Normal versus abnormal structure: considerations in morphologic responses of teleosts to pollutants.

Consideration of newer more quantitative morphologic approaches to the study of aquatic pollutants can provide opportunity for collaborative/integrated studies with other subdisciplines in toxicology. Current commonly employed morphologic approaches result largely in subjective findings difficult to analyze statistically and often are directed at levels of structural organization inconsistent with biochemical and physiological approaches. We review some of the methods and approaches available for correlated structure/function studies and present examples from normal and altered skin, gill, and liver of teleosts.

Similarity-based categorization and fuzziness of natural categories.

The adequacy of similarity to prototype as an account of categorization in natural concepts was assessed by analyzing the monotonicity of the relation between typicality of an item in a category and the probability of a positive categorization response using data from McCloskey and Glucksberg (1978). The analysis revealed a strong underlying similarity-based threshold curve, with systematic deviations. Further data collection showed that deviations from the curve could be attributed to the effects of unfamiliarity and non-categorical associations on typicality judgments, as well as differences between the perceptual appearance of an item (which tended to boost typicality) and its underlying nature (which tended to boost categorization). The results are discussed in terms of the different presuppositions and task constraints involved in rating typicality as opposed to performing a categorization.

A transient mathematical model of oxygen depletion during photodynamic therapy.

A transient one-dimensional mathematical model is presented to help visualize the qualitative and quantitative effects on inter-capillary tissue undergoing photodynamic therapy (PDT). The model is solved by a Crank-Nicholson finite difference formulation to provide time-dependent concentrations of the Type II mechanism's photo-oxidation species in the tissue surrounding a capillary. The time-dependent solution allows educated decisions to be made as to the optimum timing of light fractionation (on/off) cycles. Qualitative and quantitative optimization of the PDT process is considered along with a case study of data in the literature, the main goal being to provide optimized light therapy regimens for eventual clinical use.

Predictions of mathematical models of tissue oxygenation and generation of singlet oxygen during photodynamic therapy.

Photodynamic therapy (PDT) is a relatively new protocol for cancer treatment which has recently been approved for limited clinical use. Traditionally, the success of treatment with PDT has been compared on the basis of total light delivery. Using the mathematical model of Henning et al. (Radiat. Res. 142, 221-226, 1995), we have determined that when oxygen is not depleted from the tissue, the concentration of singlet oxygen that is generated is directly proportional to the product of the light fluence rate (phi) and the concentration of the photosensitizer (Cs). Therefore, phiCs is an appropriate parameter for comparing the potential success of PDT protocols under these conditions. For a treatment of time t, the observed photodynamic effect resulting from singlet oxygen exposure should be directly related to phiCst. For high phiCs, the model predicts that oxygen depletion occurs within the tumor tissue. As a result, the photodynamic effect is no longer proportional to phiCst. We have expanded the model of Henning et al. to include the changes in oxygen concentration which occur within the capillary as blood flows through the tissue. Our new predictions with the mathematical model for optimal PDT treatment conditions are significantly different from those predicted by the previous models. Predictions of the model are given using parameters relevant for treatment of solid tumors with Photofrin.

Mechanisms of cell killing in photodynamic therapy using a novel in vivo drug/in vitro light culture system.

Photodynamic therapy of certain neoplasms has emerged as a promising form of cancer treatment. This type of therapy involves the exogenous administration of a photosensitizer with subsequent exposure to light. The ensuing photochemical reaction results in destruction of the tumor. Whether tumor cells are destroyed directly by the photodynamic treatment or indirectly as a result of destruction of the tumor microvascular bed is unknown. To address this question, methods were adapted to test whether combinations of a photosensitizer and light resulted in direct cell killing of precision cut tissue slices placed in culture. The major advantages of this culture system are that photosensitizers are administered in vivo, tissue slices produced in minutes, placed in culture medium, and irradiated in vitro. Any resulting cellular destruction occurs in the absence of a functioning vascular system and indicates that photodynamic therapy acts through a direct cell killing mechanism. Tissue slice viability was monitored by two standard methods: assay for intracellular potassium and morphological examination at the electron microscopic level. The effects of hematoporphyrin derivative and light were examined on tissue slices produced from a prostate adenocarcinoma transplanted into male Copenhagen rats. The data indicate that direct killing of tumor slices occurs and is dependent on the irradiation protocol used.

Photodynamic therapy: effect on the endothelial cell of the rat aorta.

Previous studies in our laboratory have demonstrated that photodynamic therapy (PDT) of experimental bladder tumors leads to rapid destruction of the endothelial lining within the tumor microvasculature. Endothelial cell death during PDT may be a consequence of direct cell injury resulting from retention of photosensitizer within the endothelial cell or, alternatively, result from intravascular activation of circulating photosensitizer with subsequent indirect endothelial damage. In the experiments described here, we investigated the possibility that photosensitizer retained within the endothelial cell was sufficient to cause endothelial cell injury in the absence of circulating drug. The experimental model was rat aorta photosensitized in vivo via the intravenous injection of tin(II) etiopurpurin dichloride (SnET2), and subsequent in situ or in vitro (in explant culture) light (670 nm) treatment from an argon pumped dye laser. Damage to the lining of the aorta was assessed morphometrically by determining the areal density of silver stained endothelial cells. Results indicate that purpurin SnET2-PDT directly damages the endothelial lining.

Iminium salt of copper benzochlorin (CDS1), a novel photosensitizer for photodynamic therapy: mechanism of cell killing.

The mechanism of cell killing by CDS1, an iminium salt of octaethylbenzochlorin with copper in the aromatic ring, in combination with light from a noncoherent light source was investigated. Using a standard clonogenic assay and the AY-27 FANFT tumor line, photoactivation of CDS1 was shown to be cytotoxic. The photodynamic cell killing ability of CDS1 required the presence of molecular oxygen. The reactive species generated by light activation of CDS1 were effectively quenched by N,N'-diphenyl-p-phenylenediamine. Additionally, the photodynamic effect of CDS1 was not enhanced by deuterium oxide. To characterize the reactive oxygen species generated by the photoactivation of CDS1 the well-characterized erythrocyte ghost model was used. Superoxide dismutase and catalase were potent inhibitors of CDS1-induced lipid peroxidation of erythrocyte membranes. Sodium azide only partially inhibited lipid peroxidation. These findings differed from the known singlet oxygen generator, tin (II) etiopurpurin dichloride (SnET2). Sodium azide was a potent inhibitor of SnET2-induced lipid peroxidation, whereas superoxide dismutase and catalase were totally ineffective. Based on these results, we conclude that CDS1 requires the presence of molecular oxygen for cell killing to occur but appears to act primarily through a non-singlet oxygen mechanism.

Iminium salt benzochlorins: structure-activity relationship studies.

An iminium salt of copper(II) octaethylbenzochlorin (CDS1) is an effective new photosensitizer despite the fact that it does not produce singlet oxygen, does not fluoresce and the triplet state lifetime can only be less than 20 ns. A number of octaethylbenzochlorin derivatives were synthesized in order to determine the structural component(s) that is(are) responsible for the photodynamic action of these new photosensitizers. Studies utilizing the N-(4-[5-nitro-2-furyl]-2-thiazolyl)formamide-induced urothelial tumor revealed that the coexistence of the copper inside the aromatic ring and the iminium group at the meso position are required for the photodynamic effect.

Copper benzochlorin, a novel photosensitizer for photodynamic therapy: effects on a transplantable urothelial tumor.

An iminium salt of octaethylbenzochlorin with copper in the aromatic ring, CDS1, was tested for its tumoricidal effects on the AY-27 N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide tumor line. CDS1 was found to be an effective photosensitizer in vivo when used in combination with either a xenon arc lamp or a pulsed alexandrite laser. Hemodynamically, CDS1 and light caused a rapid decrease in tumor blood flow. Skin photosensitization was found to be minimal when drug-injected mice were illuminated in a solar simulator.

Effects of ibuprofen on the in vitro invasiveness of a human transitional cell carcinoma.

Non-steroidal anti-inflammatory drugs, such as ibuprofen, have demonstrated significant anti-cancer activity in both animals and humans. We examined the anti-invasive effects of ibuprofen on the human UM-UC urinary bladder carcinoma cell line using a rapid in vitro tumor cell invasion assay. The inhibitory effects of ibuprofen on the invasiveness and motility of the human UM-UC transitional cell carcinoma (TCC) cell line were evaluated using Matrigel coated polycarbonate filters (8 microns pore size) from Transwell cluster plates. In addition, the potential role of prostaglandin E2 in this process was examined. Ibuprofen exposure at non-cytotoxic concentrations resulted in a significant (p < 0.05) dose-dependent reduction of invasion when compared to vehicle exposed controls. Even at the highest concentration, ibuprofen had no effect on the rate of tumor cell division. Similarly, the highest concentration of ibuprofen did not alter tumor cell motility through uncoated 8 microns-pore polycarbonate filters. Addition of both prostaglandin E2 and ibuprofen to the culture medium restored tumor cell invasiveness through Matrigel-coated membranes to levels nearly identical to vehicle exposed controls (DMSO-no ibuprofen). The results indicate that ibuprofen is effective in preventing tumor cell invasion in this in vitro model. Prostaglandin E2 reverses the anti-invasive effects of ibuprofen. The anti-invasive effect of ibuprofen warrants further study alone or in combination with other therapies used in the treatment of early stage transitional cell carcinoma of the urinary bladder.

Conjuctions of visually based categories: overextension and compensation.

The degree to which overextension effects found with conjunctions of semantic categories, such as sports and games (J. A. Hampton, 1988), would generalize to categories of visual stimuli was tested in 4 experiments. Overextension occurs when participants categorize a stimulus in the conjunction of 2 categories but fail to categorize the same stimulus as belonging to 1 of the 2 constituent categories considered individually. Stimuli for the present experiments were ambiguous colored letter shapes and cartoon faces that could vary along dimensions of happiness and either apparent intelligence or apparent age. Overextension was found with both stimulus sets, thus showing that the phenomenon is not restricted to categorization in superordinate semantic categories. There was also evidence that typicality in 1 category could compensate for borderline membership of the other. More overextension was found for faces than for letters, and there was evidence for asymmetric compensation between category dimensions.

Predicting conjunction typicalities by component typicalities.

In two studies, we investigated to what extent typicalities in conjunctive concepts phrased as relative clauses--such as pets that are also birds--can be predicted from simple functions of constituent typicalities and from extensions of such functions. In a first study, analyses of a large aggregated data set, based on seven different experiments, showed that a calibrated minimum rule model and some extensions of this model accounted for a very large part of the variance in the conjunction typicalities. The same models can also account for the so-called guppy effect. A psychological explanation is presented, which states that typicalities in contrast categories, like pets that are not birds and birds that are not pets, further improve the prediction of conjunction typicalities. This hypothesis is tested in a second study.

Photodynamic therapy: a new modality for the treatment of cancer.

Photodynamic therapy is a promising new modality for the treatment of neoplastic disease. Currently, Photofrin is the only photosensitizer approved for the treatment of human cancers. In the search for new, chemically pure second generation photosensitizing agents which absorb in the deep red region of the visible spectrum, a novel and unique photosensitizer, CDS1, an iminium salt of copper octaethylbenzochlorin, was developed. This new photosensitizer is chemically pure, cationic, and possesses a strong (epsilon = 35000 M-1.cm-1) absorption peak at 750 nm (in dichloromethane). With copper in the aromatic cavity and a triplet lifetime which is not measurable (< 20 nsec), the photodynamic activity of CDS1 was unexpected. Preliminary in vitro and in vivo animal studies with a transplantable urothelial tumor indicate that CDS1 is an effective photosensitizing agent when used in conjunction with a broad band xenon arc light source or a low frequency, high peak power pulsed alexandrite laser.

Associative and similarity-based processes in categorization decisions.

Two experiments were directed at distinguishing associative and similarity-based accounts of systematic differences in categorization time for different items in natural categories. Experiment 1 investigated the correlation of categorization time with three measures of instance centrality in a category. Production frequency (PF), rated typicality, and familiarity from category norms for British participants (Hampton & Gardiner, 1983) were used to predict mean categorization times for 531 words in 12 semantic categories. PF and typicality (but not familarity) were found to make significant and independent contributions to categorization time. Error rates were related only to typicality (apart from errors made to ambiguous or unknown items). Experiment 2 provided a further dissociation of PF and typicality. Manipulating the difficulty of the task through the relatedness of the false items interacted primarily with the effect of typicality on categorization time, whereas, under conditions of easy discrimination, prior exposure to the category exemplars affected only the contribution of PF to the decision time. The dissociation of typicality and PF measures is interpreted as providing evidence that speeded categorization involves both retrieval of associations indexed by PF and a similarity-based decision process indexed by typicality.

Conceptual combination: conjunction and negation of natural concepts.

The operation of negation on combinations of natural categories was examined in two experiments. In the first, category membership ratings of lists of items were obtained for pairs of concepts considered individually and in two logical combinations: conjunctions (e.g., "Tools which are also Weapons") and negated conjunctions--forms of those conjunctions in which the modifier noun category was negated ("Tools which are not Weapons"). For conjunctions, results supported earlier findings of overextension and the geometric averaging of constituent membership values (Hampton, 1988b). Previous findings of concept dominance and noncommutativity within conjunctions were also replicated, both for typicality ratings and for probability of class membership. For negated conjunctions, the pattern of dominance was similar but interacted with order within the conjunction. Negated conjunctions were also overextended. The second experiment explored how the attributes of negated conjunctions were derived from those of the two component concepts. Frequency of generation of attributes expressed positively (has wheels) or negatively (has no wheels) followed rated frequency in the negated category. The distinctiveness of an attribute to distinguish the complement from the head-noun class was associated with the generation of attributes, particularly when there was relatively high overlap between the two categories.

Exposure to alkyllysophospholipids inhibits in vitro invasion of transitional cell carcinoma.

Alkyllysophospholipids (ALP) are a group of synthetic analogs of a naturally occurring 2-lysophosphocholine. They are directly cytotoxic to a variety of neoplastic cell lines and can modulate the activation of macrophages against tumor cells. Moreover, recent reports have demonstrated the ability of racemic 1-octadecyl-2-methyl-sn-glycero-3-phosphocholine (ET-18-O-CH3) to prevent tumor cell invasion when given in noncytotoxic concentrations. Using an in vitro model, we studied the ability of ET-18-O-CH3 to prevent transitional cell carcinoma invasion. Cytostatic activity was determined by clonal growth assay (25,000 cells per plate). Suppression of colony growth was found at concentrations greater than 4 micrograms/ml. of ET-18-O-CH3 in rat and mouse transitional cell carcinoma (TCC) cell lines and greater than 2 micrograms/ml. in a human TCC line. Inhibition of tumor cell invasion was assessed by the effects on cell migration through Matrigel-coated 8 microns-pore polycarbonate filters (using 1 x 10(5) cells per chamber). Invasion was reduced to 50 to 70% of controls in both the mouse and rat TCC lines at the highest concentration (4.0 micrograms/ml.). In the human TCC line, invasion was reduced to less than 30% of controls at concentrations as low as 0.5 micrograms/ml. Motility (without invasion) of the human TCC line, as measured by cell migration through the micropore filter without the Matrigel coating, was inhibited at the same concentration of ET-18-O-CH3 found to inhibit invasion. The anti-invasive effect seen with noncytotoxic concentrations of ALP may prove useful in the treatment of transitional cell carcinoma.

Resident sinusoidal macrophages in the liver of the brown bullhead (Ictalurus nebulosus): an ultrastructural, functional and cytochemical study.

Ultrastructural, functional, and cytochemical characteristics of resident sinusoidal macrophages (RSM) in brown bullhead (Ictalurus nebulosus) liver were examined. Following perfusion fixation of the hepatic vascular bed, light micrographs revealed RSM that possessed multiple elongate cytoplasmic processes and frequently contained erythrocytes in various stages of degradation. Following brief perfusion fixation, light microscope examination of vibratome sections of bullhead liver reacted for peroxidase revealed intensely positive RSM. By transmission electron microscopy, peroxidase activity was localized to the nuclear envelope and cytoplasmic granules of RSM and in endothelial and perisinusoidal fat-storing cells. In cryostat sections of fresh-frozen liver, glucose-6-phosphate dehydrogenase (G-6-PDH) was uniformly distributed over hepatocytes, whereas intensely positive punctate staining for G-6-PDH was localized over RSM. To test for phagocytosis by RSM, latex beads (0.81 micron) were injected into a tributary of the hepatic portal vein 2 min prior to perfusion fixation. Latex beads appeared either singly or in dense aggregates within RSM. Ultrastructurally, RSM were characterized by an irregularly shaped, eccentrically located nucleus, electron-dense vacuoles, small patches of granular endoplasmic reticulum, a well-developed Golgi apparatus, elongated mitochondria, desmosomes or desmosome-like densities that served as a source of attachment to endothelial cells, and a centriole with radiating microtubules. Invaginations of the plasma membrane (vermiform processes) characteristic of mammalian Kupffer cells were not observed in bullhead RSM. The results indicated a resident cell population of sinusoidal macrophages in the bullhead liver with properties that partially resembled mammalian Kupffer cells. These results are important for the identification of the normal resident cells in the bullhead liver.

Functional units in rainbow trout (Salmo gairdneri, Richardson) liver: III. Morphometric analysis of parenchyma, stroma, and component cell types.

Hepatic stroma and parenchyma with its component cell types were quantitatively described in adult male and female actively-spawning 5-year-old rainbow trout (Salmo gairdneri, Richardson). Point-count morphometry of glycol methacrylate sections estimated volume compartments for stroma and parenchyma. Veins composed 85% of the stroma while arteries and bile ducts occupied approximately 6-7% each. Parenchyma accounted for 95% of hepatic volume. Point-count morphometry of transmission electron micrographs estimated volume compartments as well as numerical and surface density measurements for parenchymal components. Within the hepatic parenchymal compartment, hepatocytes occupied 85% and showed significant sex differences. Female hepatocytes were significantly more numerous but were smaller, only 60% of the volume of male hepatocytes. Since hepatocyte nuclear volume was equal in both sexes, differences were due to reduced cytoplasmic volume in females. Perisinusoidal macrophages of females occupied larger volumes of their respective parenchymal compartments, and their larger mean cytoplasmic volumes suggested activation. Biliary epithelial cells of preductules and ductules were numerous. Ratios of numerical density of hepatocytes to biliary epithelial cells were consistent with a tubular arrangement of hepatocytes. Factors possibly mediating the sexual dimorphism are discussed.

Transperineal photodynamic ablation of the canine prostate.

PURPOSE: Experiments were undertaken to determine the effects of transperineal interstitial photodynamic therapy on the canine prostate. MATERIALS AND METHODS: Mongrel dogs were injected intravenously with the photosensitizer, tin (II) ethyl etiopurpurin dichloride. Twenty-four hours later, 2 optical fibers were implanted in 1 hemisphere of the prostate, which was then treated with red light (660 nm.). RESULTS: Acutely, the treated areas showed extensive hemorrhagic necrosis. At 3 and 6 weeks, the treated lobes were largely replaced by fibrous connective tissue. CONCLUSION: Transperineal photodynamic therapy of the canine prostate is feasible. Further preclinical investigation is warranted to determine the applicability of this approach to the treatment of localized prostate cancer.