Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge.

This article gives an overview of the results of genotoxicity tests, which have been conducted within the last 5 years with the human liver cell line HepG2. It is an update of an earlier review from 1998 (by Knasmüller et al.). In addition, a number of publications are discussed which are relevant for the use of human derived liver cell lines in genetic toxicology. They concern the establishment of new endpoints, the development of new cell lines and possible pitfalls and problems. HepG2 cells have been used to test a wide variety of compounds over the last years. The most interesting observations are that the cells are highly sensitive toward polycyclic aromatic hydrocarbons and that genotoxic effects are seen with a number of carcinogenic mycotoxins, that give negative results in other in vitro assays. Carcinogenic metals such as As and Cd caused positive results as well, whereas only marginal or negative results were seen with nitrosamines. The low sensitivity toward these latter carcinogens is probably due to a lack of cytochrome P4502E1 which catalyses their activation. Also, a number of structurally different synthetic pesticides as well as bioactive plant constituents ("natural pesticides") have been tested and with some of them genotoxic effects were found. In most experiments, the formation of micronuclei was used as an endpoint; however also the single cell gel electrophoresis assay is increasingly used. Several transfectant lines of HepG2 have been constructed which express increased levels of phase I enzymes (such as CYP1A1, CYP1A2, CYP2E1 etc.); furthermore, cell lines became available which express human glutathione-S-transferases. These new clones might be particularly useful for the investigation of specific classes of genotoxicants and also for mechanistic studies. Apart from HepG2 cells, a number of other human derived liver cell lines have been isolated, but so far no data from genotoxicity experiments are available, except for Hep3B cells, which were compared with HepG2 and found to be less sensitive in general. Studies with HepG2 clones of a different origin indicate that the cells differ in regard to their sensitivity toward genotoxicants; also medium effects and the cultivation time might affect the outcome of genotoxicity studies. Overall, the results support the assumption that HepG2 cells are a suitable tool for genotoxicity testing.

Cafestol and kahweol, two coffee specific diterpenes with anticarcinogenic activity.

Epidemiological studies have found an inverse association between coffee consumption and the risk of certain types of cancers such as colorectal cancers. Animal data support such a chemopreventive effect of coffee. Substantial research has been devoted to the identification of coffee components that may be responsible for these beneficial effects. In animal models and cell culture systems, the coffee diterpenes cafestol and kahweol (C+K) were shown to produce a broad range of biochemical effects resulting in a reduction of the genotoxicity of several carcinogens including 7,12-dimethylbenz[a]anthracene (DMBA), aflatoxin B(1) (AFB(1)), benzo[a]pyrene (B[a]P) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Different mechanisms appear to be involved in these chemoprotective effects: an induction of conjugating enzymes (e.g. glutathione S-transferases, glucuronosyl S-transferases), an increased expression of proteins involved in cellular antioxidant defense (e.g. gamma-glutamyl cysteine synthetase and heme oxygenase-1) and an inhibition of the expression and/or activity of cytochromes P450 involved in carcinogen activation (e.g. CYP2C11, CYP3A2). In animal models, the C+K-mediated induction of conjugating and antioxidant enzymes has been observed in hepatic, intestinal and kidney tissues. In the small intestine, these inductions were shown to be mediated by Nrf2-dependent transcriptional activation. In vitro investigations obtained in cell cultures of human origin indicate that the effects and mechanisms observed in animal test systems with C+K are likely to be of relevance for humans. In human liver epithelial cell lines transfected to express AFB(1)-activating P450s, C+K treatment resulted in a reduction of AFB(1)-DNA binding. This protection was correlated with an induction of GST-mu, an enzyme known to be involved in AFB(1) detoxification. In addition, C+K was found to inhibit P450 2B6, one of the human enzymes responsible for AFB(1) activation. Altogether, the data on the biological effects of C+K provide a plausible hypothesis to explain some of the anticarcinogenic effects of coffee observed in human epidemiological studies and in animal experiments.

Inhibition instead of enhancement of lipid peroxidation by pretreatment with the carcinogenic peroxisome proliferator nafenopin in rat liver exposed to a high single dose of corn oil.

Oxidative stress is discussed as a possible hepatocarcinogenic mechanism of peroxisome proliferators (PP) in rodents and is suggested to result from the induction of peroxisomal beta-oxidation (PBOX) by PP. The induced PBOX is assumed to produce excessive H2O2 from the degradation of fatty acids, ultimately leading to oxidative stress and lipid peroxidation. In the present short term-study, we attempted to stimulate lipid peroxidation in male Wistar rats by (1) inducing PBOX enzymes with the peroxisome proliferator nafenopin at 90 mg/kg body weight per day in the diet for 10-11 days, and (2) by supplying the induced PBOX with an abundant amount of fatty acid as substrate, using a corn oil gavage at 20 ml/kg body weight. The corn-oil gavage alone, i.e. without preceding nafenopin treatment, enhanced liver triacylglycerol nine- to tenfold and hepatic lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), was increased 50% compared with controls. Both observations were made after 18 h when the peak elevations occurred. Upon pretreatment with nafenopin, associated with a sevenfold induction of PBOX, the corn oil gavage however caused only a threefold maximal increase in hepatic triacylglycerol, also at the 18 h time-point; TBARS remained almost at control levels, as monitored at seven time points over 24-25 h. These results suggest that nafenopin reduces rather than enhances lipid peroxidation, despite the provision, in a short term study, of high doses of substrate to the induced enzyme system that is hypothetically causing oxidative stress in the liver.

Inhibition of repairable DNA-damage in Escherichia coli K-12 cells recovered from various organs of nitrosamine-treated mice by vitamin A, phenethylisothiocyanate, oleic acid and triolein.

The influence of various dietary constituents--phenethylisothiocyanate (PEITC), oleic acid (OA), triolein (TO), and vitamin A (ROL)--on the genotoxic activity of nitrosamines (NDMA, NDELA, NPYR) was investigated. For this purpose differential DNA repair assays with Escherichia coli K-12 strains were performed in vitro and in vivo with mice. Under in vitro conditions (liquid holding), all compounds reduced nitrosamine induced DNA-damage in the indicator bacteria in the dose range 1-10 micrograms/ml, the ranking order of efficiency being PEITC greater than OA greater than ROL greater than or equal to TO. In animal-mediated assays, acute oral treatment with PEITC (17-150 mg/kg), 2 h before nitrosamine administration, resulted in a marked decrease of nitrosamine genotoxicity in liver, kidneys, lungs and in the blood. Also in other organs (spleen, testes) an increase in differential survival (which serves as a measure for repairable DNA damage) occurred. With ROL only a comparatively moderate antigenotoxic effect was obtained at a high dose level (250 mg/kg) under identical experimental conditions. OA (2000 mg/kg) and TO (16,000 mg/kg) were completely inactive. Upon repeated treatment (consecutive oral administration of the putative antigenotoxins over 4 days, a final treatment 24 h before nitrosamine administration) PEITC (150 mg/kg/day), ROL (80 mg/kg/day) and OA (2000 mg/kg/day) had no influence on the genotoxic effects of the nitrosamines. Repeated treatment with TO (4000-16,000 mg/kg/day) resulted in a moderate dose-dependent reduction of NDMA-induced DNA-damage in the indicator bacteria, whereas in combination with NPYR only a marginal effect was observed. Biochemical experiments indicated that the antigenotoxic effects of PEITC seen under in vivo conditions were due to inhibition of alpha-hydroxylation of the nitrosamines, whereas ROL and TO appeared not to interfere strongly with this metabolic activation step. Our results indicate that in vitro assays do only partly reflect the antigenotoxic properties of the different food constituents in vivo and that animal-mediated DNA repair assays with E. coli strains are an appropriate approach to study the effects of modifiers of nitrosamine genotoxicity in the living animal.