Microscopic properties of forces from ice solidification interface acting on silica surfaces based on molecular dynamics simulations.

The origin of the forces acting on a silica surface from an ice solidification interface was investigated to understand the solidification phenomenon and its impact on nanometer-scale structures using molecular dynamics simulations. The microscopic forces were determined by appropriately averaging the forces acting on the silica wall from the water molecules in time and space; the time evolutions of these microscopic forces during the solidification processes were investigated for three types of silica surfaces. The results indicate that the microscopic forces fluctuate more after the solidification interface makes contact with the wall surface. To visualize the changes in the microscopic forces and hydrogen bonds due to solidification, their differences compared to the liquid state were calculated. When the solidification interface is near the wall, the changes in these microscopic forces and hydrogen bonds due to solidification are correlated. This tendency is more significant for an amorphous wall and a wall with a structure than for a crystalline wall. The changes in the microscopic force depend on the water molecules that behave as acceptors when forming the hydrogen bonds with the wall and on the configuration of the silanol groups on the silica surfaces.

Thermal transport mechanism at a solid-liquid interface based on the heat flux detected at a subatomic spatial resolution.

Heat flux is a fundamental quantity in thermal science and engineering and is essential for understanding thermal transport phenomena. In this study, the heat flux in a solid-liquid interfacial region is obtained in a three-dimensional (3D) space at a subatomic spatial resolution based on classical molecular dynamics, yielding a 3D structure of the heat flux between the solid and liquid layers in contact. The results using the Lennard-Jones potential reveal the directional qualities of the heat flux, which are significantly dependent on the subatomic stresses in the structures of condensed phase systems. The heat flux and stress at the subatomic scale are related to the macroscopic transport quantities, which can be obtained using distribution functions; the stress and energy flux properties at the subatomic scale are comprehensively investigated using a single-interaction-based stress and energy flux to determine the origin of the thermal transport mechanism at the solid-liquid interface. The findings reveal that the density of states due to the stress caused by a single interaction exhibits a bandlike behavior. The net energy transport comprises positive and negative energy transport inside and outside the band. In addition, this is related to the intrinsic transport property of the atoms and molecules at the solid-liquid interface at the subatomic scale. The difference between the energy transport rates when a solid atom in the vicinity of the interface is near to or far from the liquid phase is the origin of the energy transport mechanism at the solid-liquid interface. 3D analysis of the heat flux and stress is carried out by varying the interaction strengths between the liquid molecules and solid atoms at the solid-liquid interface. This reveals that the directional quality of transport quantities is high at strong interaction strengths, thus indicating enhanced thermal transport. Furthermore, the influence of the temperature gradient in the system suggests that the energy transport imbalance between inside and outside the stress band in a high-stress field at the subatomic scale induces the net thermal transport across the interface in the nonequilibrium state.

Structure of the Water Molecule Layer between Ice and Amorphous/Crystalline Surfaces Based on Molecular Dynamics Simulations.

The structure of the water layer between the ice interface and the hydroxylated amorphous/crystalline silica surfaces was investigated using molecular dynamics simulations. The results indicate that the density profile in the direction perpendicular to the surface has two density peaks in the water layer at the ice-silica interface, which are affected by the silanol group density on the wall and the degree of supercooling in the system. In the two density peaks, the one facing the ice interface side has the same structure as the ice crystal, while the other density peak facing the silica surface has an icelike structure. In the solidification process, the ice and icelike structures in the layer progress more on the amorphous silica surface where the density of the silanol groups is low. The relationship between the ice crystallization and the thickness of the layer has been studied in detail; the lower the temperature, the more the ice crystallization progresses and the thinner the layer becomes.

Metabolism and disposition of oseltamivir (OS) in rats, determined by immunohistochemistry with monospecific antibody for OS or its active metabolite oseltamivir carboxylate (OC): A possibility of transporters dividing the drugs' excretion into the bile and kidney.

Among any drugs, no comparative pharmacological study on how prodrug and its active metabolite behave in animal bodies is available. Immunohistochemistry (IHCs) using newly prepared two monoclonal antibodies, AOS-96 and AOC-160, monospecific for oseltamivir (OS) and its metabolite oseltamivir carboxylate (OC) were developed, simultaneously detecting the uptake or excretion of OS and OC in the intestine, liver, and kidney of rats to which OS was orally administered. In the intestine, IHC for OS revealed OS highly distributed to the absorptive epithelia with heavily stained cytoplasmic small granules (CSGs). IHC for OC showed that OC also distributed highly in the epithelia, but without CSGs, suggesting that OS was partly converted to OC in the cells. In the liver, OS distributed in the hepatocytes and on their bile capillaries, as well as on the lumina from the bile capillaries to the interlobular bile ducts. OC distributed in the whole cell of the hepatocytes, but without CSGs nor on any lumina through the interlobular bile ducts. In the kidney, a few levels of OS distributed in the cytoplasm of almost all the renal tubule cells, but they contained numerous CSGs. In contrast, OC distributed highly in the proximal tubules, but very slightly in the lower renal tubules of the nephrons. Thus, it was concluded that the two drugs behave in completely different ways in rat bodies. This paper also discusses a possibility of the correlation of OS or OC levels in tissue cells with their known transporters.

Atomic-scale thermal manipulation with adsorbed atoms on a solid surface at a liquid-solid interface.

Modulating thermal transport through interfaces is one of the central issues in nanoscience and nanotechnology. This study examined thermal transport between atoms adsorbed on a solid surface and a liquid phase based on non-equilibrium molecular dynamics. The heat flux was detected at sub-atomic spatial resolution, yielding a two-dimensional map of local heat flux in the vicinity of the adsorbed atoms on the surface. Based on the detected heat flux, the possibility of atomic-scale thermal manipulation with the adsorbed atoms was examined by varying the interaction strengths between the liquid molecules and atoms adsorbed on the surface. The results of the local heat flux at the single-atom scale clearly showed effects of the adsorbed atoms on the thermal transport through the liquid-solid interface; they can significantly enhance the heat flux at the single-atom scale using degrees of freedom normal to the macroscopic temperature gradient. The effect was especially evident for a low wettability surface, which provides key information on local enhancement at the single-atom scale of the thermal transport through a liquid-solid interface.

Expression and distribution patterns of spermine, spermidine, and putrescine in rat hair follicle.

No expression and distribution patterns of polyamines (PAs), spermine, spermidine, and their precursor putrescine in mammalian hair follicle are available, although polyamines are known to correlate well with hair growth and epidermal tumor genesis. Immunohistochemistry (IHC) using our original two monoclonal antibodies (mAbs) ASPM-29 specific for spermine or spermidine, and APUT-32 specific for putrescine allowed us to detect immunoreactivity for polyamines in hair follicles from normal adult rats. A wide range of immunoreactivity for the total spermine and spermidine was observed in the compartments of hair follicle: The highest degree of immunoreactivity for polyamines was observed in the matrix, in the Huxley's layer, in the deeper Henle's layer, and in the cuticle of the inner root sheath/the hair cuticle, while moderate immunoreactivity existed in the lower-to-mid cortex and the companion layer, followed by lower immunoreactivity in the outer root sheath, including the bulge region and in the deeper medulla, in which the immunoreactivity was also evident in their nuclei. In addition, somewhat surprisingly, with IHC by APUT-32 mAb, we detected significant levels of putrescine in the compartments, in which the immunostaining pattern was the closely similar to that of the total spermine and spermidine. Thus, among these compartments, the cell types of the matrix, the Huxley's layer, the deeper Henle's layer, and the cuticle of the inner root sheath/the hair cuticle seem to have the biologically higher potential in compartments of anagen hair follicle, maybe suggesting that they are involved more critically in the biological event of hair growth. In addition, we noted sharp differences of immunostaining by IHCs between ASPM-29 mAb and APUT-32 mAb in the epidermis cells and fibroblast. ASPM-29 mAb resulted in strong staining in both the cell types, but APUT-32 mAb showed only very light staining in both types. Consequently, the use of the two IHCs could be extremely useful in further studies on hair cycle and epidermal tumor genesis experimentally or clinically.

High-resolution separation of neodymium and dysprosium ions utilizing extractant-impregnated graft-type particles.

An efficient method for rare metal recovery from environmental water and urban mines is in high demand. Toward rapid and high-resolution rare metal ion separation, a novel bis(2-ethylhexyl) phosphate (HDEHP)-impregnated graft-type particle as a filler for a chromatography column is proposed. To achieve rapid and high-resolution separation, a convection-flow-aided elution mode is required. The combination of 35 µm non-porous particles and a polymer-brush-rich particle structure minimizes the distance from metal ion binding sites to the convection flow in the column, resulting in minimized diffusional mass transfer resistance and the convection-flow-aided elution mode. The HDEHP-impregnated graft-type non-porous-particle-packed cartridge developed in this study exhibited a higher separation performance for model rare metals, neodymium (III) and dysprosium (III) ions, and a narrower peak at a higher linear velocity, than those of previous HDEHP-impregnated fiber-packed and commercially available Lewatit® VP OC 1026-packed cartridges.

A simple in situ preconcentration method for the simultaneous determination of major and minor alkali and alkaline-earth metals in natural waters using iminodiacetate-grafted fiber.

A simple and effective procedure was developed for the simultaneous in situ preconcentration of major and minor alkali and alkaline-earth metals (Li, Na, K, Rb, Mg, Ca, Sr, and Ba) from natural waters with iminodiacetate-grafted fiber (IDA-fiber), using soil water extracts as an example. The method was verified on mixed standard solutions of the metals at concentrations and ionic strengths reported for regular soil solutions. When complemented with routine microfiltration procedures, the method could distinguish between the dissolved in hydrated ion form and bound fractions of these metals, presumably colloidal and firmly complexed. The method was tested on water extracts from heavy clayey Vertisol and light red sandy-loam soils. The results showed that most of the Rb, Mg, Ca, Sr, and Ba of the 0.45-µm filtrate were bound to unreactive water-mobile compounds, whereas K and Na were determined predominantly in dissolved/reactive forms. The preliminary charged IDA-fiber can be used to preconcentrate the metals in situ from soil water and other types of natural waters with subsequent determination in a stationary laboratory, thus avoiding the critical step of storage and transportation of the unstable water samples. Graphical abstract A bundle of preliminary charged iminodiacetate-grafted fiber placed in a water-sample during in-field preconcentration procedure.

A Molecular Dynamics Study on Wetting Phenomena at a Solid Surface with a Nanometer-Scale Slit Pore.

Non-equilibrium molecular dynamics simulations are conducted for the liquid wetting phenomena on a solid surface with a nanometer-scale slit pore. All interactions between molecules or atoms are assumed to be 12-6 Lennard-Jones (LJ) potential in order to examine the fundamental mechanisms of the wetting phenomena qualitatively. The Lorentz-Berthelot combining rule is applied to obtain the standard parameters between fluid molecules and solid atoms, which are controlled by using relative parameters to change the interaction intensity. The energy of fluid molecules in the vicinity of the entrance of the slit pore is investigated in detail so as to elucidate the mechanism of the liquid wetting phenomena from a molecular energy point of view. The results show that the total energy per unit volume of fluid molecules in the vicinity of the solid surface inside the slit pore becomes lower than that of the bulk part of the liquid membrane which exists outside the slit pore when the wetting phenomena occur.

Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle.

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27(Kip1) was greatly increased, so its mechanism was studied in detail. Synthesis of p27(Kip1) was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5'-untranslated region (5'-UTR) in p27(Kip1) mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.

Immunocytochemistry for vancomycin using a monoclonal antibody that reveals accumulation of the drug in rat kidney and liver.

We prepared monoclonal antibodies against N-(γ-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

An in vivo role of Mrp2 in the rat hepatocytes by immunocytochemistry for amoxicillin using the transporter-deficient EHBR.

An in vivo role of the multidrug resistant-associated protein (Mrp2) in rat hepatocytes was examined by immunocytochemistry (ICC) for amoxicillin (AMPC) by the use of the transporter-deficient Eisai hyperbilirubinemic rats (EHBR). The ICC revealed that in the liver of EHBR at 3-h post-administration, amoxicillin accumulated in the cytoplasmic pools and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. However, no amoxicillin was observed on the surface of the lumina ranging from the bile capillaries to the interlobular bile ducts. The drug persisted at least for 6-h after administration. In contrast, in the control rat liver at 3-h post-administration, AMPC-adsorption occurred on such luminal surface, while AMPC accumulated to a less level in both the cytoplasm and nuclei of the hepatocytes. The drug completely disappeared in the hepatocytes at 6-h post-administration. These results strongly suggest that AMPC taken up into the cytoplasm of the hepatocytes excretes via Mrp2 into the bile flow. Furthermore, electron microscopy demonstrated that the lower electron density areas in large sizes, corresponding to the cytoplasmic pools in ICC for AMPC, occurred in the cytoplasm peripheral to the nuclei of the hepatocytes in EHBR at 3-h post-administration, and then disappeared 24 h after administration.

Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney.

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with ß-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.

Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease.

The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ( ∆F/∆F)) or knock-out for the Cl(-)/H(+) exchanger ClC-5 (Clcn5 ( Y/- )). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the drug.

A probable relationship between characteristic accumulation of doxorubicin and P-glycoprotein transporter in rat liver.

Correlations between the distribution of anthracycline antibiotics doxorubicin (DX) and daunorubicin (DR) in the liver of rats injected with a single i.v. injection of each drug and the reported distribution of P-glycoprotein transporter for the drug was histochemically examined. Immunocytochemical studies for DX or DR using monoclonal antibody that equally detects both drugs, as well as conventional electron microscopy were employed. DX persisted for more than 5 days in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries. Meanwhile, DR distributed in almost the same pattern, but more rapidly decreased to a level that almost no granular morphology in the hepatocytes was visible 24 h after the injection. Also, unknown large cells that strongly reacted with the antibody appeared in the hepatic sinusoids near the interlobular triads rather than the central vein. The accumulation sites of DX or DR on the bile capillaries seems to correspond to specific sites where the ATP-binding cassette transport protein P-glycoprotein for the anthracycline occurs, suggesting a possibility that DX or DR is actually and actively excreted at these sites, possibly through P-glycoprotein. This is supported by conventional electron microscopic studies, since no specific subcellular organelles such as lysosomes assemble in the vicinity of the bile capillaries of the hepatocytes.

[Distribution and accumulation of antibiotics in cells and tissues and toxicity studies by immunocytochemistry].

No true immunocytochemistry (ICC) for drugs nor its application to pharmacokinetic studies is available. Recently, our studies have shown that ICC for drugs is extremely useful for such studies by utilizing easy and safe techniques, and gives direct evidence of drug localization. We have therefore developed antibodies and a series of pretreatment conditions for the immunodetection of drugs and have localized sites of drug uptake or accumulation in several tissues of rats following the administration of drugs. This review describes preparation of anti-drug antibody, specificity of antibody, fixation of drug in situ in rat tissues and cells, treatment of paraffin section specimens prior to immunoreaction, precision, and their application to a variety of types of antibiotics anti-cancer anthracyclines daunorubicin, doxorubicin, and epirubicin, bleomycin analog peplomycin, antimicrobial agents gentamicin, and amoxicillin. ICC for the anti-cancer anthracyclines demonstrated that the drug accumulates in a characteristic pattern in the heart, liver, kidney, gastrointestinal tract, and hair follicles, which represent the sites targeted by the drug toxicity. Some, but not all, of these drug accumulations are associated with the induction of apoptosis. It was also noted that there are striking differences in accumulation among the anthracyclines in rat tissues, maybe contributing the mechanisms of the differences in anti-tumor activities of the anthracyclines. Both ICCs for gentamicin and peplomycin identified characteristic necrotic-like cells in the specific sites of the kidney, suggesting the sites are readily affected by some chemotherapeutic agents. ICC for amoxicillin demonstrated that the sites of the drug accumulation in small intestine, liver and kidney are closely correlated with the specific sites in which certain transporter systems for penicillin occur. Thus, an ICC method is a potential new tool for pharmacokinetic studies of wide variety types of drugs containing a primary amino group(s) in their molecules.

Distribution study of peplomycin in rat kidney revealed by immunocytochemistry using monoclonal antibodies.

Peplomycin (PEP), an anti-tumor antibiotic related structurally to bleomycin, is widely used, especially for squamous cell carcinoma but shows renal toxicity. We prepared monoclonal antibodies (mAbs) against N-(γ-maleimidobutyryloxy)succinimide-conjugated PEP. The mAbs were monospecific for PEP, but did not react with bleomycin and other anticancer antibiotics. The mAbs enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of PEP in the rat kidney. Two hours after a single i.v. administration of PEP, ICC revealed immunostaining for PEP in irregularly shaped cytoplasmic granules of the proximal tubules in which the microvilli were also stained. Also, staining occurred in the distal tubules and collecting ducts, in both of which we observed scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and cytoplasm reacted strongly with the antibody. Twenty-four hours after injection, PEP in the proximal tubules completely vanished, but yet significant amounts of PEP remained in both the distal tubules and collecting ducts. Distribution patterns of PEP in cells of the kidneys resembled, in some ways, those of our recent ICC studies for an organic cation aminoglycoside antibiotic gentamicin. This ICC suggests that PEP taken up in the proximal tubule cells is localized in the lysosomes, and organic cation transporters and bleomycin hydrolase might be involved in entrance and/or disappearance of PEP in this cell type. Furthermore, the distal tubules and collecting ducts may be the sites readily affected by some chemotherapeutic agents.

Immunocytochemistry for amoxicillin and its use for studying uptake of the drug in the intestine, liver, and kidney of rats.

Specific transport systems for penicillins have been recognized, but their in vivo role in the context of other transporters remains unclear. We produced a serum against amoxicillin (anti-AMPC) conjugated to albumin with glutaraldehyde. The antiserum was specific for AMPC and ampicillin (ABPC) but cross-reacted weakly with cephalexin. This enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of AMPC in the rat intestine, liver, and kidney. Three hours after a single oral administration of AMPC, the ICC method revealed that AMPC distributed to a high degree in the microvilli, nuclei, and cytoplasm of the absorptive epithelial cells of the intestine. AMPC distributed in the cytoplasm and nuclei of the hepatocytes in a characteristic granular morphology on the bile capillaries, and in addition, AMPC adsorption was observed on the luminal surface of the capillaries, intercalated portions, and interlobular bile ducts on the bile flow. Almost no AMPC could be detected 6 h postadministration in either the intestine or the liver. Meanwhile, in the kidney, AMPC persisted until 12 h postadministration to a high degree in the proximal tubules, especially in the S3 segment cells in the tubular lumen, in which numerous small bodies that strongly reacted with the antibody were observed. All these sites of AMPC accumulation correspond well to specific sites where certain transporter systems for penicillins occur, suggesting that AMPC is actually and actively absorbed, eliminated, or excreted at these sites, possibly through such certain penicillin transporters.

Differences in accumulation of anthracyclines daunorubicin, doxorubicin and epirubicin in rat tissues revealed by immunocytochemistry.

Although anthracycline antibiotics daunorubicin (DR), doxorubicin (DX), and epirubicin (ER) possess minor differences in their chemical structures, large differences are noted in their clinical use, as well as in cellular and plasma pharmacokinetic parameters in vivo. Immunocytochemistry for DR, DX, or ER was developed using an anti-DR monoclonal antibody (ADM-1-11), which has been demonstrated to react equally well with each of the three drugs, and therefore it was used for comparing their accumulation in several rat tissue cells after a single i.v. injection of each drug. In the kidney, immunoreactivity for each drug was distributed in essentially the same pattern and in the same strength 2 h after injection, but quite differently distributed in kidney cells thereafter, so that at 120-h post-injection significant amounts of DX and ER remained, but DR had almost completely vanished. Similar patterns of accumulation were observed in cells of other tissues including the pancreas, hair follicle, and stomach, with the exception of the intestine in which none of the three drugs remained after 120 h. These results appear to be supported by previous pharmacokinetic studies on the anthracyclines. The mechanism for such differences among the three drugs remains obscure, but the hydroxyl group at C-14 of DX and ER molecule might be related to the strong propensity of DX and ER to accumulate in tissue cells. The present results should contribute to the understanding of the mechanisms of the differences in the pharmacokinetics, as well as the possibly in anti-tumor activities of the anthracyclines.

Daunomycin accumulation and induction of programmed cell death in rat hair follicles.

The anthracycline antibiotic daunomycin (DM) is useful for the treatment of leukemia but has side-effects such as alopecia. Using immunocytochemistry, we show that, after a single i.v. injection, DM accumulates in the nuclei of matrix cells and in the outer root sheath of hair follicles. DM-positive matrix cells are detectable up to 48 h after injection and exhibit a characteristic granular morphology, which is not observed in saline-injected controls. TUNEL-staining has revealed that DM injection induces programmed cell death (PCD) in rat hair follicles. Cells undergoing PCD are detectable as late as 5 days postinjection in both the matrix and outer root sheath. Newly developed double-staining has shown that some of the DM-positive matrix cell nuclei are also TUNEL-positive. Staining for activated caspase-3 has demonstrated immunopositive cells following DM administration both in the matrix and in the outer root sheath. Ultrastructural immunocytochemistry has shown the presence of DM-positive cells with two different types of morphology. About half of the immunopositive cells exhibit a morphology typical of classical apoptosis (PCD type 1), whereas the other half show signs of autophagic cell death (PCD type 2). Interestingly, little, if any, DM accumulation or apoptosis has been detected in the dermal hair papillae. This may have a bearing on potential regeneration of the hair follicles. Thus, DM accumulates in a characteristic pattern in hair follicles. This accumulation is associated with the induction of two morphologically distinct forms of PCD.