Study of 67Ga distribution in neoplasms and in liver by a cell fractionation method]

Subcellular distribution of 67Ga was quantitatively determined to evaluate the role of lysosome in accumulation of 67Ga in malignant tumor tissue and liver. The following animals and transplanted tumors were used: rats implanted with Yoshida sarcoma and hepatoma AH109A; mice implanted with Ehrlich tumor. 67Ga-citrate were injected to the rats intravenously and to the mice intraperitoneally. Ten minutes to 48 hours after the administration of 67Ga-citrate, the animal were sacrificed, and the tumor tissues and liver were excised. Subcellular fractionation of tumor tissues and livers were carried out according to the method of Hogeboom and Schneider. Radioactivity of each fraction was counted by a well type scintillation counter, and protein of each fraction was measured according to Lowry's method. In Yoshida sarcoma and Ehrlich tumor, most of the radioactivity was localized in the supernatant fraction, and small amount of radioactivity was localized in the mitochodrial fraction (lysosome contains in this fraction). But in the liver, most of the radioactivity was concentrated in the mitochondrial fraction and the radioactivity of this fraction was increased with the passage of time after administration. Twenty-four hours later, about 50% of total radioactivity was accumulated in this fraction. In the case of hepatoma AH109A, radioactivity of mitochondrial fraction was increased with the passage of time after administration, and about 30% of total activity was concentrated in this fraction at 24 hours after administration. From these results it is concluded that lysosome doses not play an important role in the tumor concentration of 67Ga and lysosome plays an important role in the liver concentration of 67Ga. In the case of hepatoma AH109A it is presumed that lysosome plays considerably important role in the tumor concentration of 67Ga, hepatoma AH109A having some nature of liver.     

The difference between the mechanism of 67Ga accumulation and 59Fe accumulation in cultured tumor cells

It is well known that the mechanism of 67Ga accumulation into tumor cells is mediated with transferrin receptor as well as iron. The present study was designed to explore the difference between the mechanism of gallium accumulation and that of iron by using mouse leukemic cell line L5178Y. When monensin which inhibits the recycle of transferrin receptor was added to the incubated system, accumulation of 59Fe and 67Ga was clearly diminished compared with that of control. However, inhibition of 59Fe accumulation was more remarkable than that of 67Ga. Furthermore, monensin has a action of Na+ ionophore which decreases Na+ gradient between the inside and the outside of the plasma membrane. Following administration of monensin, 67Ga accumulation was diminished according to the loss of the Na+ gradient. On the other hand, following administration of valinomycin, 67Ga accumulation was not affected by the loss of the K+ gradient. From these results, it was suggested that the mechanism of 67Ga accumulation into tumor cells differed from that of 59Fe and transferrin receptor and Na+ gradient of tumor cells played an important role on 67Ga accumulation into tumor cells.     

TUMOR IMAGING WITH [111niIn]MONO-DTPA-ETHYLENEGLYCOL-Ga-DEUTEROPORPHYRIN

Abstract Mono-DPTA-ethyleneglycol-Ga-deuteroporphyrin (MDEGD) was synthesized, by coordinating non-radioactive Ga in the porphyrin ring and connecting DTPA (diethylene-triamine-N,N,N,N,N,-pentaacetic acid) to its side chain. By labeling with 111_In, chemicals for scintigraphy were developed. They were applied to Syrian golden hamsters with implanted pancreatic gland cancers and C57-black mice with Lewis lung cancer to enable tumor imaging and biodistribution examination. A comparative study was also conducted with [⁶⁷Ga]citrate. In the resultant data, [¹¹¹In]MDEGD showed larger tumor/lung, tumor/kidney and tumor/blood ratio with [⁶⁷Ga]citrate. The affinity of [ⁿIn]MDEGD in inflammatory tissue was much lower than that of ⁶⁷Ga citrate. [¹¹¹In]MDEGD lost its photosensitivity.     

Relation between location of elements in periodic table and affinity for the malignant tumor (author's transl)

Affinity of many inorganic compounds for the malignant tumor was examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. And the relations between the uptake rate into the malignant tumor and in vitro binding power to the protein were investigated in these compounds. In these experiments, the bipositive ions and anions had not affinity for the tumor tissue with a few exceptions. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate into the malignant tumor. As Hg++, Au+ and Bi+++ are soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (R-SH, R-S-) present in the tumor tissue. In many hard acids (according to classification of Lewis acids), the uptake rate into the tumor was shown as a function of ionic potentials (valency/ionic radii) of the metal ions. It is presumed that the chemical bond of these hard acids in the tumor tissue is ionic bond to hard base (R-COO-, R-PO3(2-), R-SO3-, R-NH2).     

Distribution of metal-free sulfonated phthalocyanine in subcutaneously transplanted murine tumors

Metal-free sulfonated phthalocyanine with the average number of sulfonate groups per molecule 2.4 (H(2)PcS(2.4)) was recently proved to be an efficient photosensitizer for the photodynamic therapy. Fluorescence spectral imaging microscopy was applied here to study localization and relative concentration of H(2)PcS(2.4) with micron-scale resolution in subcutaneously transplanted murine tumors: Ehrlich mammary gland carcinoma (EC), Lewis lung carcinoma (LLC), P388 lymphoid leukemia (P388) and B16 melanoma (B16). The study of cryogenic tissue sections prepared 24 h after H(2)PcS(2.4) intravenous injection revealed that H(2)PcS(2.4) was present in all tissue structures in the monomeric photoactive state. The preferential accumulation of H(2)PcS(2.4) was documented in tumor cells and adjacent non-tumor tissues (skin structures, fatty tissue, connective tissue enriched in fibrous component and infiltrated with fibroblasts and macrophages) for all the studied tumor models. P388 and B16 were stained with H(2)PcS(2.4) less than adjacent skin structures, whereas EC and LLC accumulated H(2)PcS(2.4) alike or higher than particular skin structures. Staining of EC and LLC was similar and ca. 1.4 and 2 times higher than that of B16 and P388, respectively, thus revealing the differences in ability of particular tumor strains to H(2)PcS(2.4) accumulation. The H(2)PcS(2.4) concentration in remote healthy tissues (skin, muscles and connective tissue) was 2-3 times lower as compared with the analogous tissue structures from the tumor area, whereas subcutaneous fatty tissue staining did not depend on the tissue-to-tumor distance. The tissue distribution of H(2)PcS(2.4) predefines the combined action of two photodynamic damage mechanisms: eradication of tumor due to the direct tumor cell destruction and suppression of tumor growth due to the injury of growth supporting system.     

Double tracer autoradiography of bone tumor using 3H-thymidine and radionuclides for diagnostic purpose

Whole body distribution and accumulation of radionuclide for diagnostic purpose such as 99mTc-MDP (methylene diphosphonate), 67Ga-citrate (67Ga) and 201Tl-chloride (201Tl) in implanted osteosarcoma of human origin were evaluated by macroautoradiography of Balb/c nude mouse. 3H-thymidine of which uptake has a close relation to growth pattern of tumors was used for a partner of target nuclide in double tracer technique to examine the distribution characteristics. Separated distribution pattern of coupled nuclides was imaged by a difference both of half life and energy of radiation between radionuclides. The distribution pattern of each target nuclide and 3H-thymidine were compared to clarify distribution characteristics respectively in specimen. 67Ga and 201Tl were taken around the tumor and the mode of their distribution was identical to that of 3H-thymidine. Uptake of 67Ga in tumor was much higher. 99mTc-MDP accumulated in the central zone of the tumor and its pattern was quite different from that of 3H-thymidine. From above result it was demonstrated that the accumulation mechanism of 67Ga, 201Tl and 99mTc-MDP are quite different in the tumor each other. So we concluded that our method of double tracer technique was useful for evaluation of distribution pattern of radionuclides for diagnostic purpose.     

On the67Ga-binding acid mucopolysaccharide in malignant tumor

The present study was undertaken to determine the structure of⁶⁷Ga-binding acid mucopolysaccharide in tumor tissues. It was determined from measuring neutral saccharide in the structure that the principal⁶⁷Ga-binding acid mucopolysaccharide in the tumor was keratan sulfate and/or keratan polysulfate. On the other hand, it was clarified from the results of mucopolysaccharase treatment that the main⁶⁷Ga-binding acid mucopolysaccharide in tumor was not keratan sulfate, heparan sulfate, heparin, nor chondroitin sulfate A, B, or C. Based on the present results, it was deduced that the main⁶⁷Ga-binding acid mucopolysaccharide in tumor is keratan polysulfate and that this acid mucopolysaccharide plays the most important role in tumor accumuation of⁶⁷Ga.     

Tumor vascular area correlates with photosensitizer uptake: analysis of verteporfin microvascular delivery in the Dunning rat prostate tumor

The parameters that limit supply of photosensitizer to the cancer cells in a solid tumor were systematically analyzed with the use of microvascular transport modeling and histology data from frozen sections. In particular, the vascular permeability transport coefficient and the effective interstitial diffusion coefficient were quantified for Verteporfin-for-Injection delivery of benzoporphyrin derivative (BPD). Orthotopic tumors had higher permeability and diffusion coefficients (Pd = 0.036 microm/s and D = 1.6 microm(2)/s, respectively) as compared to subcutaneously grown tumors (Pd = 0.025 microm/s and D = 0.9 microm2/s, respectively), likely due to the fact that the vessel patterns are more homogeneous orthotopically. In general, large intersubject and intratumor variability exist in the verteporfin concentration, in the range of 25% in plasma concentration and in the range of 20% for tissue concentrations, predominantly due to these microregional variations in transport. However, the average individual uptake of photosensitizer in tumor tissue was only correlated to the total vascular area within the tumor (R2 = 64.1%, P < 0.001). The data are consistent with a view that microregional variation in the vascular permeability and interstitial diffusion rate contribute the spatial heterogeneity observed in verteporfin uptake, but that average supply to the tissue is limited by the total area of perfused blood vessels. This study presents a method to systematically analyze micro-heterogeneity as well as possible methods to increase delivery and homogeneity of photosensitizer within tumor tissue.     

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells ls174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells Is174t and the growth of colon cancer tissue of tumor-bearing mice,MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro.Transmission electron microscope was used to observe the cell apoptosis.FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle,RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA.Western blot method was used to detect the expressions of bc1-2 and bax protein.Cells isi74t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor.RT-PCR method was used to detect the expressions of bc1-2 and bax mRNA in the tumor tissue.Western blot method was used to detect the expressions of bc1-2 and bax protein in the tumor tissue.Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro(P<0.01).It is able to induce the apoptosis of cells Is174t,causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice(P<0.05),causing the decrease in the expression of bc1-2 and the increase in the expression of bax.Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor.The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bc1-2/bax expression.     

Ethylene dicysteine (EC) and ethyl cysteinate dimer (ECD) complexes of Ga(III) and In(III)

Ethylene dicysteine (EC) and ethyl cysteinate dimer (ECD) complexes of⁶⁷Ga and¹¹¹In were prepared and the complexation yield and radiochemical purity estimated by paper chromatography, paper electrophoresis and solvent extraction into chloroform.¹¹¹In-EC was found to be anionic, whereas¹¹¹In-ECD was neutral. EC complexes of⁶⁷Ga and¹¹¹In were stable upto 6 hours and expectedly less lipophilic than ECD complexes. ECD complexes of⁶⁷Ga and¹¹¹In were unstable in aqueous medium, but highly stable in chloroform. The utility of the work for stabilisation of products as organic extract and the possible role and limitation for the development of new⁶⁸Ga radiopharmaceuticals and of bifunctional chelating agent (BCA) for¹¹¹In are discussed.     

Determination of vinca alkaloids in mouse tissues by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for the determination of vinblastine in various normal mouse tissues, such as lung, heart, liver, kidney and muscles, and in implanted MO4 tumours. Vincristine was used as the internal standard. Freshly obtained mouse tissue or tumour tissue was frozen at -20 degrees C and then lyophilized. After lyophilisation, the dry tissues were pulverized and homogeneously mixed, and an aliquot was suspended in 0.1 M hydrochloric acid. The drugs of interest were then isolated from this suspension using ion-pair extraction at pH 3 with octylsulphate as counter-ion. The obtained extracts were analysed on a reversed-phase system with a cyanopropyl stationary phase. The detection limit was 1 ng/l in plasma and 10 ng/g in tissue. The extraction recoveries of vincristine and vinblastine were between 45 and 67%, and there were no interferences from blank components. Preliminary pharmacokinetic data for different mouse tissues and tumour implanted in muscle tissue are presented.     

Double tracer whole body autoradiography using a short-lived positron emitter and a long-lived beta emitter

We investigated glucose and amino acid metabolism in tumors and other organs using whole body autoradiography with a short-lived positron emitter and a long-lived beta emitter. The radioactive compounds used were 2-deoxy-2-[18F]-fluoro-D-glucose (18F-FDG) with a half life of 109.8 min and L-[methyl-14C]-methionine (14C-MET) with a half life of 5,730 years. A Donryu rat weighing about 150 g was subcutaneously inoculated at the back with experimental tumors of AH109A and AH272. 74 MBq (2 mCi) of 18F-FDG and 740 kBq (20 microCi) of 14C-MET was administered and after 30 min, the rat was sacrificed. Whole body frozen sections were obtained using autocryotome. For the 18F-FDG autoradiogram, the frozen sections were exposed to an X-ray film for 6 h. After seven days, these frozen sections were again exposed to 14C-MET for a week. Cross-contamination was minimized by adjusting the exposure time, the interval of exposures and the administered dose. The accumulation of the tracers was represented as the optical density ratio of the tissue of interest to the muscle. The tumor ratios were 12.5 for 18F-FDG and 8.6 for 14C-MET showing the highest accumulation in the whole body autoradiogram. In contrast the inflammatory tissue ratios were 1.27 for 18F-FDG and 0.77 for 14C-MET showing very low amino acid metabolism. With the present double tracer whole body autoradiogram, 18F-FDG accumulation was seen in the brain and the heart but not to the liver as against 14C-MET accumulation which was seen to the liver but not to the brain and the heart.     

Accumulation mechanism of 111In in malignant tumor (author's transl)]

In order to clarify the accumulation mechanism of 111In in maligant tumor, subcellular distribution of 111In was quantitatively determined. Buffalo rats bearing Morris hepatoma 7316A were injected intraperitoneally 111In-chloride and tumor tissues were removed 24 hours later. Subcellular fractionation of tumor tissues were carried out according to the method of C. de Duve, et al. and radioactivity of each fraction was counted. Most of the total radioactivity was distributed among the soluble, nuclear and lysosomal fractions. On account of its low protein content, the relative specific radioactivity was the highest in the lysosomal fraction. The lysosomal fraction was solubilized gradually and the resultant stepwise release of 111In and acid phosphatase activity were measured. There was a close relationship between them. From these results it was concluded that 111In accumulated especially in the lysosomes. In the electron micrography the tumor lysosomes had already engulfed many foreign materials, so that the lysosomal function would be depressed.     

Mechanism of 67Ga uptake by an experimental abscess--permeability of plasma from blood vessels in abscessed tissue

In the previous paper, we reported that 67Ga was accumulated in abscess and uptake rate of 67Ga in abscess increased with time after the injection of 67Ga-citrate. The present study was undertaken to elucidate the influence of blood flow on the accumulation of 67Ga in abscess. Five days after subcutaneous injection of 0.2 ml of turpentine to the rats, 131I-human serum albumin (HSA) was injected intravenously to the rats. At an appropriate time after the injection (10 min to 6 days), uptake rates of 131I-HSA in abscess and normal tissues were measured. Similarly, 51Cr-red blood cells (RBC) were injected intravenously to the above rats and the uptake rates of 51Cr-RBC were also measured. One, three, and 24 hours after injection of 131I-HSA, the uptake rates of 131I-HSA in abscess were 1.32 %dose/g, 1.84 %dose/g, and 0.82 %dose/g, respectively. However, the uptake rates of 51Cr-RBC in abscess was very small, and the value was 0.14 %dose/g at 24 hours after the injection. In the case of abscess, blood in the tissue fluid was very little, but the permeability of 131I-HSA from the blood vessel in the tissue was much larger than that of normal tissues. From these facts, it was deduced that the accelerated permeability caused the abscess accumulation of 67Ga.     

Radioactive metal complexes with affinity for tumors. II. Biodistribution of radioactivity in cellular and subcellular fractions of tumor tissues

The 99mTc and 57Co complexes of ethylenediamine-N,N-diacetic acid (EDDA) are accumulated in tumor tissue. The complexes and related radioactive compounds were administered to experimental animals bearing Ehrlich tumor, and the blood, tumor, abscess, and other tissues were separated, fractionated and analyzed. In blood, the EDDA complexes of 99mTc and 57Co were in dialyzable forms, whereas other tumor-nonlocalizing compounds were in undialyzable or protein-bound forms. The tumor/blood and tumor/muscle ratios of the radioactivity showed that the complexes had the high affinity for tumor tissues. Density gradient centrifugation analysis of the ascites tumor tissues showed that a significant amount of the radioactivity of the complexes was present in tumor cells. Subcellular fractionation of solid tumor tissue showed that the radioactivity was present in nuclear fraction.     

Functional Mechanism of Resveratrol in Inhabiting Growth of Cells Is174t and Its Mechanism in Subcutaneously Transplanted Tumor of Nude Mice

To explore the functional mechanism of Resveratrol against colon cancer cells ls174t and the growth of colon cancer tissue of tumor-bearing mice, MTT method was used to observe the functions of resveratrol for inhibition against cells ls174t in vitro. Transmission electron microscope was used to observe the cell apoptosis. FCM assay was performed to measure the change of the cell apoptosis rate and of cell cycle. RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA. Western blot method was used to detect the expressions of bcl-2 and bax protein. Ceils ls174t were transplanted subcutaneously to nude mice to observe the effect of resveratrol on the growth of subcutaneously transplanted tumor, RT-PCR method was used to detect the expressions of bcl-2 and bax mRNA in the tumor tissue. Western blot method was used to detect the expressions of bcl-2 and bax protein in the tumor tissue. Resveratrol has an effect of inhibiting proliferation of cells ls174t in vitro（P〈0.01）. It is able to induce the apoptosis of cells ls174t, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol could inhibit the growth of subcutaneously transplanted tumor of nude mice（P〈0.05）, causing the decrease in the expression of bcl-2 and the increase in the expression of bax. Resveratrol can inhibit the growth of cells 174t and the growth of subcutaneously transplanted tumor. The mechanism is possibly related to the induction of the cell apoptosis and the regulation of bcl-2/bax expression.     

Differentiation between normal and tumor vasculature of animal and human glioma by FTIR imaging

Malignant gliomas are very aggressive tumors, highly angiogenic and invading heterogeneously the surrounding brain parenchyma, making their resection very difficult. To overcome the limits of current diagnostic imaging techniques used for gliomas, we proposed using FTIR imaging, with a spatial resolution from 6 to 10 μm, to provide molecular information for their histological examination, based on discrimination between normal and tumor vasculature. Differentiation between normal and tumor blood vessel spectra by hierarchical cluster analysis was performed on tissue sections obtained from xenografted brain tumors of Rag-gamma mice 28 days after intracranial implantation of glioma cells, as well as for human brain tumors obtained in clinics. Classical pathological examination and immunohistochemistry were performed in parallel to the FTIR spectral imaging of brain tissues. First on the animal model, classification of FTIR spectra of blood vessels could be performed using spectral intervals based on fatty acyl (3050-2800 cm(-1)) and carbohydrate (1180-950 cm(-1)) absorptions, with the formation of two clusters corresponding to healthy and tumor parts of the tissue sections. Further data treatments on these two spectral intervals provided interpretable information about the molecular contents involved in the differentiation between normal and tumor blood vessels, the latter presenting a higher level of fatty acyl chain unsaturation and an unexpected loss of absorption from osidic residues. This classification method was further successfully tested on human glioma tissue sections. These findings demonstrate that FTIR imaging could highlight discriminant molecular markers to distinguish between normal and tumor vasculature, and help to delimitate areas of corresponding tissue.     

The influences of dexamethasone and indomethacin on 67Ga uptake by normal tissues

Dexamethasone (DEX) increased 67Ga uptake by the liver and spleen at 4, 8, and even 24 h after the injection of 67Ga. These results showed that DEX influenced 67Ga accumulation as well as the initial entry of 67Ga in the liver and spleen. On the other hand, indomethacin (IM) decreased 67Ga uptake by the liver and spleen at 4 h after the injection of 67Ga but did not influence the uptake at 8 or 24 h after the injection of 67Ga. Moreover, DEX or IM little influenced 67Ga uptake by the kidney and muscle. These results suggest that the influence of DEX or IM on 67Ga uptake or accumulation is specific for the liver and spleen.     

Quantification of retinoid concentrations in human serum and brain tumor tissues

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid–liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid–liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between −5.6% and +5.4% for serum and −3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9 ng g⁻¹ (ng mL⁻¹). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.     

Fluorescence imaging and spectroscopy of ethyl nile blue A in animal models of (pre)malignancies

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor-localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue-localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB-induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin-fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665-900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8-24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.