptor is interpreted by the cell also require further investigation. An increase in antiestrogen binding sites (AEBS), which could effectively partition TAM and reduce its concentration at the ER has also been proposed as a potential mechanism. Pharmacologic mechanisms, such as a shift in metabolism toward the accumulation of estrogenic metabolites, are supported by recent data demonstrating metabolite E and bisphenol in tumors from TAM-resistant patients. Furthermore, a decrease in tumor TAM accumulation and an altered metabolite profile have been reported in TAM-resistant breast tumors grown in nude mice. These and other studies suggest that TAM resistance may be multifactorial in nature, but definitive identification of mechanisms that are operative in clinical TAM resistance requires further study.

Cisplatin rescue therapy: experience with sodium thiosulfate, WR2721, and diethyldithiocarbamate.

Cisplatin has a steep dose response curve for both antitumor and adverse effects. Therapeutic strategies aimed at reducing toxicity and allowing dose escalation of intravenous cisplatin, such as administration in hypertonic saline and pharmacokinetically based dosing schedules, have been partially successful in reducing nephrotoxicity and bone marrow suppression. However, new dose-limiting toxicities consisting of peripheral neuropathy and ototoxicity have emerged, which continue to restrict potential use of high dose cisplatin therapy. Intraperitoneal administration of high dose cisplatin also offers the potential of markedly increased local drug exposure if systemic toxicity can be avoided. Proposed chemoprotective agents, including sodium thiosulfate, WR2721, and diethyldithiocarbamate (DDTC) are being extensively examined as "rescue agents" for either regional or systemic administration of cisplatin. Although each agent offers unique advantages to be considered in developing successful rescue therapy, many questions remain regarding molecular and pharmacokinetic interactions with cisplatin, appropriate dosing schedules, and effects on antineoplastic activity. We present a review of current investigations of chemoprotectors for prevention of cisplatin-related toxicities.

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo.

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.

Fungal infections in patients with acute leukemia.

We reviewed the records of 32 patients with acute leukemia and proved invasive fungal infections to determine the clinical and pathologic characteristics of systemic mycosis in patients undergoing intensive induction chemotherapy. The incidence of invasive fungal infections among our patients was at least 27 percent, and Candida and Aspergillus accounted for the majority of these infections. Patients with systemic candidiasis generally had prolonged severe neutropenia, fever refractory to antibiotics, and evidence of mucosal colonization by fungi. At autopsy, Candida was always widely disseminated. Patients with aspergillosis generally had neutropenia, fever, and pulmonary infiltrates at the time of admission to the hospital and, at autopsy, their infections were primarily confined to the lungs. Patients infected with both Candida and Aspergillus had clinical and pathologic findings that were a combination of the features of each type of infection. A diagnosis of invasive fungal infection was established before death in only nine of the patients, all of whom had systemic candidiasis. Four of these patients were successfully treated and survived their hospitalization. The reasons for frequently misdiagnosing and unsuccessfully treating systemic mycosis in patients with acute leukemia are examined, and suggestions are made for improved management of patients at high risk for these infections. These suggestions are based upon recognition of the clinical settings in which fungal infections occur, the aggressive use of invasive diagnostic procedures, and the early empiric use of amphotericin B.

Clinical pharmacokinetics of drugs used in the treatment of breast cancer.

This article reviews the absorption, bioavailability, distribution, metabolism, and elimination patterns of the agents most commonly used for the treatment of breast cancer. Although the majority of the pharmacokinetic studies reviewed have been examined in non-breast cancer patients, the kinetic findings are not significantly altered by disease state. The anthracyclines, antimetabolites, alkylating agents, antioestrogens and mitotic inhibitors are among the classes of agents used to treat breast cancer. Although extensively examined, cancer pharmacokinetic research has resulted in very few clinically relevant findings in breast cancer.

Clinical pharmacokinetics of toremifene.

Toremifene is a chlorinated triphenylethylene derivative of tamoxifen approved for use in the treatment of patients with metastatic breast cancer. Toremifene is well tolerated in patients, and common adverse effects of this drug include vasomotor symptoms such as hot flashes and vaginal discharge. This compound is administered to patients orally at a dose of 60 mg/day, although alternative methods of administration have been investigated. Oral bioavailability is estimated to be approximately 100%. At steady state, toremifene and its metabolites are highly protein bound (>95%). Toremifene is metabolised in the liver by cytochrome P450 enzymes, and it is eliminated primarily in the faeces following enterohepatic circulation. The half-life of toremifene is approximately 5 days, and steady state is reached by 6 weeks depending on the dose given. The pharmacokinetics of toremifene have been shown to be altered by certain liver conditions, but age and kidney function do not appear to be as significant.

In vitro and in vivo biologic effects of Ospemifene (FC-1271a) in breast cancer.

Ospemifene (FC-1271a) is a novel selective estrogen receptor modulator under development for osteoporosis prevention. In the present paper, we examine both the in vitro and in vivo effects of FC-1271a in breast cancer models. In vitro, the growth inhibitory effects of FC-1271a and its main metabolite are investigated in MCF-7 and MDA-MB-231 human breast cancer cells at doses ranging from 0.1 to 10 microM. Modulation of pS2 expression, an indicator of estrogen activity, was also examined in all experiments using reverse transcription-polymerase chain reaction. In vivo, the effects of treatment with 10, 25, 50, or 100 mg/kg FC-1271a on MCF-7 and MDA-MB-231 human tumor xenografts in athymic, ovariectomized mice were determined. For MCF-7 cells, FC-1271a and its main metabolite, toremifene VI (TOR VI) displayed anti-estrogenic effects in vitro as shown through growth inhibition and decreased expression of pS2. Treatment with FC-1271a in vivo inhibited MCF-7 tumor growth, compared with control (P< or =0.05). FC-1271a and TOR VI did not inhibit the growth of MDA-MB-231 cells in vitro, and no clear effects of FC-1271a treatment were seen on MDA-MB-231 tumor growth in vivo. In conclusion, FC-1271a appears to exert anti-estrogenic effects dependent on estrogen receptor positivity in vitro and in vivo on the growth of MCF-7 cells.

Tumor and serum tamoxifen concentrations in the athymic nude mouse.

The athymic nude mouse has been used as an in vivo model for pharmacologic studies of the antiestrogen, tamoxifen. Serum and tumor tamoxifen and metabolite concentrations were examined during and after 100 and 1000 micrograms/day doses injected s.c. Tamoxifen and tamoxifen metabolites were quantitated by high-performance liquid chromatography. Tamoxifen was detected in tumors after a dose of 100 micrograms/day, although serum concentrations were not detected. At a dose of 1000 micrograms/day, tumor tamoxifen and its active metabolites were detected in high concentrations ranging up to greater than 6 mmol/g tissue. Serum tamoxifen metabolites were not detected at either dose. In summary, high doses of tamoxifen were required in the nude mouse to obtain clinically relevant serum concentrations, and significant tumor levels were achieved at doses that resulted in undetectable serum levels. The relationship between serum tamoxifen concentrations, tumor tamoxifen levels, and the biologic activity of the drug requires further study.

Serum tamoxifen concentrations in the athymic nude mouse after three methods of administration.

The athymic nude mouse has been used as an in vivo model for pharmacologic studies of the antiestrogen, tamoxifen. We have examined the steady-state serum tamoxifen concentrations achieved in mice with s.c. slow-release pellets, s.c. injections, and i.p. injections, in an attempt to identify a method that would yield serum levels similar to those observed in patients receiving tamoxifen therapy. Tamoxifen and tamoxifen metabolites were examined by a high-performance liquid chromatography assay which has a sensitivity of 8 ng/ml. Tamoxifen metabolites were not observed with any dose or schedule. After slow-release pellets containing 5 or 25 mg tamoxifen no tamoxifen was detectable, even after 2 weeks of treatment. Very low levels (0.07 microM) were found with 50-mg pellets. Tamoxifen was also not detected either with daily s.c. injections of 500 micrograms/mouse or with i.p. injections of 2.5 mg/kg. However, daily s.c. injections of 1000 micrograms or i.p. injections of 25, 50, or 100 mg/kg resulted in tamoxifen concentrations ranging from 0.21 to 0.51 microM which are similar to those observed in patients. Thus, clinically relevant tamoxifen concentrations can be achieved in the nude mouse with either of these methods of administration.

Preliminary observations of intraperitoneal carboplatin pharmacokinetics during a phase I study of the Northern California Oncology Group.

The pharmacokinetic behavior of carboplatin administered by the i.p. route at a dose of 200 mg/m2 was studied during five courses of therapy in four patients with ovarian cancer. A regional pharmacologic advantage was noted with carboplatin administered by this route, with peak peritoneal fluid concentrations 18-fold those in plasma, and area under the curve (AUC) for the peritoneum showing a 18-fold and 6-fold increase over plasma AUC at 4 and 24 h, respectively. The mean residence time of total platinum in the peritoneum was 4.7 h. Approximately 10% and 40% of plasma platinum was protein bound at 4 and 24 h after treatment, respectively, whereas peritoneal fluid platinum showed minimal protein binding. Peak plasma platinum levels were comparable to those recorded in previous studies with i.v. doses of carboplatin. Peritoneal clearance of carboplatin in these four patients appeared to be less than that previously reported for cisplatin. Further studies are in progress with higher doses of i.p. carboplatin.

Characterization of cellular lipids in doxorubicin-sensitive and -resistant P388 mouse leukemia cells.

The purpose of this study was to determine whether changes in cellular lipid composition accompanied the selection of cells that are resistant to the anthracycline doxorubicin. Total cellular lipid extracts from doxorubicin-sensitive and doxorubicin-resistant P388 murine leukemia cells were prepared and separated into neutral glycosphingolipids, gangliosides, phospholipids, and neutral lipid families. No significant quantitative differences in total cholesterol, lipid-bound sialic acid, neutral hexose, and lipid-bound phosphate were found between the two cell lines. Gas-liquid chromatographic analysis of the fatty acids derived from each lipid class demonstrated that sensitive and resistant cells had essentially identical fatty acid compositions. Qualitative evaluation of the four lipid classes by high-performance thin-layer chromatography revealed only minor differences in lipid composition between the resistant and the sensitive cells. Results from this study indicate that although minor differences between the two cell lines are present, no major cellular lipid differences are evident to account for the marked differences in the cellular pharmacokinetics and cytotoxic effects of doxorubicin between doxorubicin-sensitive and doxorubicin-resistant P388 murine leukemia a cells.

Targeting chemosensitizing doses of toremifene based on protein binding.

Toremifene is currently being evaluated as a chemosensitizing agent in doxorubicin-resistant patients. Although concentrations of > 2 microM reverse resistance in vitro, target concentrations required to reverse multidrug resistance (MDR) in vivo may be highly influenced by variables such as protein binding in serum. We examined the effects of high serum concentrations on the cellular accumulation of toremifene in an MDR MDA-MB-A-1 human breast-cancer cell line. We then examined the cellular accumulation of doxorubicin at various toremifene concentrations in 5% - 100% serum. We also measured the concentrations of toremifene and its major metabolites in plasma specimens obtained from two patients receiving 360 mg/day for 5 days in a phase I study. Our results show that (1) high serum concentrations decrease toremifene accumulation, (2) toremifene concentrations of < or = 2.5 microM enhance doxorubicin accumulation, and (3) patients achieve plasma toremifene concentrations of 10-15 microM following doses of 360 mg/day x 5 days. Our findings suggest that in vivo toremifene concentrations well above those used to reverse resistance in vitro are required to overcome the effect of high serum-protein binding.

Uptake, metabolism, and cytotoxicity of doxorubicin in human Ewing's sarcoma and rhabdomyosarcoma cells.

Uptake, metabolism, and cytotoxicity of doxorubicin (DOX) in human Ewing's sarcoma (ES) and rhabdomyosarcoma (RS) cells were examined. Cellular uptake of DOX was determined by liquid scintillation spectrometry, and intracellular metabolism was examined by high performance liquid chromatography. The cytotoxicity of DOX was assessed by two different methods: an extracellular matrix detachment assay (ECM-D) and 3H-thymidine incorporation. The uptake of DOX by ES cells was 1.5-3.0 times greater than RS cells, even though both cell types achieved intracellular steady-state though both cell types achieved intracellular steady-state concentrations between 6-8 h. No significant intracellular metabolism ( less than 5%) was seen after 8-h incubations with the drug. The cytotoxic effects of DOX in both cell lines were concentration-dependent, with the RS cells being more sensitive. Measurement of 14C-DOX appears to be a reliable method for quantitating intracellular DOX. In addition, the ECM-D and 3H-thymidine assays used for assessing cytotoxicity produced similar results, showing that the ECM-D can be reliable and easily performed test of cell death.