Essential oils from mint plants, including peppermint and pennyroyal
oils, are used at low levels as
flavoring agents in various foods and beverages.
Pulegone is a component of these
oils. In a 2-year bioassay,
oral administration of
pulegone slightly increased the urothelial
tumor incidence in female rats. We hypothesized that its mode of action (MOA) involved urothelial cytotoxicity and increased cell proliferation, ultimately leading to
tumors.
Pulegone was administered by gavage at 0, 75, or 150 mg/kg
body weight to female rats for 4 and 6 weeks. Fresh void urine and 18-h urine were collected for crystal and metabolite analyses. Urinary bladders were evaluated by light microscopy and scanning electron microscopy (SEM) and
bromodeoxyuridine (
BrdU) labeling index.
Pulegone and its metabolites,
piperitenone,
piperitone,
menthofuran, and
menthone, were tested for cytotoxicity in rat (
MYP3) and human (1T1) urothelial cells by the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide assay. No abnormal urinary crystals were observed by light microscopy. Urine samples (18-h) showed the presence of
pulegone,
piperitone,
piperitenone, and
menthofuran in both treated groups. By SEM, bladders from treated rats showed superficial
necrosis and exfoliation. There was a significant increase in the
BrdU labeling index in the high-dose group. In vitro studies indicated that
pulegone and its metabolites, especially
piperitenone, are excreted and concentrated in the urine at cytotoxic levels when
pulegone is administered at high doses to female rats. The present study supports the hypothesis that cytotoxicity followed by regenerative cell proliferation is the MOA for
pulegone-induced urothelial
tumors in female rats.