Data from an expanding body of carcinogenesis studies in experimental models demonstrate that resveratrol is an attractive candidate for efficacy evaluation in chemoprevention clinical trials. In addition to its anticarcinogenic activity, resveratrol has a number of cardioprotective, anti-aging, and other biological activities that suggest its possible utility in the prevention of cardiovascular and other chronic diseases. The present oncogenicity study in p53(+/-) mice addresses critical scientific and regulatory steps in the preclinical development of resveratrol for possible clinical study as a cancer chemopreventive agent, and may also support the initiation of Phase II and Phase III clinical trials to evaluate its utility in the prevention of other diseases.
An essential part of the preclinical development of any potential chemopreventive agent to which humans may receive chronic exposure is the demonstration that the agent is not carcinogenic in experimental model systems. Bacterial mutagenesis studies (Ames Tests) with resveratrol have produced negative results, and thus provide no evidence to support the hypothesis that resveratrol is likely to be mutagenic and/or carcinogenic in humans. By contrast, however, genotoxic effects of resveratrol have been reported in mutagenesis and DNA damage studies conducted using mammalian cell models. In studies conducted using L5178 mouse lymphoma cells and Chinese hamster V79 cells, Schmitt and colleagues (2002
) reported that resveratrol increases the incidence of sister chromatid exchanges, induces micronuclei, and increases the incidence of spindle distortions and dislocated chromosomes. In considering the sum of the available genetic toxicology data, resveratrol must be considered to be an agent with, at a minimum, equivocal genotoxic activity. In consideration of this demonstrated activity in well-studied genetic toxicology model systems, in vivo
studies of the possible carcinogenicity of resveratrol become an increasingly important component of its preclinical data base.
The results of the present study demonstrate that resveratrol is not oncogenic in TSG-p53(+/-) (p53 knockout) mice when the agent is administered at its maximum tolerated dose for six months. Because the six-month oncogenicity study in the p53 knockout mouse is accepted by the United States Food and Drug Administration and other regulatory agencies as a suitable preclinical model for carcinogenicity evaluations, the lack of oncogenicity of resveratrol in the p53 knockout mouse is generally accepted as adequate evidence of lack of carcinogenicity in murine model systems.
The maximum dose of resveratrol that could be administered to mice was limited by a physical factor (impaction of drug substance in the gastrointestinal tract) rather than by any chemical-specific toxicity. Differential mortality patterns associated with impaction of the test article appeared to be related to animal body weight. At each dose level, animals with higher body weights received a greater total mass of resveratrol than did animals with lower body weights. Because the male mice in both studies were larger than the female mice, the males received a larger mass of test article each day; this could explain the sex difference in mortality observed in the range-finding study. Similarly, the female TSG-p53(+/-) (p53 knockout) mice used in the oncogenicity study were larger than the female C57BL/6 mice used in the range-finding study, and therefore received a greater mass of the test article at each dose. Essentially all of the mortality due to resveratrol impaction in females in the oncogenicity study was seen in mice whose body weights were in the upper portion of the range.
Although resveratrol did not increase the incidence of any neoplasm in the present study, a number of non-neoplastic effects were seen in resveratrol-treated animals. In evaluating the possible oncogenicity of resveratrol, perhaps the most significant of these was the finding of urothelial hyperplasia (generally of minimal to mild severity) in more than one-third of surviving animals of both sexes in the group exposed to the mid dose of resveratrol. Urothelial hyperplasia was not identified in animals in the low dose group. Because urothelial hyperplasia could be secondary to physical factors such as hydronephrosis or urinary calculi, a post-hoc analysis of individual animal data was performed in the attempt to identify such an association. No link between hydronephrosis and urothelial hyperplasia was found, and urinary calculi were not commonly seen in any experimental group. Because no physical mechanism can be proposed to explain the increased incidence of urothelial hyperplasia in mice receiving the mid dose of resveratrol, this is considered to be a direct, dose-related effect of the test article.
The Cmax of free resveratrol measured in mouse plasma in the present PK study ranged from 20 to 40 μM, and plasma levels of resveratrol conjugates in mice were more than 10-fold higher than levels of free resveratrol. The Cmax for free resveratrol in mice was reached at 1 to 2 hours after dosing, and plasma resveratrol levels remained above 10 μM for at least at 8 hours. By comparison, the Cmax of free resveratrol measured in the plasma of human volunteers enrolled in a Phase I clinical trial ranged from 0.5 to 2 μM (Royalmount Pharma, unpublished data). As in mice, Cmax in humans was reached between 1 and 2 hours post-dosing. The Cmax for resveratrol in humans also increased in a reasonably linear fashion with dose. On this basis, the low and mid dose levels of resveratrol administered in the murine oncogenicity study could be expected to yield plasma levels of free resveratrol that are at least 5- to 20-fold higher than the levels that were measured in humans enrolled in the Phase I clinical trial. These data suggest that plasma drug levels in mice in the low dose group (where no urothelial hyperplasia was seen) are likely to exceed those achieved in the clinic by a factor of at least 5 to 10. Similarly, plasma drug levels in mice in the middle dose group (the lowest dose group in which urothelial hyperplasia was seen) are likely to exceed those achieved in the clinic by a factor of at least 10, and perhaps by as much as 20. In consideration of these data, it is concluded that urothelial hyperplasia in p53 knockout mice was seen only at plasma drug levels that substantially exceed those that are likely to be reached in humans.
Clinical pathology studies identified small but statistically significant elevations in serum cholesterol in both sexes of mice exposed to resveratrol, and a mild anemia (as indicated by decreases in RBC count, hematocrit, and hemoglobin) in male mice exposed to the high dose only. In consideration of the small magnitude of the increases in serum cholesterol seen in resveratrol-treated mice, it is considered unlikely that these increases are of toxicologic significance. The mechanisms underlying the mild anemia induced by resveratrol in male mice are unknown; however, the observed anemia could reflect a minimal toxic effect of resveratrol, or could result from the activity of resveratrol as a phytoestrogen (Schmitt et al., 2002
Statistically significant, dose-related increases in liver weight were seen in surviving animals exposed to the mid and high doses of resveratrol. This hepatomegaly was not associated with any microscopic changes in the liver, but could reflect the induction by resveratrol of hepatic Phase II enzymes involved in carcinogen detoxification (Jang et al., 1997
). As discussed above, resveratrol-treated animals also demonstrated dose-related hydronephrosis and epithelial cell hyperplasia of the urinary bladder. Although neither hydronephrosis nor urothelial hyperplasia were linked to any functional alteration, these findings do identify the kidney and urinary bladder as possible targets for resveratrol toxicity. In this regard, the renal and urinary bladder toxicity of resveratrol identified in the present study is consistent with the findings of Crowell et al.
) who conducted a 4-week toxicity study of resveratrol in rats. By contrast to these data from rodent models, a recently completed study conducted in our laboratory did not identify hydronephrosis, urothelial hyperplasia, or any other significant histopathologic changes in the urinary tract of beagle dogs receiving daily oral exposure to resveratrol for 4 weeks. To our knowledge, no toxicology studies of resveratrol have been conducted in non-human primates.
The results of the present study demonstrate that chronic oral administration of resveratrol at its maximum tolerated dose does not increase the incidence of any malignant or benign neoplasm in TSG-p53(+/-) (p53 knockout) mice. Chronic oral administration of resveratrol to mice at a dose of 2000 mg/kg/day did induce a statistically significant increase in the incidence of urothelial hyperplasia, a proliferative lesion in the urinary bladder. However, mice exposed to resveratrol at 1000 mg/kg/day demonstrated no evidence of urothelial hyperplasia, and pharmacokinetic data suggest that this effect was seen only at plasma resveratrol levels that exceed targeted human plasma levels by a factor of at least 10. In consideration of the demonstrated chemopreventive activity of resveratrol in animal models for cancer in the breast, colon, and other sites; its relative lack of toxicity at doses generating plasma drug levels that are similar to targeted drug levels in humans; and its lack of oncogenicity in predictive animal model systems, resveratrol clearly merits further consideration for possible evaluation in human cancer chemoprevention trials.