Johannes Westendorf

Genotoxicity of naturally occurring hydroxyanthraquinones

A variety of structurally related hydroxyanthraquinones (HA) were investigated in a test battery for the evaluation of mutagenicity and cell-transforming activity. The tests were: (1) the Salmonella typhimurium mutagenicity assay, (2) the V79-HGPRT mutagenicity assay, (3) the DNA-repair induction assay in primary rat hepatocytes and (4) the in vitro transformation of C3H/M2 mouse fibroblasts. In Salmonella, most of the tested compounds were mutagenic in strain TA1537, but only a few were active in other strains. Among these were HA with a hydroxymethyl group, such as lucidin and aloe-emodin. In V79 cells, only HA with 2 hydroxy groups in the 1,3 positions (1,3-DHA, purpurin, emodin) or with a hydroxymethyl sidechain (lucidin and aloe-emodin) were mutagenic. The compounds found to be active in V79 cells were also active in the DNA-repair assay and in the C3H/M2 transformation assay. Thus, it appears that the genotoxicity of HA is dependent on certain structural requirements.

Toxicological profile of pollutants in surface water from an area in Taihu Lake, Yangtze Delta

The environment in urbanized areas is often contaminated with a complex mixture of toxic compounds originating from industries, agriculture and private households. Most of the contaminants end up in surface waters, such as lakes, rivers and finally the sea. Toxic contaminants may disturb the biological condition of aquatic ecosystems and be harmful for humans, if they are transported to human food or drinking water. A variety of biological tests have been introduced for monitoring the toxicological profile of aquatic ecosystems. In the present investigation, genotoxic, hormone disrupting and Ah-receptor activity was analyzed in water collected in January 1999 at Meiliang Bay, Taihu Lake, China, near to the city of Wuxi in the Shanghai area. Significant mutagenic activity could be detected in water extracts with the Salmonella/microsome assay and the arabinose resistance test. Frame shift mutations were the predominant mode of action. Ah-receptor active compounds were detected by using a luciferase reporter gene assay (CALUX-assay). The estimated toxic equivalent factor-values ranged between 134 and 232 pg TCCD-equivalents per liter lake water. The estrogen-like potential of Taihu water was estimated with two luciferase reporter gene assays using transgenic human cell lines expressing estrogen receptor alpha. Estradiol equivalents obtained with water extracts were in the range of 2.2-12.1 ng/l. We also analyzed the concentrations of 17-beta-estradiol and ethinylestradiol in the extracts using a high-pressure liquid chromatography-method. The values obtained correlated with the biologically determined estradiol equivalents, indicating that the estrogenic activity is mainly due to natural and synthetic hormones rather than xenoestrogens.

Genotoxicity assessment of suspended particulate matter in the Elbe river: comparison of Salmonella microsome test, arabinose resistance test, and umu-test

This study evaluates the applicability of three bacterial short-term genotoxicity test systems to aquatic suspended particulate matter of the Elbe river. This material was sampled in sedimentation vessels after deposition periods of one month. It was extracted in a Soxhlet apparatus with toluene and methanol. Aqueous elutriates were prepared additionally. A solid phase method was developed that enables to incubate bacteria in contact with the particulate material. The test battery consists of two mutagenicity assays (the Ames-test and the Ara-test) and an SOS induction assay (the umu-test). Both mutagenicity assays came to nearly the same assessment of the samples of particulate matter of the Elbe. The quantitative response, however, was higher in the Ara-test. The particulate river material generally induced lower genotoxic potencies in the umu-test than in the mutagenicity assays. This lead to a completely different outcome of the umu-test; 29 out of 35 mutagenic samples were not SOS inducing. No quantitative or rank correlations between the concentrations of anthropogenic contaminants (PAHs, chlorinated hydrocarbons and metals) and the observed effects could be established on a 90% confidence limit. However, there is obvious correspondence between more contaminated regions of the river system and mutagenic effects (Ames- and Ara-test) in the samples from this region, as well as correspondence between low contaminated regions and the absence of mutagenicity. For this reason, the mutagenicity assays appear more favourable to describe the anthropogenic contamination with genotoxins in complex mixtures than the umu-test. The authors recommend the Ara-test for a first genotoxicity screening of complex environmental mixtures. This forward mutagenicity assay is advantageous due to higher effects and lower costs compared to the Ames-test. The development and use of a solid phase version of Ames- and Ara-test revealed the occurrence of a major part of particle-bound mutagens. The hydrophobic nature of these mutagens was also confirmed by the gradually decreasing effects with decreasing lipophilicity of the solvents. The results suggest that the solid phase test and the use of extracts complement each other in detecting mutagens of different lipophilicity. Both versions should be used in order to include a broad variety of compounds.

Analgesic and antiinflammatory activity of Morinda citrifolia L. (Noni) fruit

M. citrifolia is a tropical plant with a long tradition of medicinal use in Polynesia and tropical parts of eastern Asia and Australia. One of its favorite uses is the treatment of painful inflammatory conditions, such as arthritis. The analgesic activity of Noni fruit puree on mice was investigated using the hot plate test. A 10% solution of freeze concentrated Noni fruit puree in the drinking water of mice reduced the pain sensitivity comparably to the central analgesic drug tramadol. This effect was only partly reversed by the application of the morphine antagonist naloxone. An alcohol extract of noni fruit puree also caused an inhibition of MMP‐9 release from human monocytes after stimulation with LPS. This effect was comparable to hydrocortisone (10−5 m). The findings suggest that preparations of noni fruits are effective in decreasing pain and joint destruction caused by arthritis. Copyright

Induction of DNA strand breaks in rainbow trout Oncorhynchus mykiss under hypoxic and hyperoxic conditions

Abstract Rainbow trout (Oncorhynchus mykiss) were exposed to various degrees of oxygen saturation (3.3–21.1 mg O2/l). After 5 h of exposure, trout were killed and DNA single-strand breaks (DSSB) were determind in the gills by the alkaline unwinding method. Significant increases of DSSB compared to normoxic conditions (11 mg O2/l) were observed under hypoxic and particularly hyperoxic conditions. The highest rate of DSSB occurred when the fish were kept under hypoxic conditions followed by a rapid increase of the oxygen concentration. DSSB formation under these conditions was comparable to that achieved by treatment with 200 μM of the potent carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These data demonstrate that natural influences, such as varying oxygen concentrations in the water, can be responsible for the induction of DSSB. These findings call into question the usefulness of the measurement of DSSB in fish as an indicator of water pollution by genotoxic chemicals.

Formation of genotoxic metabolites from anthraquinone glycosides, present in Rubia tinctorum L.

Rubia tinctorum L., a medicinal plant used for the treatment of kidney and bladder stones, contains a characteristic spectrum of 9,10-anthraquinone derivatives, which are substituted in only one of the aromatic benzo rings. The majority of the anthraquinones present in the plant itself or in plant extracts are glycosides. We investigated the metabolism of two such glycosides, alizarinprimeveroside (AlP) and lucidinprimeveroside (LuP). AlP given orally to rats was metabolized to alizarin (Al) and 1-hydroxyanthraquinone (1-HA). The reductive cleavage of AlP was also observed after treatment of this compound with rat liver enzymes (S9) and NADPH. 1-HA has been reported to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes (PRH) and intestinal and liver tumors in rats after chronic treatment. The in vitro genotoxicity of 1-HA was confirmed by our present investigations. We also observed that the glycoside AlP was active at inducing UDS in PRH, but the compound was inactive in the Salmonella/microsome assay. Oral administration of LuP to rats resulted in the excretion of lucidin and rubiadin. When LuP was treated with rat liver extract and NADPH, the compound was reduced to rubiadinprimeveroside (RuP), which was hydrolyzed to rubiadin. We have recently shown that lucidin is highly genotoxic in a battery of short-term tests. We now report that rubiadin is also highly genotoxic in Salmonella typhimurium. However, in contrast to lucidin, it requires metabolic activation. In the UDS assay in PRH, rubiadin was even more potent than lucidin and equal to the positive control DMBA. In addition, the glycoside LuP is active in the Salmonella/microsome assay as well as in the UDS assay. The present work demonstrates that the uptake of the anthraquinone glycosides AlP and LuP leads to the rodent carcinogen 1-HA, and to the highly genotoxic compounds lucidin and rubiadin. This extends our previous studies and supports our suggestion that the therapeutic use of Rubia tinctorum may involve a carcinogenic risk.

Regulation of osteoblastic phenotype and gene expression by hop-derived phytoestrogens.

Certain plant-derived compounds show selective estrogen receptor modulator (SERM) activity and may therefore be an alternative to the conventional hormone replacement therapy, which prevents osteoporosis but is also associated with an increased risk of breast and endometrial cancers. In the current study, we tested the effects of the hop-derived compounds 8-prenylnaringenin, 6-prenylnaringenin, xanthohumol and isoxanthohumol (1) to modulate markers of differentiation and gene expression in osteoblasts and (2) to regulate proliferation in MCF-7 breast cancer cells. Additionally, we analyzed the ER-binding affinities of these hop compounds as well as the ER-mediation of their effects. Bone-forming activity and ER-subtype specificity were investigated by measuring alkaline phosphatase (AP) activity in hFOB/ERalpha cells and regulation of gene transcription for AP, interleukin-6, pS2 and von Willebrand factor (VWF) in U-2 OS/ERalpha and U-2 OS/ERbeta cells. Our results demonstrate that AP, pS2 and VWF mRNA levels are significantly increased by the compounds in an estrogen-like manner via both ERalpha and ERbeta, while IL-6 is down-regulated in U-2 OS/ERalpha cells. Consistently, AP enzymatic activity is up-regulated by all compounds in hFOB/ERalpha9 cells. Depending on their concentration, all compounds show proliferative effects in MCF-7 cells. Except for 8-PN the hop constituents display an ERbeta-preference. Reversal of estrogen-specific AP-induction in Ishikawa cells indicates an ER-regulated mechanism. Finally, the flavonoids display cytotoxic effects only at high concentrations (> or =10(-4)M). In summary, we have demonstrated for the first time that specific phytoestrogen compounds found in hop extracts exert estrogen-like activities on bone metabolism. Regarding a potential for use in osteoporosis-prevention therapy, the dosage of a phytoestrogen, which is taken, will play an important role concerning a desired in vivo profile.

The genotoxicity of lucidin, a natural component of Rubia tinctorum L., and lucidinethylether, a component of ethanolic Rubia extracts.

The genotoxic activity of lucidin (1,3-dihydroxy-2-hydroxymethyl-9,10-anthraquinone), a natural component of Rubia tinctorum L., was tested in a battery of short-term tests. The compound was mutagenic in five Salmonella typhimurium strains without metabolic activation, but the mutagenicity was increased after addition of rat liver S9 mix. In V79 cells, lucidin was mutagenic at the hypoxanthine-guanine phosphoribosyl transferase gene locus and active at inducing DNA single-strand breaks and DNA protein cross-links as assayed by the alkaline elution method. Lucidin also induced DNA repair synthesis in primary rat hepatocytes and transformed C3HI M2-mouse fibroblasts in culture. We also investigated lucidinethylether, which is formed from lucidin by extraction of madder roots with boiling ethanol. This compound was also mutagenic in Salmonella, but only after addition of rat liver S9 mix. Lucidinethylether was weakly mutagenic to V79 cells which were cocultivated with rat hepatocytes. The compound did not induce DNA repair synthesis in hepatocytes from untreated rats, but positive results were obtained when hepatocytes from rats pretreated with phenobarbital were used. We conclude that lucidin and its derivatives are genotoxic.

Formation of DNA-adducts and induction of DNA-crosslinks and chromosomal aberrations by the new potent anthracycline antitumor antibiotics: morpholinodaunomycin, cyanomorpholinodaunomycin and cyanomorhpolinoadriamycin

The DNA-interaction of three newly developed semisynthetic anthracyclines with high antitumor potency MoDNM3, CNMoDNM, and CNMoADM, was investigated. When primary rat hepatocytes were incubated with tritium labeled MoDNM and CNMoDNM and their DNA was purified and enzymatically hydrolized, the formation of DNA-adducts could be demonstrated by the HPLC chromatography of the resulting mononucleoside mixtures. The parent compound, daunomycin (DNM), also formed covalent adducts with hepatocyte DNA, but to a lesser extent. These findings correlate well earlier observaitons that MoDNM and CNMoDNM are potent inducers of DNA-repair in primary rat hepatocytes, whereas DNM is only weakly active in this regard. Aklaline elution studies were performed with L 1210 mouse leukemia cells and V79 Chinese hamster fibroblasts. The cyanomorpholinyl derivatives showed dose-dependant DNA crosslinking activities in both cell lines at concentrations ≥ 5 nMol/l. The formation of crosslinks began a few minutes after treatment of the cells and reached a maximum after 1 hr. In contrast, MoDNM, at concentrations of up to 10 μMol/l, had only a limited capacity to induce single strand breaks in L 1210 cells but did not induce DNA-crosslinks. In addition, chromosomal aberrations (chromatid breaks and translocations) were induced by the treatment of Friend and L 1210 leukemia cells with CNMoADM at concentrations between 0.07–0.6 n Mol/l. At higher doses, chromosome clumping was observed. These results indicate that the high capacity of MoDNM, CNMoDNM and CNMoADM to induce DNA repair in primary rat hepatocytes is due to the formation of covalent adducts with DNA. The cyanomorpholino compounds have alkylating capacities also in cell lines such as L 1210 and V79, whereas MoDNM requires rat hepatocytes for activation. The ready formation of DNA crosslinks and chromosomal aberrations could be responsible for the high cytotoxicity of these compounds.

In vitro suppression of thymocyte apoptosis by metal-rich complex environmental mixtures: potential role of zinc and cadmium excess

Excessive amounts of heavy metals (e.g. Zn, Cu, Mn, Cr) are accumulated in river bottom sediments (RBS), being available to humans and animals along food chains. Increased exposure of mammals to certain metals (Cr, Cu) induces immunosuppresion, due to DNA damage and decreased survival of lymphoid cells. By contrast, excess of Zn and Cd causes inhibition of apoptosis thus suggesting increased survival of genetically mutated cells and higher cancer risks in exposed populations. Rat thymic lymphocytes represent a well-established model for apoptosis testing. The primary goal of our study was to assess the degree of apoptosis modulation with a number of RBS extracts differing in their metal contents. A series of freshly deposited RBS was collected at nine sampling stations along the Elbe River. All sediments were rich in Fe, Mn and Zn. The contents of Cu, Cr, Ni, Cd, Hg, Pb and As were much lower and interrelated. The short-term cytotoxicity of aqueous sediment extracts was assessed, using the following criteria: total cell counts; incidence of apoptosis and necrosis (morphological detection by fluorescent microscopy); and nuclear chromatin decay (by DNA flow cytometry). RBS extracts produced both apoptosis and necrosis of thymocytes. High contents of zinc and other heavy metals in the samples correlated with decreased thymocyte apoptosis (r= -0.543 to -0.608, P <0.01). The rates of thymocyte damage showed a distinct dependence on the time and region of sampling. Apoptosis modulation was also tested with pure salts of Mn(II), Zn(II), Cu(II), Cr(III) and Cd(II), at the test concentrations of 1, 10 and 100 microM. Cu(II) and Cr(III) proved to induce marked dose-related apoptosis, whereas Zn(II) ions caused significant suppression of apoptosis. These effects were similar to those trends observed with metal-rich sediments. In the present study. DNA flow cytometry proved to be a less sensitive index of cell death than morphological assay of apoptosis and/or necrosis. In summary, inhibition of lymphocyte apoptosis by RBS extracts and pure metals is associated with excess of zinc and, probably, cadmium. The proposed model of lymphoid cell apoptosis is a promising tool for screening cytotoxic effects of complex environmental samples.