Carmen M. Wilmot

Spin traps: in vitro toxicity and stability of radical adducts.

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3). The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.

In Vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy

Abstract In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)–induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 ± 25 nm emission band relative to the fluorescence intensity integrated over the entire 400–600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.

Comparison of EPR oximetry and Eppendorf polarographic electrode assessments of rat brain PtO2.

EPR oximetry is a promising, relatively non-invasive method for monitoring the partial pressure of oxygen in tissue (PtO2) that has proved useful in following changes under various physiologic and pathophysiologic conditions. Optimal utilization of the method will be facilitated by systematic comparisons with other available methods. Here we report on the absolute values of rat brain PtO2 using EPR and the more widely used Eppendorf polarographic microelectrode system in the same brain. EPR used an L-band (1.2 GHz) spectrometer and implanted lithium phthalocyanine (LiPc) as the oxygen-sensitive paramagnetic material. Eppendorf measurements were made by a needle probe moved vertically through the cortex at 0.5 mm intervals in three tracks including one adjacent to the location of the LiPc. Several conclusions were drawn, including, (1) the average PtO2 measured by the two methods was similar but EPR reported a significantly higher average PtO2, (2) there was poor correlation between the values in the same animal on the same side of the brain, (3) the Eppendorf reported a larger range of values and (4) the heterogeneity of oxygen levels in the brain and the areas sampled by the two methods provide an adequate explanation for the observed differences.

Spatial heterogeneity and temporal kinetics of photosensitizer (AlPcS2) concentration in murine tumors RIF-1 and MTG-B

In this study we compared the photosensitizer concentration in two experimental murine tumors using an in situ fluorescence detection instrument to examine temporal and spatial variations, after intravenous versus intratumor injection. Also, the variations in the estimate as detected by large area sampling and micro-region sampling are compared, in order to determine what the inter-tissue and inter-animal variations are, and how the method of sampling affects this estimate. The latter study was carried out ex vivo in the same tumors, which had been harvested and frozen after in vivo measurements were made. The photosensitizer, disulphonated aluminum phthalocyanine (AlPcS2) was injected either intravenously (IV) or directly into the tumor (ITu), using two murine models, MTG-B (mammary adenocarcinoma) and RIF-1 (radiation-induced fibrosarcoma) grown subcutaneously on the flank. An in situ microsampling fluorescence probe was used to assess photosensitizer concentration, through real-time measurement of the remitted intensity. The photosensitizer concentration was evaluated at 8 time endpoints between 15 min and 48 h post-injection. Inter-tumor and intra-tumor variations were assessed by repeated samples from the tumor tissues. The average photosensitizer level reaches a peak between 3 to 6 h in both tumor and normal tissues using IV administration, but peaks within 1 h following ITu administration. MTG-B tumors demonstrated a factor of 2 higher uptake than RIF-1 tumors. The pharmacokinetic uptake rates of the RIF-1 tumor were 3 times faster than for MTG-B, while there was no statistical difference in their clearance rates. Preferential uptake of AlPcS2 by both tumors compared to contra-lateral flank subcutaneous normal tissue was documented, with ITu injection exceeding IV injection by a factor of 10 in the tumor to normal tissue ratio. Inter-animal standard deviation in the mean fluorescence was near 76% for both routes of administration, but estimates of the variation within tumor were near 16% standard deviation when a large sampling volume was used. In contrast, microscopic intra-tumor standard deviation in the mean estimate was near 76%, with IV injection, indicating that high heterogeneity exists in the photosensitizer concentration on a smaller distance scale. The inter-tumor variation was reduced by ITu injection, but at the expense of increasing intra-tumor variation.

Tumor pO2 assessments in human xenograft tumors measured by EPR oximetry: location of paramagnetic materials.

Radioantibody immunotherapy (RAIT) is a promising treatment modality but the effectiveness of this targeted low dose radiation varies from tumor to tumor. Since RAIT is an oxygen dependent treatment, baseline pO2 or growth-induced changes in the microenvironment may alter treatment response. In this pilot work we monitored tumor pO2 in untreated human xenograft tumors growing s.c. in nude mice. These data will be used to plan a study of the relationship between the effectiveness of RAIT and tumor pO2. Growth or treatment-induced changes in the microenvironment may alter the tumor pO2 and thus affect the response to therapy but may also affect location and microenvironment of the particulate oxygen sensor. We monitored tumor pO2 during growth and also examined the tumor histological structure overall and in the region of the paramagnetic material in the tumor at the time of necropsy.

Increasing oxygenation and radiation sensitivity following photodynamic therapy with verteporfin in the RIF-1 tumor

The combination of verteporfin-based photodynamic therapy (PDT) wiht radiaiton therapy from an orthovoltage device has been examiend in the radiation induced fibrosarcoma tumor model. PDT with verteporfin using a 3 hour delay between injection and the time of optical irradiation has been shown to cause a significant rise in overlal tumor oxygenation. It was huypothesized that this mechanism arises from the reduced oxygen consumption from cells where the PDT has targeted the mitochondria and shut down cellular respiration. Tumor blood flow was measured and found to be still be patent immediately following therapy. This increasing oxygenation was thought to provide an opportunity to increase the radiation sensitivity of the tumor immediately following PDT. When this type of treatment was combined with radiation therapy, a delay in the tumor regrowth time demonstrated that the combined effect was greater than additive. Further study of this phenomenon will provide a more complete mechanistic understanding of the effect and possibly provide a viable pre-treatment for radiation therapy of tumore that increases the therapeutic ratio. This effect could be used to either increase the radiaton dose without increasing the side effects or decrease the dose needed for the same effect on the tumor.