Use of human derived liver cells for the detection of genotoxins in comet assays.

One of the problems of in vitro genotoxicity testing is the inadequate representation of drug metabolizing enzymes in indicator cells which are currently used. An alternative are human derived liver cell lines which retained the activities of enzymes that catalyze the activation and detoxification of genotoxins. Several cell lines were identified which were used in comet experiments. The most frequently employed line is HepG2, i.e. more than 400 individual compounds have been tested; furthermore, it was also used for the detection of combined effects in mixtures as drug metabolizing and antioxidant enzymes are represented in inducible form. One of the shortcomings of these cells are the strong inter-laboratory variation of the results. Recently it was postulated that HepaRG cells are an ideal model for human liver studies, but comet experiments were only partly successful and failed to detect genotoxins such as cadmium chloride, styrene and etoposide, as well as compounds that require activation via N-actetyltransferases (IQ, 2,4-DAT, 2-AAF). Furthermore, these cells are relatively insensitive towards ROS. Hep3B cells were used in a few studies but failed to detect representatives of important genotoxic carcinogens (AFB1, B(a)P, NDMA, IQ, PhiP), the line HCC1.1 was sensitive towards these chemicals but possesses an instable karyotype and a mutated p53. A more promising line is Huh6, but further validation of the usefulness for routine testing is needed. Recent developments which may lead to a better metabolic capacity of liver cells include improvement of the growth conditions (e.g. increase of serum levels, use of differentiated cells and of 3D-cultures), use of differentiated stem cells with hepatocyte like characteristics or of transformed proliferating hepatocytes.

Gallic acid, a common dietary phenolic protects against high fat diet induced DNA damage.

PURPOSE: Aim of the study was to find out if gallic acid (GA), a common phenolic in plant foods, prevents obesity induced DNA damage which plays a key role in the induction of overweight associated cancer. METHODS: Male and female C57BL6/J mice were fed with a low fat or a high fat diet (HFD). The HFD group received different doses GA (0, 2.6-20 mg/kg b.w./day) in the drinking water for 1 week. Subsequently, alterations of the genetic stability in blood and inner organs were monitored in single cell gel electrophoresis assays. To elucidate the underlying molecular mechanisms: oxidized DNA bases, alterations of the redox status, lipid and glucose metabolism, cytokine levels and hepatic NF-κB activity were monitored. RESULTS: HFD fed animals had higher body weights; increased DNA damage and oxidation of DNA bases damage were detected in colon, liver and brain but not in blood and white adipose tissue. Furthermore, elevated concentrations of insulin, glucose, triglycerides, MCP-1, TNF-α and NF-κB activity were observed in this group. Small amounts of GA, in the range of human consumption, caused DNA protection and reduced oxidation of DNA bases, as well as biochemical and inflammatory parameters. CONCLUSIONS: Obese animals have increased DNA damage due to oxidation of DNA bases. This effect is probably caused by increased levels of glucose and insulin. The effects of GA can be explained by its hypoglycaemic properties and indicate that the consumption of GA-rich foods prevents adverse health effects in obese individuals.

Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins: a new hope for laboratory animals?

Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.

Investigations of the genotoxic properties of two synthetic cathinones (3-MMC, 4-MEC) which are used as psychoactive drugs.

Synthetic cathinones (SCAs) are consumed worldwide as psychostimulants and are increasingly marketed as surrogates of classical illicit drugs via the internet. The genotoxic properties of most of these drugs have not been investigated. Results of earlier studies show that amphetamines which are structurally closely related to these compounds cause damage to the genetic material. Therefore, we tested the genotoxic properties of two widely consumed SCAs, namely, 3-MMC (2-(methylamino)-1-(3-methylphenyl) propan-1-one) and 4-MEC (2-(ethylamino)-1-(4-methylphenyl) propan-1-one) in a panel of genotoxicity tests. We found no evidence for induction of gene mutations in Salmonella/microsome assays, but both drugs caused positive results in the single cell gel electrophoresis (SCGE) assay which detects single and double strand breaks of DNA in a human derived buccal cell line (TR146). 3-MMC induced similar effects as 4-MEC and also caused significant induction of micronuclei which are formed as a consequence of structural and chromosomal aberrations. Negative results obtained in SCGE experiments with lesion specific enzymes (FPG and Endo III) show that these drugs do not cause oxidative damage of DNA. However, moderate induction of TBARS (which leads to the formation of DNA-reactive substances) was observed with 4-MEC, indicating that the drug causes lipid peroxidation while no clear effect was detected with 3-MMC. Results obtained with liver homogenate in SCGE-experiments show that phase I enzymes do not lead to the formation of DNA reactive metabolites. Taken together, our findings indicate that consumption of certain SCAs may cause adverse health effects in users as a consequence of damage to the genetic material.

Xanthohumol, a prenylated flavonoid contained in beer, prevents the induction of preneoplastic lesions and DNA damage in liver and colon induced by the heterocyclic aromatic amine amino-3-methyl-imidazo[4,5-f]quinoline (IQ).

Xanthohumol (XN) is a hop derived prenylated flavonoid contained in beer. Earlier findings indicated that it has promising chemopreventive properties and protects cells against DNA damage by carcinogens via inhibition of their activation. Furthermore, it was found that XN inhibits DNA synthesis and proliferation of cancer cells in vitro, inactivates oxygen radicals and induces apoptosis. Since evidence for its chemoprotective properties is restricted to results from in vitro experiments, we monitored the impact of XN on the formation of amino-3-methyl-imidazo[4,5-f]quinoline (IQ)-induced preneoplastic foci in livers and colons of rats (9/group). Additionally, we studied its effects on IQ-induced DNA damage in colonocytes and hepatocytes in single cell gel electrophoresis assays and on the activities of a panel of drug metabolising enzymes. Consumption of the drinking water supplemented with XN (71 microg/kg b.w.) before and during carcinogen treatment led to a significant reduction of the number of GST-p+ foci in the liver by 50% and also to a decrease of the foci area by 44%. DNA migration was decreased significantly in both, colon mucosa and liver cells, but no alterations of the activities of different phases I and II enzymes were found in hepatic tissue. Our findings indicate that XN protects against DNA damage and cancer induced by the cooked food mutagen. Since the effects were observed with low doses of XN which are reached after consumption of brews with high XN levels, our findings may be relevant for humans.

Effects of coffee and its chemopreventive components kahweol and cafestol on cytochrome P450 and sulfotransferase in rat liver.

Coffee drinking appears to reduce cancer risk in liver and colon. Such chemoprevention may be caused by the diterpenes kahweol and cafestol (K/C) contained in unfiltered beverage. In animals, K/C treatment inhibited the mutagenicity/tumorigenicity of several carcinogens, likely explicable by beneficial modifications of xenobiotic metabolism, particularly by stimulation of carcinogen-detoxifying phase II mechanisms. In the present study, we investigated the influence of K/C on potentially carcinogen-activating hepatic cytochrome P450 (CYP450) and sulfotransferase (SULT). Male F344 rats received 0.2% K/C (1:1) in the diet for 10 days or unfiltered and/or filtered coffee as drinking fluid. Consequently, K/C decreased the metabolism of four resorufin derivatives representing CYP1A1, CYP1A2, CYP2B1, and CYP2B2 activities by approximately 50%. For CYP1A2, inhibition was confirmed at the mRNA level, accompanied by decreased CYP3A9. In contrast to K/C, coffee increased the metabolism of the resorufin derivatives up to 7-fold which was only marginally influenced by filtering. CYP2E1 activity and mRNA remained unchanged by K/C and coffee. K/C but not coffee decreased SULT by approximately 25%. In summary, K/C inhibited CYP450s by tendency but not universally. Inhibition of CYP450 and SULT may contribute to chemoprevention with K/C but involvement in the protection of coffee drinkers is unlikely. The data confirm that the effects of complex mixtures may deviate from those of their putatively active components.