Indole-3-carbinol (I3C) is a well-established chemopreventive agent in animals, and is particularly effective against mammary cancer. We have completed a Phase I trial of I3C in 18 women, 17 premenopausal and one postmenopausal, from a high-risk breast cancer cohort. Chronic I3C dosing, performed after a 4-week placebo run-in period, consisted of a 4-week period of 200 mg I3C twice daily and a subsequent 4-week period of 400 mg I3C twice daily. These chronic doses were well tolerated by all subjects. Various hormonal parameters were measured during the placebo and dosing periods, including determination of the urinary 2-hydroxy-estrone:16a-hydroxy-estrone ratio. All measurements were made during the follicular phase of the estrus cycle for premenopausal women. Serum estradiol, progesterone, LH, FSH, and sex hormone binding protein showed no consistent or significant changes in response to administration of I3C. Activities of cytochrome P450 1A2 (CYP1A2), N-acetyltransferase-2 (NAT2), xanthine oxidase, and flavin monooxygenase-3 (FMO3) were examined using a single 100 mg oral caffeine dose as a probe. Enzymatic activities were inferred from validated ratios of urinary caffeine and caffeine metabolites. Comparing the results from the placebo period to those from the 800 mg daily dose period, CYP1A2 was found to be elevated by I3C in 90% of the subjects, with a mean increase of 5.3-fold. In contrast, FMO3 was decreased in 90% of subjects by I3C, with a mean decrease of 73%. Xanthine oxidase and NAT-2 activities were not affected by I3C administration. The apparent inductive effects on CYP1A2 were mirrored by a near doubling of the urinary 2-hydroxy-estrone/16a-hydroxy-estrone ratio in response to I3C. Of interest, however, is that the maximal increase was observed with the 400 mg daily dose of I3C, with no further increase found at the 800 mg daily dose. If the ratio of hydroxylated estrone metabolites is a biomarker for chemoprevention, as has been suggested, then a dose of 400 mg I3C daily will elicit a maximal protective effect. Gregory Reed, Sarah Moreland, Kirstin Peterson, Holly Smith, James Crowell, Aryeh Hurwitz. Proceedings of the AACR, Volume 44, March 2003.