A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells.

Low extracellular zinc concentrations have been associated with the induction of apoptosis. To assess the relationship between intracellular zinc concentration and the rate of apoptosis, cells were grown in media containing 0.5, 25, or 50 microM zinc and analyzed by flow cytometry or fluorescence microscopy. Cells grown in 0.5 microM zinc medium over 48 h showed a successive decrease in intracellular zinc concentration measured by the zinc-specific fluorophore, zinquin. After 18 h in 0.5 microM zinc medium, rhodamine 123 retention decreased. However, the addition of 10 microM zinc to the 0.5 microM medium before 16 h in culture restored rhodamine retention in the cells. After 30 h there was an increase in the number of cells cultured in 0.5 microM zinc medium that bound annexin V-FITC. These data indicated that decreased intracellular zinc concentration preceded early markers of apoptosis, with alterations in mitochondrial transmembrane potential preceding the loss of polarity in the cell membrane.

Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype.

Dendritic cells (DC) are highly specialized antigen-presenting cells located in many nonlymphoid tissues, and Langerhans cells (LC), a specialized form of DC, are found in the skin. LC as antigen-presenting cells play a critical role in the induction of allergic contact dermatitis. LC research is difficult because few LCs can be isolated from human skin, so efforts have focused on obtaining DCs from alternative sources. Mononuclear cells from peripheral blood and CD34+ stem cells from human cord blood and marrow can be induced to form phenotypic and functional DCs, but experiments of this type are expensive and the DC yield is low. We report here the induction of the myeloid leukemia cell line (KG-1) to a DC morphology and phenotype by culturing the cells in a defined cytokine cocktail. Morphologically, the KG-1-derived DCs are large irregularly shaped cells with prominent dendritic processes and hair-like cytoplasmic projections. Phenotypically, the KG-1-derived DCs lack lineage-specific markers, and express MHC class II, costimulatory molecules CD80 and CD86, and CD83. Functionally, KG-1-derived DCs are capable of phagocytosing latex microspheres and are able to induce a potent allogeneic T-cell response. Within the KG-1-derived DCs, a subpopulation maintains the DC phenotype and morphology described above but further develops CD1a+ marker expression similar to that of resident skin-derived LCs. These findings illustrate that phenotypic, morphologic and functional DCs can be derived from the KG-1 cell line.

MK-801 neurotoxicity in cupric silver-stained sections: lesion reconstruction by 3-dimensional computer image analysis.

Routine histopathologic evaluation of the brain (paraffin embedding, hematoxylin and eosin staining) makes it difficult for an investigator to identify the overall location and relative extent of lesions as they relate to neural substructures. Moreover, it is very difficult to convey this information to others who are less familiar with neuroanatomy. This study combined a 3-dimensional imaging program with a cupric silver stain for neuronal degeneration in order to determine the location and extent of a focal lesion produced by MK-801 (dizocilpine maleate), a glutamate receptor antagonist that induces necrosis in a small population of neurons in the cortex of rats. A male Sprague-Dawley rat was treated with a subcutaneous dose of MK-801 (10 mg/kg) and was perfused with fixative through the left ventricle 3 days after treatment, a time point known to reveal maximal neurotoxic effects. The brain was embedded in a gelatin matrix, frozen, and serially sectioned at a thickness of 40 microm. The cupric silver method of de Olmos was used to stain frozen sections at 320-microm intervals. Using a color charged-couple device (CCD) camera and a macro lens, a series of 2-dimensional images, which encompassed the entire rostral to caudal extent of the brain, was captured. A computer program was written to define internal and external boundaries in these 2-dimensional images. Then, 3-dimensional reconstructions were generated on a Silicon Graphics workstation using IRIS "Explorer." The quality of the 3-dimensional reconstructions allowed for easy identification of various neural substructures while clearly revealing the exact location and extent of the resulting necrotic neurons that were positively identified by the cupric silver stain. This 3-dimensional lesion reconstruction method provides a powerful tool for conveying spatial information about the nature of neurotoxic lesions in the brain. In addition, it may be used to investigate further dose-response relationships and the effects of other neurotoxicants.

Selective modulation of B-cell activation markers CD86 and I-Ak on murine draining lymph node cells following allergen or irritant treatment.

It is well known that T cells are key effector cells in the development of allergic contact dermatitis. However, we and others have shown that mice exposed to contact allergens show a preferential increase in B lymphocytes in the draining lymph nodes (DLN) as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. The purpose of the present investigation was to determine whether chemical allergens, in contrast to irritants, would modulate B-cell activation markers, CD86 and I-Ak, on B cells isolated from DLN of treated mice using the local lymph node assay (LLNA) protocol. Mice were treated on the ears for 3 consecutive days with concentrations of allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one, and trinitrochlorobenzene), or irritants (benzalkonium chloride and sodium lauryl sulfate), which caused an increase in the number of DLN cells. The DLN were excised 72 h following the final chemical treatment, and the cells were prepared for analysis by flow cytometry. In mice treated with allergens an increase in the median intensity of I-AK and CD86 on B220+ or IgG/IgM+ B cells was observed compared to mice treated with irritants or vehicles. Mice treated with allergens demonstrated an increase in the median intensity of CD86 on B220+ B cells that was dose dependent and peaked at 72 h following the final allergen treatment. The increase in the median intensity of I-AK also was dose dependent but peaked at 96 h. Finally, T and B cells isolated from both allergen- and irritant-treated mice demonstrated an increase in [3H]thymidine incorporation compared to vehicle-treated and naïve mice at 72 h following the final chemical treatment. The results suggest that B cells isolated from DLN of allergen-treated mice are activated and proliferating. Analysis of B-cell activation markers may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing.

Since the p53 gene function is critical to how a cell responds to DNA damage, we investigated the p53 status in Chinese hamster cell lines commonly used in genotoxicity tests for cytogenetic damage around the world. These included: Chinese hamster ovary K1 (CHO-K1), Chinese hamster ovary WBL (CHO-WBL), and Chinese hamster lung (CHL) cells. The results of DNA sequencing, protein analysis, and cell cycle analysis demonstrate that the CHO-K1 and CHO-WBL cell lines have mutant p53 sequence [a mutation in codon 211 in exon 6 resulting in a change from Thr (ACA) to Lys (AAA)], mutant protein (high spontaneous levels that are non-inducible after X-irradiation), and mutant function (lack of G1 checkpoint). Interestingly, the CHL cell line has a completely wild-type p53 DNA sequence. However, the CHL cells have an abnormally high spontaneous level of wild-type p53 protein expression that is not inducible after X-irradiation, yet there is some evidence of G1 delay after irradiation. The protein data suggests that p53 in CHL cells is not being regulated normally, and thus is probably not functioning normally. The mechanism leading to this abnormal regulation of p53 in CHL cells clearly does not involve mutation in the p53 gene. Overall, the CHL cell line may be similar to the CHO cell lines, in that they all appear to have abnormal p53 function. Further work is needed to determine whether the presence of spontaneously high levels of wild-type p53 in CHL cells results in a difference in response to DNA damage (quantitatively or qualitatively) compared to the p53 mutant CHO cell lines.

Computerized image analysis of morphologically transformed and nontransformed Syrian hamster embryo (SHE) cell colonies: application to objective SHE cell transformation assay scoring.

We have developed an automated image analysis system that provides comparable classification of morphologically transformed SHE cell colonies to the current visual classification method used in the in vitro SHE cell transformation assay. Visual classification of morphologic transformation in this assay has been shown to accurately predict the carcinogenic potential of chemical, biological and physical agents. The image analysis system is quantitative, based on measuring features of colony color, texture and growth patterns. A linear combination of feature measurements produces a classification process that agrees with visual assessment 93% of the time. All identifiable sources of error are explored and the method is found to be robust in analyzing nearly 500 colonies from a variety of studies performed over a one year period. The high degree of correlation between the visual classification and the objective measurements of the image analysis system validates the reproducibility of the visual scoring process and serves as a basis for automation of the assay.

Selective modulation of T cell memory markers CD62L and CD44 on murine draining lymph node cells following allergen and irritant treatment.

Naive and activated T cells are known to express different adhesion molecules and are thought to exhibit different migratory patterns that result from their expression of discrete adhesion molecules. Two adhesion molecules that have been associated with differentiating naive and activated/memory T cells are CD62L (L-selectin) and CD44 (H-CAM). It has been demonstrated previously that naive T cells express a CD62LhiCD44lo phenotype, whereas memory T cells exhibit a CD62LloCD44hi phenotype. The purpose of the present investigation was to determine whether chemical allergens, in contrast to irritants, would induce a CD62LloCD44hi phenotype on CD4 and/or CD8 T cells isolated from draining lymph nodes (DLN) of treated mice. Mice were treated on the ears for 3 consecutive days with concentrations of allergens or irritants which caused an increase in the number of DLN cells. The DLN were excised 72 hr following the final chemical treatment and cells prepared for analysis by flow cytometry. In mice treated with the allergen trinitrochlorobenzene an increase in the percentage of CD4+ cells expressing CD62LloCD44(hi) was observed compared to cells isolated from mice treated with the irritant benzalkonium chloride or vehicle treated mice. Mice treated with dintrochlorobenzene had an increase in the percentage of CD4+ cells expressing CD62LloCD44(hi) that was dose dependent and peaked at 72 hr following the final allergen treatment. Concomitant with changes on CD4+ cells, increases in the percentage of CD8+ cells expressing CD62LloCD44hi were observed with allergens, but not with irritants. Increases in the percentage of CD4+ and CD8+ cells expressing CD62LloCD44(hi) were observed with other allergens including oxazolone and alpha-hexylcinnamaldehyde, but not the irritant sodium lauryl sulfate. These data demonstrate that allergens, but not irritants, cause a selective and reproducible increase in the percentage of CD4+ and CD8+ cells expressing the T cell activation/memory phenotype CD62LloCD44hi. Analysis of T cell activation/memory markers may be useful in differentiating allergen and irritant responses in the draining lymph nodes of chemically treated mice.

The combination of epidermal growth factor and transforming growth factor-beta induces novel phenotypic changes in mouse liver stem cell lines.

Mouse liver stem cell (oval cell) lines were investigated in order to determine the role which two families of growth and differentiation factors (GDFs), epidermal growth factor (EGF) family and transforming growth factor beta (TGF-beta) family, play in liver regeneration. EGF family members, including EGF, amphiregulin, betacellulin, heparin-binding epidermal growth factor, and TGF-alpha, were mitogenic for oval cell lines while TGF-beta family members, including TGF-beta1, TGF-beta2 and TGF-beta3, inhibited mitogenesis and induced apoptosis in oval cell lines. Surprisingly, the combination of EGF family members and TGF-ss family members resulted in neither proliferation nor apoptosis but instead in a novel cellular response, cellular scattering in tissue culture and morphological differentiation in Matrigel. Analysis of the signal transduction pathways activated by exposure of oval cell lines to either EGF, EGF+TGF-beta, or TGF-beta indicated that novel combinations of intracellular signals result following stimulation of the cells with the combination of EGF+TGF-beta. These data reveal that the dynamics of synergistic GDF action following tissue injury and regeneration results in a new level of complexity not obvious from the study of individual GDFs.

Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

Phenotypic analysis of lymphocyte subpopulations in lymph nodes draining the ear following exposure to contact allergens and irritants.

The murine local lymph node assay (LLNA) measures in vivo proliferation in draining lymph nodes (DLN) following topical exposure to chemicals to assess contact sensitization potential. However, proliferation has also been observed with some irritants. To further characterize events in the DLN during the LLNA and distinguish allergens from irritants, phenotypic analysis of lymphocyte subsets was made following topical exposure. In preliminary studies, mice were treated on the ears for 3 consecutive days, and 48 hr following the final application, analysis of CD3, CD4, CD8, and B220 expression was evaluated by flow cytometry. The allergens oxazolone (OXAZ) and picryl chloride (TNCB) and the irritant benzalkonium chloride (BC) increased cell number compared to vehicle. The increase in lymph node cellularity for these materials was due to an increase in the total number of T and B lymphocytes. Interestingly, even though contact sensitization is a cell-mediated immune response (Th1), mice exposed to the contact allergens showed a preferential increase in B lymphocytes in the DLN as seen by an increase in the percentage of B220+ cells. The percentage of B220+ cells was 13.1 and 36.1% for OXA and TNCB, respectively, compared to percentages of 7.4 and 9.3% for irritant and vehicle, respectively. With some allergens, a concomitant decrease in the percentage of CD3+ cells was seen. Time course studies demonstrated the increase in the percentage of B220+ cells was seen in allergen treated mice by 24 hr after the final application of material, plateaued by 48 hr, and was still elevated by 96 hr. In allergen-treated mice, percentages of B220+ cells increased dose dependently. Further studies were performed to evaluate additional contact allergens and irritants and determine if evaluation of flow cytometric parameters could potentially identify contact allergens and differentiate them from irritants. Analysis of data from these studies, which examined a total of five contact allergens and six irritants, showed that the modifications to the LLNA improved the identification of irritants and allergens in individual experiments by including both phenotypic analysis of the DLN and cell number per node as endpoints rather than either endpoint alone.

Calcium functions as a transcriptional and mitogenic repressor in Syrian hamster embryo cells: roles of intracellular pH and calcium in controlling embryonic cell differentiation and proliferation.

We have investigated the role played by the growth and differentiation factor (GDF)-induced calcium ion second messenger signal in the control of cellular differentiation and proliferation in Syrian hamster embryo (SHE) cells. Blocking the platelet-derived growth factor (PDGF)-induced calcium ion signal with either extracellular/intracellular acidification or pharmacological agents resulted in increased immediate early gene expression and mitogenesis. The increase in immediate early gene expression resulted from a change in the level of immediate early gene transcription and not immediate early gene mRNA stability. Analysis of the promoter elements that control immediate early gene expression indicated that the calcium ion effect is mediated through the CaRE/CRE and AP1 promoter elements. The calcium signal-mediated reduction in PDGF A/B-stimulated SHE cell immediate early gene expression resulted in a reduction in PDGF A/B-induced cellular proliferation. These results demonstrate that in SHE cells, calcium functions to suppress mitogen-induced proliferation at the level of immediate early gene expression, an effect related to the control of cellular proliferation and differentiation by GDFs through the calcium ion second messenger.

Evaluation in rats of the dose-response relationship among colonic mucosal growth, colonic fermentation, and dietary fiber.

The dose-response relationship among dietary fiber, colonic fermentation, fecal weight, and mucosal growth were evaluated in this study. The morphometric parameter of total mucosal volume was used to assess diet-induced differences in colonic mucosal growth. Dietary fibers with a wide range of fermentability and that have previously been shown to inhibit the development of colonic neoplasia in rats were used. Sprague-Dawley rats were fed Purina Rodent Chow, AIN-76a fiber-free diet, or an AIN-76a diet supplemented with three different dietary fibers, (cellulose, guar gum, or wheat bran) at 2, 5, 10, or 15% of the diet. Diets were fed for 28 days. Total colonic mucosal volume was determined using stereologic principles and computerized image analysis; 48-hr fecal weight was measured; and the concentration of short-chain fatty acids (SCFA) in colonic contents was determined at study termination. Each type of fiber induced a dose-dependent increase in total mucosal volume of the colon and fecal weight. Mucosal volume and fecal weight were closely correlated (R2 > 0.95). Total mucosal volume was not correlated with the concentration of total SCFA or butyrate in the colon. These results indicate that diet-induced change in colonic mucosal growth, as measured by total mucosal volume, is positively correlated with fecal weight and not related to alterations in colonic fermentation. Enhanced colonic mucosal growth occurs in rats fed dietary fibers that have previously been shown to inhibit the development of genotoxin-induced colonic neoplasia in rats.

The interactions of diet and colonic microflora in regulating colonic mucosal growth.

The colonic mucosa can adapt its growth to alterations in diet. Metabolites from colonic microflora are frequently implicated as the primary factor in mediating the colonic mucosal response to diet; however, there is also evidence indicating that diet may have a direct effect in mediating this response. The aim of this study was to determine the role of diet, microflora, and microflora metabolites in altering the growth of the colonic mucosa. Two 28-day feeding studies were conducted using Sprague-Dawley rats. The first study compared the growth of the colonic mucosa in germ-free and conventional rats fed 6 different diets. The second study compared the growth of the colonic mucosa to the concentration of bacterial-derived short-chain fatty acids (SCFs), bile acids, and ammonia. The diets that were fed consisted of (1) AIN-76a diet without dietary fiber; (2) standard AIN-76a diet, which contained 5% cellulose; (3) AIN-76a diet with 5% guar gum; (4) a "Western" human diet with 20% fat and 10% cellulose; (5) AIN-76a diet formulated to mimic Diet 4 in fat content but with 2.5% cellulose; and (6) Purina Rodent Chow. Quantitative volumetric and stereologic analysis was used to assess changes in total colonic mucosal volume as a measure of mucosal growth. In germ-free rats, Diets 2-4 and 6 induced a significant increase (18-38%) in mucosal volume compared to Diet 1. In conventional animals, only Diets 4 and 6 induced a significant increase (up to 63%) in mucosal volume compared to Diet 1. Relative to the germ-free animals, only conventional animals on Diets 4 and 6 had an increase in mucosal volume. The increases in mucosal volume in Diets 4 and 6 were not consistently associated with increased SCFAs, ammonia, or bile acids. There was a wide range in the colonic concentrations of SCFAs (2-fold), ammonia (6-fold), and bile acids (10-fold). The presence of colonic microflora in and of itself does not lead to enhanced colonic mucosal growth. Rather, there are unique interactions between specific types of diet and microflora that lead to a growth-promoting effect. This effect could not be explained by alterations in the concentration of SCFAs, ammonia, or bile acids in colonic contents.

Modulation of the platelet-derived-growth-factor-induced calcium signal by extracellular/intracellular pH in Syrian hamster embryo cells. Implications for the role of calcium in mitogenic signalling.

Studies have been performed to understand the interactions and the role which intracellular calcium and intracellular pH have in mediating mitogen-stimulated cellular proliferation. Stimulation of Syrian hamster embryo (SHE) cells with the mitogen platelet-derived growth factor A/B (PDGF) results in intracellular acidification and capacitative calcium entry involving the intracellular release of calcium via the inositol trisphosphate gamma receptor calcium channel, followed by an extracellular influx of calcium through a dihydropyridine-sensitive plasma membrane calcium channel. Chronic extracellular/intracellular acidification results in the inactivation of both these calcium channels due to slowly reversible protein alterations. Paradoxically, transient intracellular acidification, like that following PDGF stimulation, could not stimulate the activation of either calcium channel. In addition, even though intracellular calcium fluxes by themselves could intiate intracellular acidification, loss of the PDGF-induced calcium signal did not result in the loss of the PDGF-induced transient intracellular acidification. Importantly with regard to the role intracellular calcium and pH have in mediating the mitogenic signal leading to cellular proliferation, chronic extracellular/intracellular acidification, which leads to a complete loss of the PDGF-induced calcium signal, did not result in the loss of PDGF-induced mitogenesis. These results indicate that the PDGF-induced calcium signal is not essential for PDGF-stimulated mitogenesis in Syrian hamster embryo cells. In contrast, blocking the PDGF-induced transient intracellular acidification completely blocks PDGF-induced mitogenesis, indicating that the mitogen-induced transient intracellular acidification, unlike the intracellular calcium ion signal, is indispensible for cellular proliferation in Syrian hamster embryo cells.

Increases in human epidermal DR+CD1+, DR+CD1-CD36+, and DR-CD3+ cells in allergic versus irritant patch test responses.

In an attempt to differentiate an allergic patch test response from an irritant response, we evaluated by flow cytometry the percentages of various epidermal cell populations isolated from allergen and irritant-treated patch test sites. Nine allergic individuals were patch tested with various allergens (Rhus, dinitrochlorobenzene [DNCB], or nickel chloride) and a vehicle control for 48 h. Eight additional individuals were patch tested with irritating chemicals (sodium lauryl sulfate or nonanoic acid) and with a vehicle control for 48 h. Epidermal cells, isolated from suction blisters, were double labeled for CD1/HLA-DR, CD3/HLA-DR, or CD36/HLA-DR cell surface markers and analyzed by flow cytometry to determine the percentage of various cell populations. A mean increase of 0.91 +/- 0.3 in the percentage of DR+CD1+ Langerhans cells over the vehicle control patch test site was detected in allergen-positive patch test sites in allergic individuals, whereas a decrease of 0.19 +/- 0.2 in the percentage of DR+CD1+ Langerhans cells from the vehicle control patch test site was detected in irritant-treated patch test sites. Epidermal cells from allergen-positive patch test sites also exhibited an increase of 5.2 +/- 1.8 in percentage of DR+CD1- cells over the vehicle control patch test site compared to an increase change of 0.8 +/- 0.4 in epidermal cells isolated from irritant-treated patch test sites. We also found that DR+ cells that lacked the CD1 determinant expressed the macrophage/monocyte antigen CD36 (OKM5). Finally, a 2.3 +/- 0.8 increase in the percentage of DR-CD3+ cells over the vehicle control patch test site was observed in allergen-positive patch test sites compared to an increase of 0.2 +/- 0.2 observed in irritant-treated patch test sites. These results demonstrate a significant increase in DR+CD1+, DR+CD1-CD36+, and DR-CD3+ epidermal cells in allergen-positive patch test sites compared to irritant patch test sites.

Induction of protein phosphorylation, protein synthesis, immediate-early-gene expression and cellular proliferation by intracellular pH modulation. Implications for the role of hydrogen ions in signal transduction.

In Syrian hamster embryo cells, intracellular acidification (but not alkalization) results in proliferation, immediate-early-gene expression and tyrosine phosphorylation. In addition, both intracellular acidification and alkalization result in serine/threonine phosphorylation and de novo protein synthesis of specific proteins. Calcium is not mobilized in response to either intracellular alkalization or acidification. Neither intracellular acidification nor alkalization altered the serum proliferative signal while intracellular alkalization (but not acidification) reduced the epidermal-growth-factor-induced proliferative signal, tyrosine phosphorylation and immediate-early-gene expression. Finally, intracellular acidification (but not alkalization) could induce immediate-early-gene expression in cells growing in the presence of serum, indicating that the pH signalling pathway is not down modulated by the serum signalling pathway. These results, while indirect, indicate that hydrogen ions may play an important role in mitogen-signal transduction in Syrian hamster embryo cells.

Age, sunlight, and facial skin: a histologic and quantitative study.

Quantitative methods were developed to assess the interrelation between age and sunlight on the facial skin of healthy women living in the same sunny area. The women were grouped into the following categories: young versus old and low versus high solar exposure. The features evaluated were perceived age, amount of facial wrinkling, skin color, and skin elasticity. A punch biopsy specimen of cheek skin was obtained and prepared histologically for evaluation of solar elastosis. The histologic examination was complemented by quantification of collagen and elastin by computer-assessed image analysis. Perceived age was estimated by untrained women viewing high quality photographs. As expected, those with greater sun exposure looked older and had more wrinkles, more severe elastosis, increased elastin, and decreased collagen.

Examination of tetrachlorosalicylanilide (TCSA) photoallergy using in vitro photohapten-modified Langerhans cell-enriched epidermal cells.

Lymphocytes from BALB/c mice photosensitized in vivo to tetrachlorosalicylanilide (TCSA) were investigated to determine whether they could be stimulated to proliferate when cultured with Langerhans cell-enriched cultured epidermal cells (LC-EC) photohapten-modified in vitro with TCSA + UVA radiation. Cultured LC-EC were photohapten-modified in vitro by irradiation in TCSA-containing medium using a 1000-watt solar simulator equipped with filters to deliver primarily UVA radiation (320-400 nm). Lymphocytes from TCSA-photosensitized mice were incubated with LC-EC that had been treated in vitro with 0.1 mM TCSA and 2 J/cm2 UVA radiation (TCSA + UVA). Responder lymphocytes demonstrated a significant increase in their blastogenesis response compared to lymphocytes that were incubated with LC-EC irradiated with UVA prior to treatment with TCSA (UVA/TCSA) or with LC-EC that had received no treatment. Lymphocytes from naive mice or mice photosensitized with musk ambrette (MA) demonstrated a significantly lower response to LC-EC modified with TCSA + UVA, indicating the specificity of the response. Maximum blastogenesis response was achieved when LC-EC were treated with 0.1 mM TCSA and a UVA radiation dose of at least 0.5 J/cm2. Epidermal cells depleted of LC by treatment with anti-Ia antibody plus complement or by an adherence procedure were unable to stimulate this blastogenesis response. Epidermal cells treated in vitro with TCSA + UVA demonstrated enhanced fluorescence compared to control cells. The fluorescence observed was not restricted to any specific epidermal cell type; however, fluorescence microscopy studies revealed that dendritic Ia-positive cells, presumably LC, were also TCSA fluorescent. Flow cytometry showed that Ia-positive epidermal cells demonstrated the greatest UV fluorescence when treated with TCSA + UVA compared to both cells irradiated with UVA and subsequently treated with TCSA and untreated cells. This is consistent with the enhanced antigen presentation capability of TCSA + UVA treated LC-EC, which leads to the conclusion that LC photohapten-modified in vitro with TCSA + UVA demonstrate enhanced TCSA fluorescence and are capable of stimulating lymphocytes from TCSA photosensitized mice in an antigen-specific manner.

Increased hyaline droplet formation in male rats exposed to decalin is dependent on the presence of alpha 2u-globulin.

A peculiar decalin-induced male rat nephropathy associated with the altered renal handling of filtered protein appears limited to the accumulation of the protein, alpha 2u-globulin. Several strains of male rats that produce alpha 2u-globulin (Fischer-344, Sprague-Dawley, Buffalo, and Norway Brown) demonstrate spontaneous renal cortical hyaline droplets which are exacerbated after exposure to decalin. In all cases, a close correlation exists between hyaline droplet formation observed histologically and alpha 2u-globulin accumulation measured biochemically. In stark contrast, the NCI-Black-Reiter strain, which does not produce measurable quantities of alpha 2u-globulin, neither forms hyaline droplets nor accumulates any filtered protein in its kidney cortex either spontaneously or after exposure to decalin. Also, female rats injected ip with male rat alpha 2u-globulin exhibit increased hyaline droplet formation and alpha 2u-globulin accumulation when treated with decalin. These data provide evidence that the presence of alpha 2u-globulin is key in understanding why this nephropathy appears unique to the male rat.

Acute and subchronic nephrotoxicity of d-limonene in Fischer 344 rats.

In the studies described here, we have examined the sex-specific sensitivity of rat kidney to d-limonene. At 24 hr after an acute dose of 200 mg d-limonene/kg body weight administered to adult male and female Fischer 344 rats by oral gavage, an increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2u-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene. In a separate subchronic study, groups of 5-wk-old male rats were administered d-limonene in a corn oil vehicle at 0 (control), 2, 5, 10, 30, or 75 mg/kg body weight by single daily gavage (5 days/wk) for 13 wk. Rats from selected dose groups received interim necropsies from days 8-29, while all groups were necropsied at the end of the study. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg d-limonene/kg body weight. Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. The no-observable-effect level for these effects was 5 mg d-limonene/kg body weight. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose. It is concluded that treatment with d-limonene caused an increase in the formation of hyaline droplets in male rats only, that this increase was associated with an accumulation of alpha 2u-globulin, that d-limonene (or its metabolite) accumulated significantly in male rat kidney compared with that in females and that subchronic dosing produced a triad of morphological changes in the male rat kidney. These observations suggest that d-limonene caused nephrotoxicity specific to the male rat and that this toxicity may not be predictive of a similar response in humans.