In vivo pH MEASUREMENT AND IMAGING OF TUMOR TISSUE USING A pH-SENSITIVE FLUORESCENT PROBE (5,6–CARBOXYFLUORESCEIN): INSTRUMENTAL AND EXPERIMENTAL STUDIES

Abstract This study evaluated the effectiveness of dual-wavelength ratio fluorescence imaging using a pH-dependent indicator (5,6–carboxyfluorescein, 5,6–CF) for in vivo pH mapping of tissue. A prototype version of a highly sensitive fluorescence imaging device consisting of a modified xenon lamp, an image-intensified camera and a digital imageprocessing system has been developed. 5,6–Carboxyfluorescein was used because its fluorescence emission increases as a function of pH in the physiological (6.0–7.4) pH range. The ratio of fluorescence intensities obtained with the imaging system has been calibrated using aqueous 5,6–CF standards at various pH values. Because the pH of interstitial fluid of malignant tumors tends to be lower than that of normal tissue and can be depressed by glucose administration, experiments were performed on 10 CDF mice bearing lymphoid leukemia P388 grafted subcutaneously. The range of linearity of the calibration curve was obtained between 5.3 and 6.7 with a measured pK, value of 5.93. Consequently the maximum sensitivity was observed in this range. The calculated pH from ratio images was 6.21 ± 0.12 in tumorous tissue. This value was equivalent to those obtained at the same time using microelectrodes (6.2 ± 0.3).
These experiments showed that a dose of 5 mg/kg 5,6–CF and an excitation power density of 2.5 mW/cm² are sufficient to give a fluorescent pH image of tumors. The limitation of 5,6–CF for the in vivo mapping of tissue results from its low pK_a and consequent range of sensitivity. The advantages of this imaging technique compared to microelectrodes are that it (1) is noninvasive, (2) displays a two-dimensional pH image with high resolution (profile distribution of pH in tissue) and (3) can be used to monitor pH over a few hours.     

EFFICACY OF PHOTODYNAMIC THERAPY ON ORIGINAL AND RECURRENT RAT MAMMARY TUMORS

Abstract— Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of ˜ 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [¹⁴C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.     

CAROTENOID PIGMENT ADMINISTRATION and DELAY IN DEVELOPMENT OF UV-B-INDUCED TUMORS

—The results of two studies are reported. In the first study, the carotenoid pigments, β-carotene. canthaxanthin and phytoene were administered to mice after one UV-B-induced skin tumor had developed, to see if carotenoid administration would delay the development of subsequent tumors. Canthaxanthin significantly delayed the development of subsequent tumors. In the second study, the same pigments were administered starting 10 weeks before, and for 24 weeks after, exposure to a large single fluence (8 × 10⁴J/m²) of UV-B radiation, to determine the effect of the pigments on tumor development. Phytoene significantly prevented the development of tumors to this fluence of radiation.     

IMPROVING THE EFFICIENCY OF HEMATOPORPHYRIN PHOTOTHERAPY OF OCULAR TUMORS

Malignant melanoma of the choroid is the most common primary intraocular tumor in adult humans. Controversy exists over which is the most effective therapy. One therapeutic modality that has not been thoroughly investigated is hematoporphyrin derivative phototherapy (HPdPRT), a technique used successfully in the clinic on many non-ocular tumors.
The effect of HPd PRT on an ocular, amelanotic melanoma was evaluated using Greene melanoma implanted on pigmented rabbit iris. The parameters used clinically on non-ocular tumors (intravenous 2.5 mg HPd kg^-1 body weight and irradiation at 633 nm 48 h later) were totally ineffective in killing Greene melanoma implanted on the iris. The dose of dye (2.5–5 mg kg^-1), wavelength of light (500-700 nm), and illumination intensity were varied to determine the most efficient parameters for treating this tumor. The most important parameter was dye dose; increasing it to 5 mg kg^-1 resulted in some control of tumor growth. Administering 100% O₂ prior to and during irradiation also improved HPd PRT cytotoxicity. The use of pulsed light (pulses of 1 or 2 min) further enhanced killing and reduced the length of irradiation needed. These studies suggested that HPd PRT might be used efficiently on ocular melanomas.     

DRUG AND LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR CELL CLONOGENICITY AND HISTOLOGIC CHANGES

Abstract— The dependence of photodynamic therapy (PDT) on changes in drug and light doses was determined in C3H/HeJ mice bearing the RIF tumor. Measurements of tumor clonogenicity were determined 24 h after PDT over a range of drug and light doses. Representative histological samples were prepared at each of these doses. Both the drug and light dose dependence experiments showed an exponential decrease in clonogenicity after an initial shoulder region. Reciprocity of drug and light dose was established from those clonogenicity curves. Histological examination of tumors gave information concerning the localization of gross damage within tumors. Increases of light dose in PDT were shown to extend the depth of necrosis within tumors. Increases of drug dose produced enlargements in the area of necrotic spots produced by PDT     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

IDENTIFICATION OF A TRANSFORMING ras ONCOGENE IN AN ULTRAVIOLET RADIATION-INDUCED CORNEAL TUMOR OF Monodelphis domestica

Chronic exposure of the gray, short-tailed oppossum, Monodelphis domestica to ultraviolet radiation (UVR) induces mesenchymal tumors of the cornea. High molecular weight DNA samples from 6 UVR-induced corneal tumors were assayed for their ability to transform NIH 3T3 cells to tumorigenicity. NIH 3T3 cells transfected with DNA from 5 of the corneal tumors produced 14 tumors in nude mice. Cell lines were established from these tumors. DNA from 13 of 14 tumor cell lines contained repetitive opossum DNA sequences. Southern blot analysis revealed that DNA from 3 of 4 cell lines derived from tumorigenic NIH 3T3 cells transfected with DNA from a single oppossum tumor contained opossum Ki-ras oncogene sequences in addition to the murine Ki-ras gene. Northern blot analysis of mRNA from a mouse tumor cell line containing opossum Ki-ras gene sequences showed mRNA species identical in size to opossum Ki-ras mRNA, as well as murine Ki-ras mRNA species. These results suggest that an activated Ki-ras oncogene was present in one of the original opossum corneal tumors tested. Thus, activation of Ki-ras may play a role in the development of UVR-induced corneal tumors in Monodelphis domestica. Further characterization of ras oncogenes in these opossum tumors may provide information on the molecular mechanisms by which UVR induces corneal tumors in this species.     

CAROTENOIDS AFFECT DEVELOPMENT OF UV-B INDUCED SKIN CANCER

One large dose of UV-B (8 × 10¹⁴ J m^-2: 290-320 nm) has been found to cause the development of skin tumors in hairless mice, and carotenoid pigments prevent or delay tumor development in this system. This model was used to determine if the protective effect of carotenoid pigments against UV-B induced skin tumors occurred during the induction phase or progression phase of UV-B carcinogenesis. The results indicate that the carotenoids canthaxanthin and (J-carotene interfered with the progression phase in this system; whether they also had an effect during induction could not be definitely determined from our data.     

In vivo TUMOR OXYGEN TENSION MEASUREMENTS FOR THE EVALUATION OF THE EFFICIENCY OF PHOTODYNAMIC THERAPY

Among the sequence of events which occur during photodynamic therapy (PDT) are depletion of oxygen and disruption of tumor blood flow. In order to more clearly understand these phenomena we have utilized transcutaneous oxygen electrodes to monitor tissue oxygen disappearance. These results provide, for the first time, non-invasive real-time information regarding the influence of light dose on tissue oxygenation during irradiation. Measurements were conducted on transplanted VX-2 skin carcinomas grown in the ears of New Zealand white rabbits. Rabbits were treated with Photofrin II and tumors were irradiated with up to 200 kJ/m2 (500 W/m2) of 630-nm light. Substantial reductions in tumor oxygen tension were observed upon administration of as little as 20 kJ/m2. For a series of brief irradiations, oxygen tension was modulated by the appearance of laser light. Tissue oxygen reversibility appeared to be dependent upon PDT dose. Long-term, irreversible tissue hypoxia was recorded in tumors for large (200 kJ/m2) fluences. These results suggest that transcutaneous oxygen tension may be useful as a general indicator of the effectiveness of PDT and as an in situ predictor of the energy required to elicit tumor damage.     

SYSTEMIC INFLUENCE OF PRE-IRRADIATION OF A LIMITED SKIN AREA ON UV-TUMORIGENESIS

Abstract— It has been established that chronic UV irradiation of mice produces a systemic effect. The animals become incapable of rejecting an implanted UV-induced tumor. A possible consequence of the induction of this systemic effect could be an enhancement of the de novo formation of tumors by chronic UV irradiation. We have therefore carried out an investigation to determine whether such an effect is demonstrable in an animal model. Hairless mice (Skh hr 1) were pre-irradiated for a few months with UV radiation while certain skin areas of the animals were shielded from the radiation. Subsequently, the initially shielded skin areas were chronically exposed to UV radiation, which resulted in the development of tumors in these skin areas. It was found that the formation of tumors in the initially shielded skin areas was enhanced by the pre-irradiation of the other skin areas. Thus, a systemic effect appeared to have influenced the development of tumors in the initially shielded skin areas.     

Insulin binding and glucose transport in the R3230AC mammary adenocarcinoma.

Cells dissociated from the R3230AC mammary adenocarcinoma from intact and diabetic rats were examined for insulin binding and glucose transport. The Kd for insulin binding, approximately 10(--10) M, was similar in all tumors studied. However, the apparent number of receptor sites per cell increased in cells from diabetic rats. Kinetic analysis of 3-0-methyl glucose (3-OMG) entry showed both diffusional and passive carrier characteristics. Insulin (4 X 10(--9) M) in vitro did not affect diffusional entry, whereas the hormone altered the passive carrier system, as reflected by an increase in Km and Vmax. Insulin decreased initial velocity of glucose transport at 4--6 mM glucose levels but increased initial velocity of glucose transport at 20 mM glucose. An explanation of the role of insulin on tumor growth in vivo from effects on glucose transport in vitro is proposed.     

INDUCTION OF SKIN TUMORS IN HAIRLESS MICE BY A SINGLE EXPOSURE TO UV RADIATION*

Abstract— Skin tumors were induced in hairless mutant mice following a single exposure to ultraviolet radiation (UV). Tumors were first noted as early as 7 weeks following irradiation. The UV, emitted by FS20/40T12 fluorescent lamps, was principally in the 280–320 nm spectral region with a peak at 313 nm. Single (skin surface) doses of 3 times 10⁴ J/m² to 24 times 10⁴ J/m² were delivered in 3 h or less. The higher doses resulted in more severe acute damage as well as greater tumor yield. Most of the tumors were benign hyperplastic epithelial papillomas; 4 out of 96 tumors examined histologically proved to be squamous cell carcinomas. This appears to be the first report of experimental carcinogenesis due to a single UV exposure, not requiring exogenous chemical promotion.     

以葡萄糖为碳源合成生物降解性聚酯的研究

利用从油田土壤中筛选的菌种ＤＧ１７ 以葡萄糖为碳源通过微生物发酵法合成了具有不同结构单元的新型生物可降解性聚合物———聚羟基脂肪酸酯（ＰＨＡｓ） ．初步研究了ＤＧ１７ 以葡萄糖为碳源的生物合成规律,并借助ＧＣ、ＮＭＲ 等分析手段对合成的聚合物进行了结构的分析表征,另外还研究了ＰＨＡｓ 的活性污泥降解情况．研究表明,在限氮条件下,只有碳氮比高于５后,ＤＧ１７ 才能在其体内合成ＰＨＡｓ．在过量碳源的存在下,氮磷比低,得到的聚合物是一种具长侧链的聚（ 羟基辛酸 ｃｏ 羟基癸酸） 的共聚物,为一种热塑性弹性体．在硫酸铵浓度为０－５ｇ／Ｌ,碳氮比为２０ 条件下合成的Ｐ（ＨＯ ｃｏ ＨＤ） 热塑性弹性体的数均分子量为１－１６ ×１０ － ５ ,分子量分散指数为２－４３ ．其玻璃化温度及熔融温度分别为Ｔｇ ＝ － ５２ ℃,Ｔｍ ＝ ５０ ℃．氮磷比高,则合成热塑性塑料ＰＨＢ．结果表明培养基中氮源与磷酸盐的相对浓度是影响ＤＧ１７ 生物合成路径的重要条件．     

SYNTHESIS OF PORPHYRIN NITROGEN MUSTARDS AND THEIR ANTI-TUMOR ACTIVITIES

With the development of laser medicine and technology in light-conducted fiber and endoscope, photodynamic therapy (PDT) obtained regulatory approvals in the United States and elsewhere to cure malignent tumors in skin, esophagi, bronchi and bladder for it's selective tumoricidal effects without the serious side effects of conventional therapy in a variety of tumors. We focused our attention on both the alleviation of patient's suffering from damages of normal tissues in chemotherapy and the eliminating of cutaneous photosensitivity as well as the enhancement of the depth of tumor necrosis which is generally not more than 8-10 nm even under the maximum safe light dose in PDT. Thus, a series of new compounds porphyrin nitrogen mustards, namely, 2,7,12,18-tetramethyl-13,17,di[3-N,N-di(2-chloroethyl) amino propyl] porphin (I₁) and 2,7,12,18-tetramethyl-3,8-di(l-alkyloxyethyl)-13,17-bis[3-N, N-di(2-chloroethyl)amino propyl]porphin (I_2-8) were synthesized(I) by introducing the alkylating agent nitrogen mustard into the periphery of porphyrin(Scheme 1). The mean overall yield of I_2-8 is 13%.     

HIGH DOSE PHOTOIRRADIATION THERAPY OF HUMAN TUMORS USING THE GOLD METAL VAPOR LASER

Abstract The gold metal vapor laser (GMVL; Quentron, Adelaide, South Australia) produces up to 6.0 W of power and this has allowed the study of high dose and high dose rate photoirradiation therapy. We have undertaken 35 treatments in 31 patients with tumors in the rectum (4), esophagus (8), brain (17) and malignant ascites (2). All patients were carefully monitored for complications and, where possible, assessed for response. Dose rates of 1.2-2.2 W were used and doses were estimated to be between 72 and 245 J cm⁻². The major complications noted were in the patients with esophageal cancer and included fever 1-10 days following therapy in 5/8. There was one early death at 10 days and 3/8 patients developed mediastinitis. Of the 4 anorectal tumors treated (8 treatments) there was one bleed 10 days after treatment and no other local complications. One of 2 patients with malignant ascites developed a bowel obstruction immediately after treatment. Of the 17 patients with glioma there was 1 early death at day 10 and no local complications. We conclude that phototherapy of the gastrointestinal tract with the GMVL is tolerable using high dose rates and doses but significant complications occur. Therapy of intra-cranial tumors with similar doses and dose rates is not associated with local complications. The basis for this may be the different anatomy, the content of HpD in different sites, and use of steroids in cerebral tumors, or the better control of hyperthermia in the brain.     

THE INFLUENCE OF NUTRITION ON THE DNA CONTENT OF ESCHERICHZA COLI AND ITS RESPONSE TO ULTRAVIOLET LIGHT

Abstract— The influence of nutrition on the sensitivity of Escherichia coli 15 T- to ultraviolet light (u.v.) and the synthesis of DNA has been studied. Growth in media containing glucose or NH,⁺ has been found to endow cells with a greater resistance to lethal u.v. damage than those grown in media containing succinate or amino acids, respectively. In addition, the sensitivity of the lactose ( lac ) locus of the DNA to mutagenic damage has been found to be altered by changes in the carbon supply but not by changes in the nitrogen source, while the sensitivity of loci controlling amino acid synthesis was altered by changes in the nitrogen source but not in the carbon source. Cells fed with glucose or NH₄⁺ have been found to possess more DNA than cells fed with succinate or amino acids, respectively. The data indicate that the type of carbon and nitrogen supplied to the cells will determine whether or not set regions of the DNA will undergo more than one round of replication. The presence in the cell of identical genetic loci either in duplicate or in multiples, directed by the particular types of carbon and nitrogen supplied, is suggested to be, in part, the reason why an alteration in nutrition is able to influence the sensitivity of bacterial cells to radiation.     

THE DOSE-RESPONSE RELATIONSHIP OF TUMORIGENESIS BY ULTRAVIOLET RADIATION OF 254 nm

Abstract— Groups of albino hairless mice, Skh-hrl, were exposed daily to UVC radiation from low pressure Hg arcs (Philips TUV 40W). These lamps emit predominantly radiation of 254 nm. Three groups of animals were used in the experiments, each receiving a different daily dose.
The results were described with the Weibull probability function. As in earlier studies with UVB. the tumor induction time was proportional to a power of the daily dose. The exponent turned out to be as low as -0.2. This implies that the induction time varied only a little with the daily dose. The average number of tumors per animal was proportional to a power of time. A sample of 73 tumors of at least 4 mm in diameter were investigated histologically. The large majority were classified as squamous cell carcinomas.
A comparison was made with the results of an earlier reported experiment with Westinghouse FS40 sunlamps. Throughout the whole range of daily doses used in the present experiment, UVC was less carcinogenic than UVB. An intriguing difference between the two types of radiation was that the tumors induced by UVC appeared much more scattered over the irradiated parts of the animals than the UVB-induced tumors.     

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

PROTECTIVE ACTION OF CAROTENOID PIGMENTS AGAINST PHOTODYNAMIC DAMAGE TO LIPOSOMES*

Abstract— When liposomes (a model membrane system) are subjected to a dye-sensitized photo-oxidation, lysis, as measured by glucose leakage or a change in light scattering, results. Before lysis occurs, the membrane lipids undergo peroxidative damage, as determined by the appearance of malondialdehyde. Carotenoids incorporated into the liposomal membrane protect against both lipid peroxidation and liposomal lysis. Other ¹O₂ quenchers and free radical absorbers also protect liposomes from photodynamic damage.