Antitumor effects of hydrogen peroxide in vivo

Abstract

Glucose oxidase covalently coupled to polystyrene microspheres (GOL) produced H2O2 at an average rate of 3.6 nmol/min per 109 beads under standard assay conditions. Injection of 1.3 .times. 1010 to 1.1 .times. 1011 GOL i.p. prolonged the survival of mice by 27% after injection of 106 P388 lymphoma cells in the same site, consistent with destruction of 97.6% of the tumor cells. Placing mice for several hours in 100% O2, the probable rate-limiting substrate for GOL, afforded a 42% prolongation of survival from P388 lymphoma, consistent with destruction of 99.6% of the tumor cells. When the P388 inoculum was 105, 104 or 103 cells, GOL led to long-term survival (presumed cure) of 23, 77 and 92% of the mice, respectively, consistent with reduction of the injected tumor dose to < 10 cells. S.c. growth of 105 P388 cells (.apprx. 300 mouse LD50) was suppressed in 83% of mice by admixture of GOL with the tumor cell inoculum. GOL alone had no effect against a more peroxide-resistant tumor, P815 mastocytoma. However, P815 cell glutathione reductase could be inhibited in vivo by well-tolerated doses of the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). BCNU alone cured few mice with P815. Together, BCNU and GOL apparently cured 86% of mice injected with 106 P815 cells i.p. The protective effect of GOL was abolished by boiling it to inactivate the enzyme, by co-injection of catalase coupled to latex beads or by delaying the injection of tumor cells for 3 h, by which time the beads had formed aggregates. Soluble glucose oxidase, in doses 3-fold higher than that bound to GOL, had no detectable antitumor effect. A single injection of preformed H2O2 readily killed P388 cells in the peritoneal cavity, but only at doses nearly lethal to the mice. GOL had very little toxicity, as judged by the normal appearance of the mice for 400 days, gross and microscopic findings at autopsy and various blood tests. GOL injected i.p. remained in the peritoneal cavity, where it was gradually organized into granulomata by macrophages, without generalized inflammation. Thus, an H2O2-generating system confined to the tumor bed exerted clear-cut antitumor effects with little toxicity to the host.