Gerlinde Scharf

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.

Effects of cruciferous vegetables and their constituents on drug metabolizing enzymes involved in the bioactivation of DNA-reactive dietary carcinogens

Epidemiological studies give evidence that cruciferous vegetables (CF) protect humans against cancer, and also results from animal experiments show that they reduce chemically induced tumor formation. These properties have been attributed to alterations in the metabolism of carcinogens by breakdown products of glucosinolates, which are constituents of CF. The present article gives an overview on the present state of knowledge on the impact of CF and their constituents on enzymes that are involved in the metabolism of DNA-reactive carcinogens. The development of in vitro models with metabolically competent cell lines led to the detection of potent enzyme inducers contained in CF such as sulforaphane. Recently, we showed that Brassica juices induce glutathione-S-transferases (GST) and cytochrome P-450 1A2 in human hepatoma cells (HepG2) and protect against the genotoxic effects of B(a)P and other carcinogens. Earlier in vivo experiments with rodents indicated that indoles and isothiocyanates, two major groups of glucosinolate breakdown products, attenuate the effects of polycyclic aromatic hydrocarbons (PAHs) and nitrosamines via induction of GST and inhibition of cytochrome-P450 isoenzymes, respectively. Our own investigations showed that CF are also protective towards heterocyclic amines (HAs): Brussels sprouts- and garden cress juices attenuated IQ-induced DNA-damage and preneoplastic lesions in colon and liver of rats. These effects were paralleled by induction of uridine-di-phospho-glucuronosyl transferase (UDPGT) which is very probably the mechanism of protection against HAs by cruciferous vegetables. There is also evidence that consumption of CF might protect humans against cancer. In matched control intervention studies with these vegetables, it was shown that they induce GST-activities in humans but overall, results were inconclusive. Recently, we carried out crossover intervention studies and found pronounced GST-induction upon consumption of Brussels sprouts and red cabbage, whereas no effects were seen with white cabbage and broccoli. Furthermore, we found that the isoenzyme induced was GST-pi which plays an important role in protection against breast, bladder, colon and testicular cancer. No induction of the GST-alpha isoform could be detected. Urinary mutagenicity experiments gave further evidence that CF affect drug metabolism in humans. Consumption of red cabbage led to changes in the pattern of meat-derived urinary mutagenicity. Overall, CF are among the most promising chemopreventive dietary constituents and further elucidation of their protective mechanisms and the identification of active constituents may contribute to the development of highly protective Brassica varieties.

Enhancement of Glutathione and γ-Glutamylcysteine Synthetase, the Rate Limiting Enzyme of Glutathione Synthesis, by Chemoprotective Plant-Derived Food and Beverage Components in the Human Hepatoma Cell Line HepG2

Glutathione (GSH) is an important antioxidant and cofactor of detoxifying metabolism. Therefore, elevation of GSH as achieved by inducing γ-glutamylcysteine synthetase (GCS), the limiting enzyme of GSH synthesis, may contribute to chemoprevention against cancer. In previous animal studies, increases in GCS were mainly found in liver and other organs that are not easily accessible in humans. Thus, employment and evaluation of alternative systems such as human-derived cell lines are encouraged. In the present experiment, we used the hepatoma cell line HepG2 to investigate the response of GCS and GSH to five plant-derived chemoprotectants contained in regularly consumed foodstuffs and beverages (kahweol/cafestol [K/C] [15.5-62.0 μM], α-angelicalactone [100-400 μM], benzyl isothiocyanate [1.7-5.0 μM], diallyl sulfide [175-700 μM], and quercetin [10-50 μM]). All treatments led to dose-dependent increases in both GCS activity and GSH concentration. Time course studies with K/C indicated that the enhancement of GCS preceded that of GSH, suggesting a causal relationship. K/C did not enhance γ-glutamyl transpeptidase, a further enzyme that assists GSH-related chemoprotection. Although GCS induction has been suggested to require an initial short-lived GSH depletion, we did not find any decrease in GSH after 3 h of incubation with K/C. In summary, HepG2 cells were shown to be a useful model to investigate the capacity of potential chemoprotectants to enhance GCS and GSH. To our knowledge, the present study is also the first to show increases in GCS by K/C and a-angelicalactone in vitro and by diallyl sulfide and quercetin in any system.

The coffee components kahweol and cafestol induce γ-glutamylcysteine synthetase, the rate limiting enzyme of chemoprotective glutathione synthesis, in several organs of the rat

Abstract. The coffee components kahweol and cafestol (K/C) were reported to be protective against mutagenic damage by heterocylic amines and aflatoxin B1 in the rat, while in humans the consumption of coffee with a high K/C content was associated with a lower rate of colon tumors. An important mechanism of this antimutagenic effect appears to be the potential of K/C to induce glutathione-S-transferase (GST) and to enhance hepatic levels of glutathione (GSH), the co-factor of GST, which is independently involved in further protective mechanisms. In the present study, we investigated mechanisms and organ specificities (liver, kidney, lung, colon) of the K/C effect on GSH levels, and particularly the role of γ-glutamylcysteine synthetase (GCS), the rate limiting enzyme of GSH synthesis. Chows containing one of four concentrations of either a 1:1 mixture of K/C (0.012–0.122%) or of cafestol alone (0.006–0.061%) were fed to male F344 rats for 10 days. In the K/C-treated livers, a dose-dependent increase of up to 2.4-fold in the activity of GCS was observed, being statistically significant even at the lowest dose, and associated with an increase in GSH of up to three-fold. Notably, the highest dose doubled the hepatic mRNAs of the heavy and light subunits of GCS, suggesting enhanced transcription. In the extrahepatic organs, GCS activity and GSH levels were increased as well, although more moderately than in the liver. Since enhancement of GCS had also been observed as a consequence of oxidative stress, the possibility of such an involvement in the actions of K/C was examined by determining hepatic thiobarbituric acid reactive substances and the ratio of oxidized and reduced GSH. However, no evidence of oxidative stress was detected. In summary, K/C increased GSH levels apparently through the induction of the rate limiting enzyme of GSH synthesis, which may be a key factor in the chemopreventive potential of coffee components.

Search for dietary antimutagens and anticarcinogens: methodological aspects and extrapolation problems

It is well documented that dietary factors play a crucial role in the aetiology of human cancer and strong efforts have been made to identify protective (antimutagenic and anticarcinogenic) substances in foods. Although numerous studies have been published, it is problematic to use these results for the development of nutritional strategies. The aim of this article is a critical discussion of the pitfalls and problems associated with the search for protective compounds. The main obstacles in regard to the extrapolation of the data to the human situation arise from: (i) the use of inadequate experimental in vitro models, which do not reflect protective mechanisms in man and therefore give misleading results; (ii) the use of genotoxins and carcinogens that are not relevant for humans; (iii) the lack of knowledge about dose-effect relationships of DNA-protective and cancer protective dietary constituents; (iv) the use of exposure concentrations in animal models which exceed by far the human exposure levels; and finally (v) the lack of knowledge on the time-kinetics of protective effects. More relevant data can be expected from in vitro experiments with cells possessing inducible phase I and phase II enzymes, short-term in vivo models with laboratory animals which enable the measurement of effects in organs that are targets for tumour formation, and human biomonitoring studies in which endpoints are used that are related to DNA damage and cancer.

Coffee and its chemopreventive components Kahweol and Cafestol increase the activity of O6-methylguanine-DNA methyltransferase in rat liver—comparison with phase II xenobiotic metabolism

A lower rate of colon cancer was observed in consumers of coffee with a high content of the diterpenes Kahweol and Cafestol (K/C). In animal models, K/C have been found to protect against the mutagenic/carcinogenic effects of compounds such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), aflatoxin B1, and 7,12-dimethylbenz[a]anthracene. Thus far, such chemoprotection by K/C has been attributed to modifications of xenobiotic metabolism, e.g. enhanced detoxification by UDP-glucuronosyltransferase (UDPGT) and/or glutathione transferase (GST). In the present study, we investigated the potential of several coffee-related treatments (K/C [1:1], Cafestol-alone, Turkish coffee) to modify the expression level of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) which is involved in the reversal of the precarcinogenic DNA damage O(6)-alkylguanine induced by alkylating agents. The results show that, in the male F344 rat, K/C and Cafestol increase hepatic MGMT in a dose-dependent manner up to a maximum of 2.6-fold at 0.122% K/C in the feed. Turkish coffee led to enhancements of up to 16%, the more moderate increase being associated with the lower estimated K/C intake through the beverage. In the livers of the rats receiving Turkish coffee, we also found 10-30% increases in several GST-related parameters (overall GST, GST-pi, glutathione, gamma-glutamylcysteine-synthetase) and a two-fold increase in UDPGT activity. Dose-response studies with K/C revealed that MGMT increased in parallel with three of the four GST-related parameters whereas the dose-response curves of UDPGT and of GST-pi activity displayed a steeper slope. Increased expression level of MGMT may extend the antimutagenic/anticarcinogenic potential of coffee components to protection against DNA alkylating agents.

Effects of garden and water cress juices and their constituents, benzyl and phenethyl isothiocyanates, towards benzo(a)pyrene-induced DNA damage: a model study with the single cell gel electrophoresis/Hep G2 assay

The aim of this study was to investigate the chemoprotective effects of water and garden cress juices towards benzo(a)pyrene (B(a)P)-induced DNA damage using the single cell gel electrophoresis (SCGE)/Hep G2 test system. This experimental model combines the advantages of the SCGE assay with that of human derived cells possessing inducible phase I and phase II enzymes. Treatment of Hep G2 cells with small amounts of water cress or garden cress juice (0.1-1.25 microl/ml) and B(a)P reduced the genotoxic effect of the latter in a dose-dependent manner. Contrary to the results with the juices, unexpected synergistic effects were observed with benzyl isothiocyanate (BITC, 0.6 microM), a breakdown product of glucotropaeolin contained abundantly in garden cress. Although these concentrations of BITC did not cause DNA damage per se, at higher concentrations (> or = 2.5 microM), the compound caused a pronounced dose-dependent DNA damage by itself. With phenethyl isothiocyanate (PEITC), the breakdown product of gluconasturtin contained in water cress, no synergistic effects with B(a)P were seen; however, significant induction of DNA damage was observed when the cells were exposed to the pure compound at concentrations > or = 5 microM. In experiments with (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE, 5.0 microM), the ultimate genotoxic metabolite of B(a)P, and the juices, only moderate protective effects were seen indicating that detoxification of BPDE is not the main mechanism behind the protective effect of the juices against B(a)P-induced DNA damage. In conclusion, our findings show that garden and water cress juices are highly protective against B(a)P-induced DNA damage in human derived cells and that their effects can not be explained by their isothiocyanate contents.

Protective effects of Brussels sprouts towards B[a]P-induced DNA damage: a model study with the single-cell gel electrophoresis (SCGE)/Hep G2 assay

The aim of this study was to investigate the chemoprotective effects of Brussels sprouts juice towards benzo[a]pyrene (B(a)P)-induced DNA damage in the single-cell gel electrophoresis (SCGE)/Hep G2 test system. This assay combines the advantages of the SCGE assay with that of the use of human-derived cells possessing inducible phase I and phase II enzymes. Co-treatment of Hep G2 cells with small amounts of Brussels sprouts juice (0.25-2.0 microl/ml) and B(a)P reduced the genotoxic effect of the latter in a dose-dependent manner. Contrary to the results with the crude juice, unexpected synergistic effects were observed with allyl isothiocyanate (AITC, 1.0-6.0 microM), a breakdown product of sinigrin, which is the most abundant glucosinolate in Brussels sprouts. Although these concentrations of AITC did not cause DNA damage per se, at higher concentrations (> or =25 microM), the compound caused a pronounced dose-dependent DNA damage by itself. Mechanistic studies showed that Brussels sprouts juice causes induction of activities of ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) at dose levels which were protective towards B(a)P. In combined treatment experiments with (+/-)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE, 5.0 microM), the main genotoxic metabolite of B(a)P, and Brussels sprouts juice, only weak protection was found indicating that the mechanism of chemoprotection of Brussels sprouts is not mediated through inactivation of this metabolite. In conclusion, our findings show that Brussels sprouts are highly protective against B(a)P-induced DNA damage in human-derived cells.

Elevation of glutathione levels by coffee components and its potential mechanisms.

The tripeptide glutathione (L-γ-glutamyl-L-cysteinylglycine) is found ubiquitous in microorganisms, plants and animals. In mammalian cells, where the tripeptide fulfils numerous functions, concentrations range from 0.5 to 10 mM (Meister & Tate, 1976; Meister, 1984; Redegeld et al., 1990). Glutahione is involved, for example, in the synthesis of proteins and DNA, in the regulation of enzyme activity, in the transport and reservoir of amino acids. A very important function of glutathione is the protection of cells, for instance as an antioxidant or as a co-factor in the conjugation of xenobiotics (Meister & Anderson, 1983; Redegeld et al., 1990).