Steve Tuttle

MIBG inhibits respiration : Potential for radio- and hyperthermic sensitization

INTRODUCTION Meta-iodobenzylguanidine (MIBG) in its 131I-labeled form is clinically used as a tumor-targeted radiopharmaceutical in the diagnosis and treatment of adrenergic tumors. This well established drug may have additional clinical applications as a radiosensitizer or hyperthermic agent, ie., MIBG reportedly inhibits mitochondrial respiration in vitro. The mechanism for MIBG inhibition of cellular oxygen consumption is uncertain. Moreover, MIBG reportedly stimulates glycolysis both in vitro and in vivo. Our studies show the effect of MIBG on 9L glioma oxygen consumption and redox status with tumors cells in vitro and in vivo. MATERIALS AND METHODS The effects on electron transfer were determined by following oxygen consumption with a Clark oxygen electrode. Fluorescence measurements were used to determine effects of MIBG on intracellular electron acceptors, NADPH and flavoproteins, in vitro and in vivo. 31P-NMR was used to determine alterations in tumor cell pH in vivo. RESULTS Our results show the inhibition of oxygen utilization with MIBG for cell suspensions in vitro. The same results were demonstrated for tumor cell suspensions rapidly isolated from tumors grown in rats. Moreover, NAD(P)H and flavoprotein (Fp) fluorescence changes were observed to rapidly occur following MIBG addition in vitro. Changes in intracellular pH measured with 31P-NMR, in vivo, precede the changes in fluorescence of NAD(P)H and Fp obtained with frozen sections of tumor. CONCLUSIONS We conclude that 31P-NMR measurements and fluorescence changes, following MIBG injection, can be used as criterion for selecting the proper time to treat tumors with ionizing radiation or hyperthermia.

Role of Guanosine Triphosphate in Ferric Ion-Linked Fenton Chemistry

We measured the production of reactive hydroxyl radical (OH.) by Fe2+ itself or complexed with nucleotide triphosphates or tripolyphosphate (TPP). Coumarin-3-carboxylic acid (3-CCA) reacts with the OH. produced by Fe2+, Fe3+ or Cu2+ plus ascorbate and with various iron complexes. We measured in real time the increased fluorescence of 3-CCA after hydroxylation to 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). Phosphate-buffered solutions do not affect the yield of Fe(2+)-linked OH. as do other organic buffer solutions. Our results show that guanosine triphosphate enhances the Fe(2+)-linked production of OH.. We also tested inosine triphosphate, adenosine triphosphate and xanthine triphosphate for their capacity to produce OH. with Fe2+. Inosine triphosphate is the most effective nucleotide in the production of OH.. However, the Fe(2+)-mediated yield of OH. is greater in the presence of TPP compared to the nucleotide triphosphates. Organic buffers as well as the purine and ribose portion of nucleotides compete for OH. and decrease the yield of fluorescent 7-OHCCA. We also decreased the yield of OH. by adding guanosine to the Fe2+/TPP-generating system. Adenosine, ribose and deoxyribose also react with Fe(2+)-generated OH.. The decreased yield of 7-OHCCA occurs because the ribose and purine part of the molecule reacts with OH.. The maximal production of reactive OH., compared to all nucleotides and phosphates tested, occurs with a ratio of 2 TPP/Fe2+ complex. In conclusion, the real-time measurement of the production of fluorescent 7-OHCCA provides a convenient means for measuring chemically generated OH.. The TPP/Fe(2+)-generating mixture, in the presence of 3-CCA, can be used to study the scavenging ability of other competing molecules.

Cellular Protection Against Damage by Hydroperoxides: Role of Glutathione

Radicals produced by X-rays may react with oxygen, producing intermediates that are believed to be toxic to cells. We propose a hypothesis that glutathione (GSH) may protect against radiation damage in four ways, as shown in Figure 1.1

Glutathione Depletion or Radiation Treatment Alters Respiration and Induces Apoptosis in R3230Ac Mammary Carcinoma

Glutathione depletion by L-buthionine sulfoximine inhibits the growth of Ehrlich mouse mammary carcinoma, R3230Ac rat mammary carcinoma and the PC3 human prostrate carcinoma cells, in vitro. Inhibition of growth occurs within the first 24 hours after exposure to the drug. The cell density does not increase over the initial cell density over 7 days. A549 human lung carcinoma and the DU145 human prostrate carcinoma cells show no inhibition of growth under the same treatment conditions. A comparative study of the R323OAc and A549 cells demonstrated a marked increase in apoptosis following L-BSO treatment in R3230Ac, which was dependent on L-BSO concentration and incubation time. L-BSO did not induce apoptosis in A549 cells at any of the concentrations tested. The incidence of apoptosis for R323OAc cells following exposure to 0.1 mM L-BSO was similar to the incidence of radiation-induced apoptosis observed after exposure to 10 Gy. Treatment with L-BSO or radiation alone inhibited O2 utilization in of R323Oac, while no effect on O2 utilization was observed in A549 cells. LBSO altered the bioreductive capacity of both the R323OAc and A549 cells. These results suggest that the ability of L-BSO to block mitochondrial O2 utilization may be involved in the apoptotic response in R3230Ac cells.

Factors Controlling Oxygen Utilization

We demonstrate, theoretically, that oxygen diffusion distance is related to the metabolic rate of tumors (QO2) as well as the oxygen tension. The difference in QO2 rate between tumors can vary by as much as 80-fold. Inhibition of oxygen utilization by glucose or chemical inhibitors can improve the diffusion distance. Combining respiratory inhibitors with increased availability of oxygen will further improve the oxygen diffusion distance for all tumors. A simple means for inhibiting oxygen consumption is the use of glucose (the Crabtree effect). The inhibition of tumor oxygen utilization by glucose occurs in R323OAc mammary carcinoma and 9L glioma cells. However, stimulation of oxygen consumption is observed with glucose in the Q7 hepatoma cell line. MIBG, a known inhibitor of oxygen utilization, blocks oxygen consumption in 9L, but is weakly inhibitory with the Q7. Q7 tumor cells demonstrate an anomalous behavior of glucose and MIBG on oxygen consumption. Our results clearly demonstrate the necessity for comparing effects of different agents on different tumor cells. Generalizations cannot be made with respect to the choice of inhibitor for in vivo use. Our work shows that oxygen consumption also can be inhibited with malonate and chlorosuccinate. These substrates may be effective in vivo, where glucose is low and glutamine is the major substrate. Our results indicate that information about individual tumor substrate-linked metabolic controls may be necessary before attempting to inhibit oxygen utilization in vivo for therapeutic benefit.