Photoradiation therapy of cutaneous angiosarcomas in mice.

Cutaneous angiosarcoma is a relatively rare but devastating malignant vascular tumor. It has a high incidence of recurrence following conventional therapeutic modalities applied either singly or in combination. The increased vascularity of cutaneous angiosarcomas, facilitating selective uptake and retention of a photosensitizing agent, such as hematoporphyrin derivative (HPD), suggests that these tumors would respond well to photoradiation therapy. To study the feasibility of this treatment modality, transplantable hemangiosarcomas were implanted in B6C3F1 female mice. Within 2.5 to 3.5 hours after intraperitoneal administration of HPD, fluorescence was recorded in the tumor as compared with surrounding normal skin. When these photosensitized tumors were exposed to 70 J/cm2 of laser energy from an argon-pumped dye laser at 630 nm, the tumors showed marked necrosis within 24 hours. In another series, the tumors were initially photosensitized with HPD for 3 hours and then treated with laser energy ranging from 0 to 96 J/cm2. A dual labeling procedure demonstrated a dose-related decrease in DNA synthesis rate in tumors that were exposed to 0 to 30 J/cm2 at 24 hours after treatment. Furthermore, tumor tissue exposed to laser energy in excess of 30 J/cm2 showed no significant cellular DNA synthesis. These data, supported by histologic evidence of tissue destruction, suggest that photoradiation therapy has a great potential as a therapeutic modality for cutaneous angiosarcomas.

Phototoxicity of brain tissue in hematoporphyrin derivative treated mice.

Photoradiation therapy conditions which have been used to treat subcutaneous and breast tumors are lethal when applied to the head of mice. Treatment of control mice with laser light at 631 nm over an energy density range of 0-90j/cm2 had no measurable effect but mice photosensitized with 5 mg HPD/kg 72 hrs prior to laser treatment showed a threshold for brain damage at 56j/cm2, above which the mice developed cerebral edema and died. Laser treatment caused the same rate and magnitude of temperature rise in both control and HPD-photosensitized mice. Moreover, studies using mice whose brain temperature was kept below 37 degrees C during laser treatment showed a greater phototoxicity than mice without temperature regulation. Therefore, temperature rise in cerebral tissue was not associated with phototoxicity in the brain. In contrast the oxygen consumption rate in a brain cell suspension from an HPD-treated mouse was only 54% of that from a control mouse following treatment with laser light. This observation, when taken with supporting data from other investigations, suggests that one mechanism for the phototoxic response in brain tissue is oxygen deprivation resulting from mitochondrial damage.

Development of a protocol for photoradiation therapy of malignant brain tumors: part 1. Photosensitization of normal brain tissue with hematoporphyrin derivative.

The successful application of phototherapy to subcutaneous tumors has suggested that a similar procedure should be developed for treating gliomas. As a result, attempts are being made to determine a set of conditions that would optimize the destruction of tumor cells while minimizing injury to surrounding brain tissue. To initiate this task, we developed a novel assay method to assess the amount of phototoxicity induced in normal brain by light exposure of mice treated with hematoporphyrin derivative (HPD). The application of this procedure demonstrated that a sufficient amount of HPD was retained in brain tissue, even 72 hours after injection, to cause severe cerebral damage in light-treated mice.

Ozone sensitivity in aging WI-38 cells based on acid phosphatase content.

Young [16-19 population doubling level (PDL)] and senescing (50-53 PDL) WI-38 cell populations were exposed to 1 ppm ozone for 2 hr and the resultant extracellular and intracellular acid phosphatase concentration was measured. Dose-response curves were also determined for surviving populations of young and old cells after a 1 hr ozone exposure ranging in concentration from 0 to 1.00 ppm. Senescing cells released 8 times more acid phosphatase per million cells than the young cells. Both old and young cells showed a clear dose-response to the 1 hr ozone gradient exposure. However, the older cells demonstrated a consistent 17% average lower survival rate than the young cells. The higher acid hydrolase level in older WI-38 cells is probably related to the lower survival rate observed in the older cells in vitro.

Bioassay procedure for the detection of mutagenic metabolites in human urine with the use of sister chromatid exchange analysis.

A short-term bioassay system for the detection of activated mutagenic metabolites in urine from humans exposed to promutagens was described. Human diploid fibroblasts were grown in medium containing 5--20% urine from smokers, from nonsmokers, and from individuals undergoing cyclophosphamide (Cp) chemotherapy for treatment of cancer. The cells were then subjected to sister chromatid exchange (SCE) analysis. Activated Cp metabolic products in urine specimens produced up to a ten-fold increase in SCE's over preinjection SCE levels for the same individuals. Linear dose-response curves over a urine concentration range from 5 to 20% in culture medium were obtained from cells grown in urine specimens from 7 nonsmokers and 8 cigarette smokers. This test system proved to be sensitive to ambient exposure levels of environmental mutagens and demonstrated that urine from smokers was significantly more mutagenic than was urine from nonsmokers. Replicate experiments showed highly reproducible SCE values for each individual as well as for average SCE values for each group of subjects. The ability of this bioassay system to detect trace mutagenic activity in human urine reproducibly makes it an attractive choice for the monitoring of humans who have been exposed to environmental and/or industrial mutagens.

An improved organ culture method for adult mammalian lung.

An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated with agar and 200-micron-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface. Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can be identified in the living tissue. The explants were studied with time-lapse cinematography, light microcoscopy, histology, and erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased from 1 week to 1 month.

Consideration of atherosclerotic plaques as benign neoplasms.

The monoclonal nature of atherosclerotic plaques, as well as their altered cell size, proliferation rate and function have led to the hypothesis that plaque tissue is analogous to benign tumors. To test this hypothesis, we have studied rates of DNA release by histologically-normal human arterial wall tissue and by early atherosclerotic plaques during incubation in a maintenance medium. No DNA was detected in media conditioned by normal arterial tissue after a 24 h incubation period, but plaque tissue (like benign tumors) released 10-100 ng DNA/ml during this period. These data offer additional support for the thesis that atherosclerotic plaques are similar to benign tumors.

Light- and electron-microscopic characteristics of artrial smooth muscle cell cultures subjected to hypoxia or carbon monoxide.

Smooth muscle cells from the tunica media of piglet aortae grown under hypoxic conditions undergo the following changes: First, they become modified by partial loss of myofilaments and proliferation of organelles, which are characteristics of young primitive cells. Second, an increase in number of pinocytotic vesicles at and below the cell surface, indicating increased extracellular uptake of material, can be detected. This is followed by accumulation of Oil Red O positive intracytoplasmic granules and vacuoles as well as the subsequent formation of mount-like protrusions. The latter consist of a core of extracellular material and necrotic debris covered with a cap of viable cells. A third feature of the cells subjected to hypoxia is a conspicuous rise in the number of lysosomes. This is considered to be a manifestation of a defense mechanism of the cells to remove undesirable material from cytoplasm. Cells exposed to an atmosphere rich in carbon monoxide exhibit basically the same alterations as those grown under hypoxic conditions; however, formation of mound-like aggregates is less prominent, while the rise in the number of lysosomes is more evident than in the hypoxic cells. The above alterations are similar to changes observed in smooth muscle cells of rabbit with experimental atherosclerosis. It is suggested that whereever the arterial smooth muscle cell is subjected to adverse conditions basically the same mechanism, consisting of dedifferentiation, increased permeability and lysosomal defense reaction, takes place.

Measurement of the cell migration index with a HeNe laser.

Human diploid fibroblasts (WI38) were inoculated into Rose multipurpose culture chambers, using a high population density to develop confluency. After 24 hours, cells from a 1 mm swath were scraped from the center of the chamber. This cleared path was positioned to permit an unobstructed transmission of a 0.9 X 20 mm beam of light from a 1 mW HeNe laser. As cells migrated, at 37 degrees C, into the path of the laser beam the light scatter was recorded, using a photomultiplier tube. Because the amount of light scatter was proportional to the number of cells migrating into the beam, the system measured the migration index of the cells. Slight variations in the design of this device could facilitate data collection during surveys of toxic agents, fertility tests, and delayed hypersensitivity.

Production of ring-shaped particles by normal and metaplastic tissue. I. Human skin.

Maintenance media incubated with biopsy specimens of human skin tissues contained minute (10-12 nm wide), ring-shaped particles (RSP) similar to those described previously in culture media of mammalian cell lines. In addition to the qualitative demonstration of the particles by electron microscopy, a quantitative method was applied to estimate in media the amount of DNA that could be attributed primarily to RSP content. The amounts of DNA, obtained with 146 test specimens, varied with the pathologic condition of the tissue in the following ascending order: normal skin, verruca vulgaris, seborrheic verruca, actinic keratosis, basal cell carcinoma, and squamous cell carcinoma.

The induction of atherosclerotic plaque-like mounds in cultures of aortic smooth muscle cells.

Smooth muscle cells harvested from the tunica media of piglet aortae were maintained in continous culture for 10 months. When grown in the presence of 95% air and 5% CO2 they maintained a mature morphology as evaluated ultrastructurally. As these populations became confluent, the cells became oriented parallel to each other. When grown in the presence of 4% O2, 91% N2, and 5% CO2, this polarized pattern was disrupted. Focal areas of lipid accumulation were observed, succeeded by mound formation at these sites. The mound stained positive with PAS, aldehyde fuchsin, and oil red O. They were surrounded by 2-4 layers of intact cells. The centers of the mound were composed of extracellular material and cell debris.