The bioavailability time of commonly used thymidine analogues after intraperitoneal delivery in mice: labeling kinetics in vivo and clearance from blood serum.

Detection of synthetic thymidine analogues after their incorporation into replicating DNA during the S-phase of the cell cycle is a widely exploited methodology for evaluating proliferative activity, tracing dividing and post-mitotic cells, and determining cell-cycle parameters both in vitro and in vivo. To produce valid quantitative readouts for in vivo experiments with single intraperitoneal delivery of a particular nucleotide, it is necessary to determine the time interval during which a synthetic thymidine analogue can be incorporated into newly synthesized DNA, and the time by which the nucleotide is cleared from the blood serum. To date, using a variety of methods, only the bioavailability time of tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) have been evaluated. Recent advances in double- and triple-S-phase labeling using 5-iodo-2'-deoxyuridine (IdU), 5-chloro-2'-deoxyuridine (CldU), and 5-ethynyl-2'-deoxyuridine (EdU) have raised the question of the bioavailability time of these modified nucleotides. Here, we examined their labeling kinetics in vivo and evaluated label clearance from blood serum after single intraperitoneal delivery to mice at doses equimolar to the saturation dose of BrdU (150 mg/kg). We found that under these conditions, all the examined thymidine analogues exhibit similar labeling kinetics and clearance rates from the blood serum. Our results indicate that all thymidine analogues delivered at the indicated doses have similar bioavailability times (approximately 1 h). Our findings are significant for the practical use of multiple S-phase labeling with any combinations of BrdU, IdU, CldU, and EdU and for obtaining valid labeling readouts.

Recolonization of Mutans Streptococci after Application of Chlorhexidine Gel

Streptococcus mutans is specifically suppressed by intensive treatment with chlorhexidine gel, but the time for recolonization and the effect on other oral bacteria are not totally clear. In this study, recolonization of mutans streptococci was evaluated in nine healthy adult volunteers, who were highly colonized with this microorganism. Stimulated saliva was collected before (baseline) and at 1, 7, 14, 21 and 28 days after application of 1% chlorhexidine gel on volunteers' teeth for two consecutive days. On each day, the gel was applied using disposable trays for 3 x 5 min with intervals of 5 min between each application. Saliva was plated on blood agar to determine total microorganisms (TM); on mitis salivarius agar to determine total streptococci (TS) and on mitis salivarius agar plus bacitracin to determine mutans streptococci (MS). Chlorhexidine was capable of reducing the counts of MS and the proportion of MS with regard to total microorganisms (%MS/TM) (p<0.05), but these values did not differ statistically from baseline (p>0.05) after 14 days for MS and 21 days for %MS/TM. The counts of TM and TS and the proportion of MS to total streptococci did not differ statistically from baseline (p>0.05) after chlorhexidine treatment. The results suggest that the effect of chlorhexidine gel treatment on suppression of mutans streptococci is limited to less than a month in highly colonized individuals.

Streptococcus mutans é especificamente suprimido pelo tratamento intensivo com clorexidina em gel, mas o tempo de recolonização e o efeito em outras bactérias orais não está totalmente claro. Nesse estudo, a recolonização de estreptococos do grupo mutans foi avaliado em nove voluntários adultos saudáveis, os quais eram altamente colonizados por esse microrganismo. Saliva estimulada foi coletada antes (baseline) e 1, 7, 14, 21 e 28 dias após a aplicação de clorexidina em gel a 1% nos dentes dos voluntários por dois dias consecutivos. Em cada dia, o gel foi aplicado utilizando moldeiras descartáveis por 3 x 5 min com intervalos de 5 min entre cada aplicação. A saliva foi inoculada em ágar sangue para determinação dos microrganismos totais (MT); em mitis salivarius ágar para determinação dos estreptococos totais (ET) e em meio mitis salivarius com bacitracina para determinar a contagem de estreptococos do grupo mutans (EGM). O tratamento com clorexidina foi capaz de reduzir as contagens de EGM e a proporção de EGM em relação aos microrganismos totais (%EGM/MT) (p<0,05), mas esses valores não diferiram estatisticamente do baseline (p>0,05) após 14 dias para EGM e 21 dias para %EGM/MT. As contagens de MT e ET e a proporção de EGM em relação a estreptococos totais não difereriram estatisticamente do baseline (p>0,05) após o tratamento com clorexidina. Os resultados sugerem que o efeito do tratamento com clorexidina em gel na supressão de estreptococos do grupo mutans é limitado a menos de um mês em indivíduos altamente colonizados. .

Flow cytometric method for estimation of 5-bromo-2´-deoxyuridine content in rat serum.

Labelling of DNA in replicating cells using 5-bromo-2´-deoxyuridine (BrdU) is widely used, however the rapid clearance and metabolisation of BrdU in the living organism is a critical issue. Although the pharmacokinetic of BrdU in experimental animals is empirically approximated, the exact time-curve remains unknown. Here we present novel method for estimation of the BrdU content in the blood serum. The application is based on the in vitro cocultivation of tumour cells with the examined serum and the subsequent quantification of the incorporated BrdU in the DNA using flow cytometry analysis. Our results demonstrate that this approach can quantify the BrdU concentration in serum at 1 micromol.dm(-3) and might represent an attractive alternative to conventional chromatographic analysis. The employment of tumour cells as "detectors" of the BrdU content in serum provides an advantage over high pressure liquid chromatography (HPLC), as this approach allows us to approximate not only the concentration of BrdU, but also to determine, whether BrdU is present in the blood serum in effective concentration to reliable label all cells undergoing the S-phase of the cell cycle. The presented application might be a helpful tool for studies on pharmacokinetics of BrdU or other thymidine analogues when testing various administration routes or protocols.

Flow cytometric determination of 5-bromo-2'-deoxyuridine pharmacokinetics in blood serum after intraperitoneal administration to rats and mice.

5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

A new method for in vitro detection of bromodeoxyuridine in serum: a proof of concept in a songbird species, the canary.

Systemic injection of a thymidine analogue such as bromodeoxyuridine (BrdU) in vertebrates is commonly used to detect and study cell production during development, adulthood, and pathology, particularly in studies of adult neurogenesis. Although researchers are applying this technique to multiple species in various physiological conditions, the rate of BrdU clearance from the serum remains unknown in most cases. Changes in this clearance rate as a function of the species, sex or endocrine condition could however profoundly affect the interpretation of the results. We describe a rapid, sensitive, but simple bioassay for post-injection detection and quantification of BrdU in serum. This procedure was shown to be suitable for determining the length of time a thymidine analogue remains in the bloodstream of one avian species and seems applicable to any vertebrate provided sufficiently large blood samples can be collected. This technique was used to demonstrate that, in canaries, BrdU injected at a dose of 100 mg/kg is no longer available for incorporation into DNA between 30 and 60 min post-injection, a delay shorter than anticipated based on the available literature. Preliminary data suggest a similar fast clearance in Japanese quail and mice.

Rapid development of Th2 activity during T cell priming.

The paradigm of T helper-1 (Th-1) and Th-2 cells developing from non-committed naive precursors is firmly established. Th1 cells are characterized by IFNgamma production and, in mice, the selective switching to IgG2a. Conversely IL-4 production and selective switching to IgG1 and IgE characterize Th2 cells. Analysis of Th2 induction in vitro indicates that this polarization develops gradually in T cells activated by anti-CD3 in the presence of IL-4; conversely anti-CD3 and IFNgamma induce Th1 cells. In this report, we explore evidence that indicates that the T helper cell polarization in vivo cannot solely be explained by the cytokine environment. This is provided by studying the early acquisition of Th1 and Th2 activities during responses to a mixture of Th1 and Th2-inducing antigens. It is shown that these divergent forms of T cell help can rapidly develop in cells within a single lymph node. It is argued that early polarization to show Th-1 or Th-2 behavior can be induced by signals delivered during cognate interaction between virgin T cells and dendritic cells, in the absence of type 1 or type 2 cytokines. This contrasts with the critical role of the cytokines in reinforcing the Th-phenotype and selectively expanding T helper clones.

[76Br]Bromodeoxyuridine PET in tumor-bearing animals.

5-bromodeoxyuridine (BUdR) provides in vitro measures of tumor cell proliferation. We used positron emission tomography to study tissue and plasma kinetics of [76Br]BUdR in tumor-bearing animals. In order to account for the slow washout of the major plasma metabolite, [76Br]bromide, a mathematical correction for the distribution volume of [76Br]bromide was applied. However, following correction specific tumor tracer retention was low or even zero and did not correlate with independent measures of proliferation. The kinetic characteristics of [76Br]BUdR make this tracer unsuitable for proliferation imaging.

Analysis of 76Br-BrdU in DNA of brain tumors after a PET study does not support its use as a proliferation marker.

76Br-bromodeoxyuridine has previously been suggested as a PET tracer to characterize proliferation potential. However, in animal studies a large fraction of the tissue radioactivity is due to 76Br-bromide, which remains extracellular for extensive periods and contributes significantly to the level of radioactivity. The present project aimed at investigating whether in human brain tumors, sufficient amounts of 76Br-bromodeoxyuridine would be incorporated into DNA, to motivate further attempts with this tracer. Eight patients with brain tumors: 3 meningiomas, 2 astrocytoma grade IV, 1 astrocytoma oligodendroglioma grade II-IV and 2 metastases, were examined with PET and 76Br-BrdU on three occasions: immediately after injection of the tracer, at 4-6, and at 18-20 hours after administration. After the first PET study, diuresis was introduced and maintained for about 12 hours. About 20 hours after tracer administration, 200 mg/m(2) bromodeoxyuridine was administered to 7 patients median 5.8 (range 1-22) hours prior to operation allowing the immunohistochemical analysis of the proliferation potential. During the operation, tumor samples were taken and radioactivity in DNA extracted and measured. The uptake of radioactivity was higher in the tumors than in brain parenchyma. However, in the operative samples only 1-27% (average: 9%) of the radioactivity was found in the DNA fraction. The plasma radioactivity remained high throughout the study with only minimal signs of elimination by the diuresis. 76Br-BrdU is extensively metabolized to 76Br-bromide, and only a minor fraction of the radioactivity is found in the DNA fraction, making it unlikely that this tracer can be used for assessment of proliferation potential.

Quantification of proliferating lymphocyte subsets appearing in the intestinal lymph and the blood.

Lymphocyte emigration from the intestinal wall via lymphatics is necessary to maintain gastrointestinal immunity and also to connect the different parts of the mucosal immune system. In the present study the numbers and time kinetics of proliferating lymphocyte subsets leaving the gut wall via intestinal lymphatics were analysed in mesenteric lymph node adenectomized minipigs (n = 8). After cannulation of the major intestinal lymph duct, afferent lymph was collected under non-restraining conditions. In four pigs lymphocytes taken from the intestinal lymph and blood were incubated in vitro with the thymidine analogue bromodesoxyuridine (BrdU) to label all lymphocytes in the S-phase of the cell cycle. The other four pigs received a single i.v. injection of BrdU 1 week after cannulation. The initial percentage of BrdU+ lymphocyte subsets in the intestinal lymph 15 min after BrdU injection was comparable to that after the in vitro labelling (1.5 +/- 0.7% in T cells, 10.6 +/- 1.6% in IgM+ cells and 30.0 +/- 11.9% in IgA+ cells). From this level onwards, the percentage of in vivo labelled BrdU+ lymphocyte subsets reached a maximum at 12 h after BrdU application. A different pattern of BrdU+ subsets was seen in the blood. After an early peak at around 3-4 h, the frequency of BrdU in vivo labelled cells decreased. Each subset had a maximum between 12 h and 48 h after BrdU application (maximum of BrdU+ CD2+ T cells at 12 h, 4.6 +/- 1.5%; IgM+ BrdU+ at 48 h, 8.8 +/- 3.3%). The present results provide a basis to determine the time necessary for induction of specific intestinal immunity during oral vaccination studies.

Determination of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma and urine by capillary electrophoresis.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.

Development and validation of a bioanalytical assay for (E)-5-(2-bromovinyl)-2'deoxyuridine in plasma by capillary zone electrophoresis.

A capillary zone electrophoretic method for the quantification of (E)-5-(2-bromovinyl)-2'-deoxyuridine in plasma has been developed and validated. Separation was performed with a 25 mmol/l borate buffer, pH 9.0, after an initial rinsing step with sodium hydroxide. The rinsing step was necessary for reproducible analyses of aqueous samples and plasma extracts obtained by C18 solid-phase extraction after deproteination with perchloric acid. No interferences with plasma compounds were observed. The calibration graph was linear over the range of 30 to 3000 ng/ml using 5-fluorouracil as external standard. The limit of quantification was 24 ng/ml. The CZE method is fast, reproducible, linear and is therefore a good alternative for the already established HPLC methods.

Mutual kinetic interaction between 5-fluorouracil and bromodeoxyuridine or iododeoxyuridine in dogs.

The pharmacokinetics of 5-fluorouracil (FUra) were studied alone and in the presence of either bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in five mixed breed hounds. FUra was administered intravenously over 1 min as a 5 mg/kg dose in all three treatments. BrdUrd or IdUrd was infused intravenously at 2.5 mg/hr.kg x 6 hr and FUra was given 3 hr into the 6-hr infusion of thymidine analog. Serial blood samples were obtained for 3 hr prior to and after FUra dosing. Plasma concentrations of FUra, BrdUrd and bromouracil (BrUra), and IdUrd and iodouracil (IUra) were measured by reversed-phase HPLC. Concomitant administration of BrdUrd or IdUrd resulted in substantial changes in the kinetics of FUra. In particular, the total plasma clearance of FUra was decreased and area under the plasma concentration-time curve was increased. There were also significant changes in the volume of distribution steady-state, and in the mean residence time and half-life of FUra. The data show that FUra had little or no effect on the disposition of BrdUrd or IdUrd. In contrast, FUra had a significant effect on the disposition of BrUra and IUra going to form catabolic products. The observed pharmacokinetic interaction in dogs is probably due to a competition between BrUra (or IUra) and FUra for the rate-limiting catabolic enzyme, dihydropyrimidine dehydrogenase. This kinetic interaction was mutual and of a smaller magnitude for FUra + BrdUrd than for FUra + IdUrd.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels.

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.

High-performance liquid chromatographic determination of 5-bromodeoxyuridine in human plasma.

A new method is described for the determination of submicromolar concentrations of 5-bromodeoxyuridine in human plasma. Sample pretreatment involves cold methanol deproteinization, freezing-thawing and lyophilization. The sample is then analysed by reversed-phase high-performance liquid chromatography. This method is very reproducible and has a detection limit of 0.1 microgram/ml (0.32.10(-6) M). Comparison with other procedures indicates that the method is advantageous as regards sensitivity and specificity and can be readily applied in clinical pharmacological investigations.

Steady-state arterial and hepatic venous plasma concentrations of 5-bromo-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in animals--drugs which are subject to both splanchnic and extra-splanchnic elimination.

We have previously shown that 5-fluorouracil (FUra) in humans obeys Michaelis-Menten elimination kinetics. In this article we show that the related bromine and iodine-containing analogs in animals obey similar kinetics. Steady-state arterial (CssA) and hepatic venous plasma (CssV) concentrations of 5-bromo-2'-deoxyuridine (BrdUrd) are reported for 7 rabbits given 5 different infusion rates of BrdUrd and 5 dogs given 4 or 5 different infusion rates of BrdUrd. Steady-state arterial and hepatic venous plasma concentrations of 5-iodo-2'-deoxyuridine (IdUrd) are reported for 5 rabbits and 2 dogs given 5 different infusion rates of IdUrd. Each set of data could be fitted by a nonlinear least squares method to the equation: (equation; see text) where Vm/Q is the maximum difference, (CssA) - (CssV), and Km is the Michaelis constant. The estimated parameter Vm/Q and Km are compared for the two drugs and different species and also with the same parameters derived in the same manner from previously published data on fluorouracil in 8 cancer patients. The infusion rate needed to saturate the splanchnic elimination system (Rs in mumol/kg/min) was also estimated. For BrdUrd the mean value of Rs in the rabbit, namely 1.23, and in the dog, namely 1.25 mumol/kg/min are essentially the same.

Improved high-performance liquid chromatographic assay for the quantification of 5-bromo-2'-deoxyuridine and 5-bromouracil in plasma.

A sensitive and specific procedure using high-performance liquid chromatography (HPLC) was developed for the quantification of 5-bromo-2'-deoxyuridine (BUdR) and 5-bromouracil (BU) in plasma. BUdR and BU were first extracted with a mixture of ethyl acetate and 2-propanol from plasma presaturated with solid ammonium sulfate. Following evaporation of the organic extract, the remaining residue was reconstituted in saturated ammonium sulfate solution, washed with a mixture of n-pentane-methylene chloride and re-extracted with the original solvent mixture. The organic extract was evaporated, reconstituted in mobile phase and chromatographed on a regular-bore ODS HPLC column using ultraviolet absorbance detection. The BUdR and BU quantification limits were both 0.1 microM, the mean intra-assay coefficients of variation were 5.0 and 5.6%, respectively, and the mean inter-assay coefficients of variation were 5.4 and 10.7%, respectively. This method was used to determine steady-state femoral arterial and hepatic venous plasma concentrations of BUdR and BU in a patient receiving a continuous intravenous infusion of BUdR (20 mg/kg per day).

Intra-arterial bromodeoxyuridine radiosensitization and radiation in treatment of malignant astrocytomas.

Bromodeoxyuridine (BUdR), a nonhypoxic radiosensitizing drug, is a halogenated pyrimidine analog that is incorporated into the deoxyribonucleic acid of dividing cells in a competitive process with thymidine; BUdR also sensitizes these cells to radiation therapy. Neurons and glial cells have a very low mitotic rate. They will not incorporate BUdR and will not be sensitized. Bromodeoxyuridine is best delivered intra-arterially because of its regional advantage, calculated to be between 6 and 16. An 8-week BUdR infusion is delivered before and during radiation therapy through a permanently implanted pump with a catheter placed retrograde into the external carotid artery. Eighteen patients with malignant glioma (15 grade IV, and three grade III) were entered into a Phase I dose-escalation protocol with BUdR dosages ranging from 400 to 600 mg/sq m/day. The maximum dose that can be tolerated appears to be 400 mg/sq m/day for 8 weeks. The 18 patients entered in this study have a median Kaplan-Meier estimated survival time (+/- standard error of the mean) of 22 +/- 5 months with 11 patients still alive. Three patients are alive at 30, 29, and 21 months after diagnosis with no evidence of tumor on computerized tomography. There have been no vascular complications. Side effects in all patients have included anorexia, fatigue, ipsilateral forehead dermatitis, blepharitis, iritis, and nail ridging. Myelosuppression requiring dose reduction occurred in one patient. One patient had a Stevens-Johnson syndrome requiring termination of BUdR. It is concluded that intra-arterial BUdR may improve survival times in patients with malignant gliomas.

Bioanalysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine is an antiviral drug that is experimentally used for modulation of the antitumour effect of fluoropyrimidines, such as ftorafur and 5-fluorouracil. The isolation of the analyte, in the presence of 5-fluorouracil, from the matrix is performed either by means of a simple protein precipitation (plasma) or by means of a liquid-liquid extraction with ethyl acetate (urine). Following pretreatment, the analyte is analysed by reversed-phase chromatography and quantified by absorbance detection at 307 nm. The minimum detectable concentration in plasma and urine samples is ca. 6 ng/ml. The recovery after deproteination of plasma samples is 75%, while after liquid-liquid extraction of urine the recovery amounts 92%. The degree of protein binding of the analyte, measured by ultrafiltration, is found to be 97%. These data allow the bioanalysis of (E)-5-(2-bromovinyl)-2'-deoxyuridine for pharmacokinetic studies.

Serum and urine concentrations of oral bromovinyldeoxyuridine in humans as monitored by a bioassay system based on varicella-zoster virus focus inhibition.

A simple and sensitive bioassay method for measuring (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) concentrations in human serum and urine has been established. This method is based on the inhibitory effect of BVDU on varicella-zoster virus (VZV) focus formation in vitro. The minimal concentration of BVDU that could be detected in serum by this method was 0.2 microgram/ml. Following a single oral administration of 250 mg BVDU, serum BVDU concentrations of 1.2-2.2 micrograms/ml were attained 1 hr later; at 5 and 7 hr, serum BVDU levels were below 0.2 microgram/ml. Upon repeated administration of 125 mg BVDU at 8 hr intervals, the serum BVDU concentrations reached 0.7-1.1 microgram/ml at 2 hr after the fourth administration. These concentrations are approximately 300-450-fold higher than the 50% inhibitory dose of BVDU for VZV in vitro. Urinary BVDU concentrations were on average 10 to 20 times higher than the serum BVDU concentrations.

Immunohistochemical detection of proliferating cells in vivo.

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.