Investigations into the genotoxic potential of olive extracts.

The phenolic anti-oxidant 3-hydroxytyrosol (HT) is a major constituent of olives and olive oil. Published data showed it was negative in the Ames test at concentrations up to 5 µL per plate, but did induce chromosomal aberrations in human lymphocytes. HIDROX, an olive extract containing approximately 2.4% HT, was reported as both positive and equivocal in an Ames test in different papers from the same laboratory. Negative results for micronucleus induction in vivo in both an acute study and as part of a 90-day rat toxicity study were also reported for HIDROX. Given the widespread use and consumption of olives, olive oil and olive extracts, it was important to obtain more data. Here we confirm that pure HT, and an olive extract containing 15% HT, both induced micronuclei in cultured cells in vitro, but show that these responses were either due to high levels of cytotoxicity or to reaction of HT with culture medium components to produce hydrogen peroxide. Another extract (H40) containing 40% HT also induced micronuclei in vitro, probably via the same mechanism. However, both extracts were negative in robust Ames tests. The 15% HT formulated extract did not induce micronuclei in rat bone marrow after 4 weeks of dosing up to 561 mg HT/kg/day. H40 produced increased rat bone marrow micronucleus frequencies at 250 and 500 mg HT/kg/day in a 90-day toxicity study, but the results were questionable for various reasons. However, when two different batches of this extract were tested in acute micronucleus studies at doses up to 2000 mg HT/kg, giving plasma exposures that exceeded those in the 90-day study, negative results were obtained. Based on weight of evidence it is concluded that the olive extracts tested are not genotoxic at high doses in vivo, and any genotoxic risks for human consumers are negligible.

Black pepper constituent piperine: genotoxicity studies in vitro and in vivo.

Piperine is responsible for the hot taste of black pepper. Publications on genotoxicity of piperine are reported: negative Ames Tests and one in vitro micronucleus test (MNT). In vivo tests were mainly negative. In the majority of the data the administered dose levels did not follow the dose selection requirements of regulatory guidelines of having dose levels up to the maximum tolerated dose (MTD). The only oral high dose studies were a positive in vivo MNT in mice in contrast to a negative in vivo chromosome aberration test in rats. Thus, conflicting results in genotoxicity testing are published. To investigate this further, we administered piperine to mice up to the MTD and determined micronuclei-frequency. Piperine reduces core body temperature and interferes with blood cells both being known to result in irrelevant positive in vivo MNTs. Therefore we added mechanistic endpoints: core body temperature, haematology, erythropoietin level, and organ weights. Additionally an in vitro MNT in Chinese hamster ovary cells was performed. Piperine was negative in the in vitro MNT. It caused significant reduction of core body temperature, decrease of white blood cells and spleen weights but no increase in the micronucleus-frequency. Thus, in our studies piperine was not genotoxic.

A nonradioactive high-throughput/high-content assay for measurement of the human serotonin reuptake transporter function in vitro.

Both the tricyclic and specific serotonin reuptake inhibitor classes of antidepressants act primarily by inhibiting the reuptake of released serotonin by the human serotonin reuptake transporter (hSERT). In this article, the authors describe the use of a fluorescent substrate of the transporter (4-(4-(dimethylamino)-styrl)-N-methylpyridinium, ASP) to develop a microplate-based high-throughput screen for hSERT function. The assay is sensitive to known inhibitors of serotonin uptake, including fluoxetine (Prozac), with the correct rank order of potency and IC(50) values close to those reported in the literature for tritiated serotonin uptake. The authors also describe the validation of the assay for natural product screening using a test set of 2400 pure phyto-chemicals and 80 plant extracts. The mean Z of the screened plates was 0.53. Hit rates, confirmation rates, and validation of the hits in a "classical" assay for serotonin uptake are all reported. The assay can also be read in "high-content" mode using a subcellular imaging device, which allows direct detection of possible assay interference by acutely cytotoxic compounds. Among the compounds identified were several previously reported inhibitors of the hSERT, as well as compounds having structural similarity to the tricyclic antidepressant drugs.