The modifying effects of the two
flavonoids diosmin and
hesperidin given during the initiation and postinitiation phases of oral
carcinogenesis initiated with 4-nitroquinoline 1-oxide (4-NQO) were investigated in male F344 rats. The compounds were tested alone and in combination. At 6 weeks of age, animals were divided into experimental and control groups and fed diets containing 1000 ppm
diosmin and 1000 ppm
hesperidin and a diet containing both compounds (900 ppm
diosmin and 100 ppm
hesperidin). At 7 weeks of age, all animals except those treated with each test chemical alone and control groups were given 4-NQO (20 ppm) in the
drinking water for 8 weeks to induce
oral cancer. Starting 7 days before the 4-NQO exposure, groups of animals were fed the diets containing test chemicals for 10 weeks and then switched to the basal diet. Starting 1 week after the cessation of 4-NQO exposure, the groups given 4-NQO and a basal diet were switched to the diets containing
diosmin,
hesperidin, or
diosmin combined with
hesperidin and maintained on these diets for 22 weeks. The other groups consisted of rats given
diosmin (1000 ppm),
hesperidin (1000 ppm), and the combination regimen of these two compounds (900 ppm
diosmin with 100 ppm
hesperidin) alone, and untreated rats. All animals were necropsied at the termination of the study (week 32). The incidences of tongue lesions (
neoplasms and preneoplasms),
polyamine levels in the tongue tissue, and cell proliferation activity estimated by a 5-bromodeoxyuridine-labeling index and by morphometric analysis of
silver-stained nucleolar organizer regions
protein were compared among the groups. Feeding of both compounds singly or in combination during the initiation phase caused a significant reduction in the frequency of tongue
carcinoma [
diosmin, 68% reduction (P < 0.01);
hesperidin, 75% reduction (P < 0.005); and the combination regimen, 69% (P < 0.05)]. When fed the test compounds singly or the combination regimen after 4-NQO exposure, the frequency of
tongue cancer was also decreased [
diosmin, 77% reduction (P < 0.005);
hesperidin, 62% reduction (P < 0.05); and the combination regimen, 77% (P < 0.005)]. The incidences of oral preneoplasia (
hyperplasia and dysplasia) in these groups were also decreased when compared with
carcinogen controls (P < 0.05-P < 0.001). There were no pathological alterations in rats treated with test compounds or the combined regimen alone or those in an untreated control group. Dietary administration of these compounds significantly decreased the expression of cell proliferation
biomarkers (
5-bromodeoxyuridine-labeling index and
silver-stained nucleolar organizer regions
protein number) of the nonlesional tongue squamous epithelium (P < 0.05). Also,
polyamine concentrations in the oral mucosa were lowered in rats given the
carcinogen and test compounds, alone and in combination, compared with those of rats given 4-NQO alone (P < 0.05). These findings suggest that supplementation with the
flavonoids diosmin and
hesperidin, individually and in combination, is effective in inhibiting the development of
oral neoplasms induced by 4-NQO, and such inhibition might be related to suppression of increased cell proliferation caused by 4-NQO in the oral mucosa.