Volker Mersch-Sundermann

Genotoxic and antigenotoxic effects of catechin and tannins from the bark of Hamamelis virginiana L. in metabolically competent, human hepatoma cells (Hep G2) using single cell gel electrophoresis

The genotoxic and antigenotoxic activities of catechin, hamamelitannin and two proanthocyanidin fractions prepared from the bark of Hamamelis virginiana L. were investigated in a human derived, metabolically competent hepatoma cell line (Hep G2) using single cell gel electrophoresis (SCGE) for the detection of DNA-damage. DNA-migration was calculated as Olive tail moment (OTM). Catechin and a low-molecular weight proanthocyandin fraction (W(M)) caused only slight increases of OTM up to concentrations of 166 microg/ml whereas hamamelitannin and the proanthocyandin fraction with higher molecular weight (W(A)) led to a two-fold enhancement of OTM at the same concentrations. These effects were dose-independent. Treatment of the cells with the test compounds in a dose-range of 2-166 microg/ml prior to the exposure to benzo(a)pyrene (B(a)P, 10 microM, 2.5 microg/ml) led to a significant reduction of induced DNA damage which was dose-dependent for all test compounds, except for hamamelitannin. The inhibitory effects of proanthocyanidins were stronger than those of catechin and hamamelitannin; the lowest effective concentrations were about 2 microg/ml. In order to clarify the mechanisms of protection, possible effects of the test compounds on enzymes involved in toxification and detoxification of B(a)P were investigated. While B(a)P toxification by cytochrome P450 was not inhibited by the test compounds, detoxification by glutathion-S-transferase (GST) was induced by catechin and W(M). Combination experiments with the ultimate metabolite of B(a)P, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE; 5 microM, 1.5 microg/ml), revealed strong inhibitory effects, indicating that the observed protective effects were caused by scavenging of the ultimate mutagen by the test compounds. Exposure of Hep G2 cells to the test compounds after B(a)P treatment did not influence B(a)P induced DNA damage, demonstrating that repair mechanisms were not affected.

Benzo(a)pyrene induced micronucleus formation was modulated by persistent organic pollutants (POPs) in metabolically competent human HepG2 cells

Due to their bioaccumulation and their biological properties persistent organic pollutants (POPs) attract wide attention. In the present study we investigated the genotoxicity, cogenotoxicity and antigenotoxicity of three selected POPs (DDT, aroclor-1254 and toxaphene) in the HepG2 micronucleus assay. Exposure of HepG2 cells to DDT (17.8-60 microM) and aroclor-1254 (23-184 microM) alone did not increase the micronucleus-frequencies. A slight genotoxic effect could be observed after exposure to toxaphene (20-40 microM). Additionally, the ability of POPs to enhance/decrease the benzo(a)pyrene (BaP)-induced micronucleus formation was investigated. Exposure of HepG2 cells to 50 microM BaP alone led to a more than 2-fold increase of micronuclei (MN) compared with the background frequency. But when the cells were pretreated with 23-181 microM aroclor-1254 or 10-20 microM toxaphene, BaP exposure caused significantly more MN than BaP alone. In contrast, DDT (17.8-60 microM) reduced BaP-induced micronucleus induction by 6-38%. Mechanisms of action are discussed.