Heavy water labeling method was applied to measure the effect of
genistein on mammary gland
carcinogenesis by incoporating (2)H from (2)H(2)O into the
deoxyribose (dR) moiety of
purine deoxyribonucleotides in dividing cells. In the present study, we followed the study design of Lamartiniere group to evaluate the efficacy of (2)H(2)O labeling on the measurement of mammary gland carconogenesis. Female Sprague-Dawley rats were fed
estrogen-free AIN-93G diet starting 1 week before breeding and continuing through pregnancy and lactation. Female pups were assigned to the following groups on postnatal day 16 and fed AIN-93G diet: vehicle (
dimethylsulfoxide) (
DMSO),
genistein, and
estradiol benzoate (EB). On postnatal days 16, 18, and 20, female pups were injected subcutaneously with 500 mug
genistein/g body wt, 500 ng EB/g body wt, or an equivalent volume of the vehicle. At day 50 postpartum, half of each group were gavaged with 60 mg dimethylbenz[a]
anthracene (DMBA) in perila oil. After 1 week of DMBA treatment, all animals were labeled with (2)H(2)O by administration of 4% (2)H(2)O in
drinking water after single intraperitonial bolus injection with 99.9% (2)H(2)O until sacrifice on postnatal day 81. The time-dependent
weight gains were observed in all groups throughout the experimental period. The enrichment of body (2)H(2)O was attained at 1.84-2.47% through
oral administration of (2)H(2)O. Mammary epithelial cell proliferation was measured by enrichment (EM1) of dA from rats. DMBA-treated groups showed higher fractional synthesis than DMBA non-treated groups. The group exposed only to
genistein showed significantly lower EM1 (1.46 +/- 0.87%) than those of control groups, i.e., the DMBA non-treated group (2.28 +/- 0.29%) and the DMBA-treated group (2.32 +/- 0.28%).
Bromodeoxyuridine (
BrdU) immunostaining of mammary tissue revealed that
genistein reduced proliferation of the mammary epithelial, and the number of cells stained positive for
BrdU both in DMBA-treated groups and DMBA non-treated groups. H&E staining of mammary epithelium also showed that the exposure to
genistein decreased proliferation of the mammary epithelium. The epithelium in the rats treated with DMBA showed mostly multiple cell layers, in contrast to the mostly double layer shown in the DMBA non-treated rats. The exposure to
genistein in the prepubertal period inhibited mammary epithelial cell proliferation. In conclusion, the (2)H(2)O labeling results were in good agreement with the results of
BrdU incorporation and histomorphometry, which demonstrates that (2)H(2)O labeling can be used as a tool to measure
carcinogenesis.