Cytotoxicity and genotoxicity of xenoclauxin and desacetyl duclauxin fromPenicillium Duclauxii (delacroix)

The duclauxin derivatives xenoclauxin and desacetylduclauxin were examined for their effects on the growth of L-1210 murine leukemia cells, on the induction of DNA repair in the rat and mouse hepatocyte primary culture (HPC/DNA repair test), and on oxidative phosphorylation in mitochondria from rat livers in comparison to duclauxin. Both derivatives inhibited the growth of L-1210 culture cells as strongly as duclauxin. Duclauxin derivatives were negative in the HPC/DNA repair test. Xenoclauxin exhibited a potent uncoupling effect accompanying a marked depression of state 3 respiration of mitochondria in a similar fashion to that of duclauxin. Desacetylduclauxin significantly inhibited the state 3 respiration without causing uncoupling of oxidative phosphorylation in mitochondria. These results strongly suggest that xenoclauxin and desacetylduclauxin fromPenicillium duclauxii are not genotoxic but are cytotoxic mainly due to their potent inhibition of ATP synthesis in mitochondria.Abbreviations DNP 2,4-dinitrophenol - ETP electron transport particles - HPC hepatocyte primary culture cells - RC respiratory control - TdR thymine deoxyribonucleotide - UDS unscheduled DNA synthesis     

Induction of unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts treated with cysteine S-conjugates of chlorinated hydrocarbons

S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA aminooxyacetic acid - CEC S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase - DCE 1,2-dichloroethane - DCVC S(1,2-dichlorovinyl)-cysteine - GSH glutathione - HU hydroxyurea - IBR IBR-modified Dulbecco's Eagle's reinforced medium - MN2 micronuclei/2,000 cells - 4-NQO 4-nitroquinoline-1-oxide - SHE Syrian hamster embryo fibroblasts; ³H-Thd, ³H-thymidine - TCE 1,1,2-trichloroethylene - UDS unscheduled DNA synthesis     

Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents

The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.Abbreviations 2-AAF 2-acetylaminofluorene - 2-AF 2-aminofluorene - AFB₁ aflatoxin B₁ - ARG autoradiography - DMN dimethylnitrosamine - LSC liquid scintillation counting - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 4-NQO 4-nitroquinoline-1-oxide - PCA perchloric acid - TCA trichloroacetic acid - UDS unscheduled DNA synthesis     

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [³H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.Abbreviations NHMI N-nitrosoheptamethyleneimine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Supported by a grant and a fellowship (R.B.) from the Royal Norwegian Council for Scientific and Industrial Research.     

Microbial glucosidation of dihydroxyanthraquinones. General properties of the glucosidation system

Corresponding mono-β-d-glucosides were obtained by fermentation ofStreptomyces aureofaciens B 96 with four isomeric dihydroxyanthraquinones (alizarin, quinizarin, chrysazin and anthraflavin). The effect of some factors (sugar source, concentration of substrates, pH) on biosynthesis of 1-hydroxy-2-(β-d-glucopyranosyloxy)anthraquinone was studied and optimum conditions for the microbial glucosidation were determined.     

Identification and sequence characterization of a 1.3 Kb EcoRI repeat fragment that harbors a DNA repair site of rat pachytene spermatocytes

Introduction of well-programmed nicks and gaps and the associated DNA repair activity in the genome at the pachytene interval is a characteristic feature of the meiotic prophase in organisms as varied as lilium and mouse. In the present study we have shown that the DNA synthetic activity in rat pachytene spermatocytes is insensitive to aphidicolin, a specific inhibitor of DNA polymerase , and , suggesting DNA -polymerase-mediated repair synthesis in these cells. We have developed a novel approach for the isolation of the DNA repair sites by combining two independent techniques. Following incorporation of BrdUrd into pachytene spermatocytes in the presence of aphidicolin, the repair sites were released as ssDNA fragments by treatment of nuclei with 30 mM NaOH. Subsequently, the BrdUrd containing ssDNA fragments were specifically isolated using polyclonal anti-BrdUrd antibodies. The DNA fragments released were of two size classes, namely 4–7S (major) and 9–12S (minor) and constituted approximately 1.75% of the pachytene genomic DNA. These DNA repair fragments were distinct from Okazaki fragments and other replicative intermediates isolated from rat bone marrow cells as evidenced by (a) their different size distribution and (b) little cross-hybridization. Southern hybridization of restriction enzyme digests of rat genomic DNA with probes made against BrdUrd-ssDNA fragments revealed that although the repair sites were distributed throughout the genome, strong hybridization signals were observed in EcoRI, (1.3 kb and 2.4 kb), BamH1 (9 kb) and HindIII (5 kb) repetetive DNA fragments. The EcoRI 1.3 kb family were cloned into M13 mp19, and a repair positive (1.3 A) and a repair negative (1.3 B) were identified and sequenced. The repair positive clone contained (a) (CA)₂₂ repeat, (b) a (CAGA)₆ repeat and (c) 4 sequences sharing high homology with various hypervariable minisatellite (HVMS) sequences. One of the HVMS sequence contained a GGCAGG motif known to be responsible for germline instability. The repair negative clone had (a) (CA)₆ repeat and (b) a HVMS like sequence without GGCAGG. The significance of these motifs and their relevance to the events of DNA metabolism at pachytene interval have been discussed.     

Modulation of genotoxic and cytotoxic effects of aromatic amines in monolayers of rat hepatocytes

Cultured rat hepatocytes exposed to 2-acetylaminofl uorene (AAF), 2-aminofl uorene (AF) or N-hydroxy-2-acetylaminofluorene (N-OH-AFF) for 3 hrs resulted in an increase in DNA repair measured as unscheduled DNA synthesis, with N-OH-AAF > AAF > AF. Cytotoxic effects were only seen with N-OH-AAF above 10^–6 M. -Naphthof avone increased the unscheduled DNA synthesis and cytotoxic effects of N-OH-AAF, whereas it decreased DNA repair and the covalent binding of AAF to cellular proteins. In contrast, very little effects of paraoxon were seen on the repair synthesis elicited by AAF, AF or N-OH-AAF. The addition of ascorbate reduced the covalent binding of AAF, the DNA repair synthesis caused by AAF and N-OH-AAF, and the cytotoxic effects of N-OH-AAF. The addition of pentachlorophenol or salicylamide all resulted in similar effects as ascorbate, through reduction of sulfation. Galactosamine, an inhibitor of glucuronidation, and the nucleophile GSH caused no or only minor effects of the activation of AAF, AF or N-OH-AAF as judged from the endpoints tested. These results are consistent with an arylnitrenium ion, a sulfate ester or a free radical as the arylamine metabolite causing cellular DNA damage, whereas the sulfate ester or a radical intermediate may be responsible for the cytotoxic effects of N-OH-AAF.Abbreviations AAF 2-acetylaminofluorene - AF 2-aminofluorene - N-OH-AAF N-hydroxy-2-acetylaminofluorene - cytochrome P-450 a collective term for all forms of the cytochrome P-450 polysubstrate monooxygenase - DMSO dimethyl sulfoxide - HU hydroxyurea - S-9 9000 g supernatants - LDH lactate dehydrogenase - UDS unscheduled DNA synthesis - ANF -naphthoflavone - GSH glutathione - PCP pentachlorophenol - MET metyrapone - PAR paraoxon - DEM dimethylmaleate     

Effects of pretreatment with inducers of hepatic mixed function oxidases on DNA repair elicited by various compounds in hepatocytes from adult and neonatal rats

Studies were conducted to assess the effects of inducers of hepatic mixed function oxidases on DNA repair responses to 13 different genotoxic agents in hepatocytes from adult male mice. Phenobarbital pretreatment increased DNA repair elicited by diethylnitrosamine but had no effect on responses to the other compounds. Pretreatment with p,p-dichlorodiphenyltrichloroethane, 3-methylcholanthrene or -naphthoflavone induced the DNA repair responses to a variety of activation-dependent carcinogens. DNA repair responses to the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine were not increased by any of the pretreatments, which indicated that the pretreatment-related enhancement of responses to the other compounds was due to induction of their metabolic activation. Taken together, the findings suggest that Aroclor, or other pretreatments, may increase the sensitivity of the hepatocyte DNA repair assay for detecting the genotoxicity of certain compounds; however, the potential benefit may be limited due to specific features of the assay. In contrast, Aroclor pretreatment did not produce any enhancement of in vivo DNA repair elicited by dimethylnitrosamine, diethylnitrosamine, o-aminoazotoluene, 2-acetylaminofluorene, 3-methylcholanthrene or aflatoxin B₁, and thus does not appear to be useful for improving the sensitivity of the in vivo/in vitro assay.Whereas the amount of DNA repair produced by dimethylnitrosamine was not increased by classical inducers of liver microsomal enzymes, pretreatment with pyrazole greatly augmented in vitro and in vivo DNA repair responses to dimethylnitrosamine; responses to diethylnitrosamine were increased to a lesser degree by pyrazole pretreatment. The effects of lactational exposure to enzyme inducing agents on DNA repair in neonatal hepatocytes was also investigated.Abbreviations 2-AAF 2-acetylaminofluorene - 4-AB 4-aminobiphenyl - 6-AC 6-aminochrysene - AFB aflatoxin B₁ - ARO Aroclor 1254 - o-AT o-aminoazotoluene - B(a)P benzo[a]pyrene - B-NF beta-naphthoflavone - BZ benzidine - DDT p,p-dichlorodiphenyltrichloroethane - DDE p,p-dichlorodiphenyldichloroethylene - DEN diethylnitrosamine - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 3-MC 3-methylcholanthrene - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 2-NA 2-naphthylamine - NNG net nuclear grains - PB phenobarbital - PYR pyrazole     

DNA single stranded gaps formed during DNA repair synthesis induced by methyl methanesulfonate are filled by sequential action of aphidicolin- and dideoxythymidine sensitive DNA polymerases in HeLa cells

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases and ) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase ). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.Abbreviations ddThR dideoxythymidine - MMS methyl methanesulfonate - dNTP deoxynucleoside triphosphate     

Identification of human genes involved in repair and tolerance of DNA damage

Summary An overview is presented on strategies of cloning mammalian DNA repair genes. Complementation of human and rodent repair defects and mutagen hypersensitivities by chromosome and DNA mediated gene transfer and mRNA microinjection is described, and the features of the cloned human DNA repair genes are summarized. It is shown that transfection of repair deficient cell lines with cloned bacterial and human genes may give rise to protection from the genotoxic effects of mutagens.Abbreviations MGMT O⁶-methylguanine-DNA methyltransferase - MNNG N-methyl-N-nitro-N-nitrosoguanidine - HeCNU N-hydroxyethyl-N-chloroethylnitrosourea - MMC mitomycin C - MPA mycophenolic acid - ERCC excision-repair cross-complementing rodent UV-complementation group - UV ultraviolet light - XP xeroderma pigmentosum - CHO Chinese hamster ovary Dedicated to Prof. Dr. U. Hagen on the occasion of his 65th birthdayExtended version of an oral presentation given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Interaction of Replication Protein A and Flap Endonuclease 1 with DNA Duplexes Containing a Nick or a Flap

Nicks and flaps are intermediates in various processes of DNA metabolism, including replication and repair. Photoaffinity modification was employed in studying the interaction of the replication protein A (RPA) and flap endonuclease 1 (FEN-1) with DNA duplexes similar to structures arising during long-patch base excision repair. The proteins were also tested for effect on DNA polymerase (Pol) interaction with DNA. Using Pol, a photoreactive dTTP analog was added to the 3" end of an oligonucleotide flanking a nick or a flap in DNA intermediates. The character and intensity of protein labeling depended on the type of intermediates and on the presence of the phosphate or tetrahydrofuran at the 5" end of a nick or a flap. Photoaffinity labeling of Pol substantially (up to three times) increased in the presence of RPA or FEN-1. Various DNA substrates were used to study the effects of RPA and FEN-1 on Pol-mediated DNA synthesis with displacement of a downstream primer. In contrast to FEN-1, RPA had no effect on DNA repair synthesis by Pol during long-patch base excision repair.     

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) β, ζ or η. Base excision repair was also proficient without Polβ. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polα (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Polδ (pol3-1) or Pol (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Polδ and Pol, respectively. Deleting the polymerase catalytic domain of Pol (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Pol, but not by purified yeast Polη. These results suggest that efficient repair synthesis of yeast NER requires both Polδ and Pol in vitro, and that the low fidelity Polη is not accessible to repair synthesis during NER.     

Sensitivity of the cell cycle to TGFβ1 does not correlate with transformation of a rat liver epithelial cell line

The effects of TGF1 on cell cycle events in a rat liver derived epithelial cell line (BL9) and in two in vitro transformants of this line were studied by flow cytometry. Using either ethidium bromide staining or the incorporation of bromodeoxyuridine to evaluate DNA synthesis it was shown that TGF1 prevented the entry of G0/G1 phase BL9 cells into S phase. TGF1 did not exert its inhibitory effect(s) on DNA synthesis by the modulation of early events in the cell cycle. The tumorigenic transformed BL9 cell lines gave contrasting responses to the effects of TGF1. DNA synthesis in a BL9 cell line derived by transfection with an active N-ras oncogene was unaffected by TFG1 and thus appeared refractory to its growth controlling effects. On the other hand cells from a BL9 cell line derived by in vitro transformation with activated aflatoxin B₁ retained their sensitivity to the effects of TGF1. Thus the loss of the inhibitory effect of TGF1 on DNA synthesis is not obligatory for the malignant transformation of rat liver epithelial cells.Abbreviations TGF1 transforming growth factor 1 - BSA bovine serum albumin - FBS foetal bovine serum - BrdUrd bromodeoxyuridine - PI propidium iodide - PBS phosphate buffered saline     

DNA repair in pollen: Range of mutagens inducing repair, effect of replication inhibitors and changes in thymidine nucleotide metabolism during repair

Summary Pollen of Petunia hybrida carry out DNA repair during the first two hours of germination when certain mutagens are included in the germination medium. This repair, detected readily as unscheduled DNA synthesis, since there is no replicative DNA synthesis in Petunia pollen, can be induced by the chemical mutagens N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, azaserine and methyl methanesulphonate. These compounds are all considered to be capable of direct covalent interaction with DNA. Mutagens requiring metabolic activation before interaction with DNA did not induce DNA repair synthesis in pollen. The practice of solubilizing water-insoluble chemical mutagens with dimethyl sulphoxide did not prove practical, due to the extremely harmful effects of dimethyl sulphoxide on pollen. Pretreatment of pollen before germination with pure ether, however, had no harmful effect on either repair or pollen germination. Therefore water-insoluble, ether-soluble mutagens were tested by pretreatment of the pollen with mutagens in ether solution. By this means it was shown that the direct-acting mutagen, diethyl sulphate, would also bring about unscheduled DNA synthesis in pollen, while 2-acetylaminofluorence and dimethyl-p-aminobenzene, both requiring metabolic activation, did not do so. Inhibitors of DNA replicative synthesis, hydroxyurea, azaserine, azauridine and fluorodeoxyuridine did not inhibit unscheduled DNA synthesis brought about by N-methyl-N-nitro-N-nitrosoguanidine. On the contrary, these compounds stimulated repair synthesis to varying degrees, hydroxyurea having the greatest effect. Pollen uptake of ³H-thymidine and the amount of radioactive label subsequently appearing in dTMP and dTDP+dTTP was increased by 4-nitroquinoline-1-oxide. Partial inhibition of these increases and of 4-nitroquinoline-1-oxide induced repair synthesis by 3,5-cyclic AMP suggested that thymidine:AMP phosphotransferase rather than thymidine kinase was responsible for thymidine phosphorylation in pollen. Enzyme assays on pollen extracts confirmed this.     

Protective devices of early developmental stages in Pyrrhalta viburni (Coleoptera,Chrysomelidae)

Summary Larvae and eggs of the leaf beetle Pyrrhalta viburni were investigated for protective devices against predators. The eggs are covered with faeces, which appeared to have no feeding deterrent activity against the ant Myrmica sabuleti. Chemical analyses of the material covering the eggs by gas chromatography-mass spectrometry (GC-MS) showed the triterpenes -amyrin and -amyrin as main components. Both compounds are also present in the hostplant Viburnum lantana. GC-MS analyses of eggs and larvae showed that 1,8-dihydroxy-3-methylanthraquinone (=chrysophanol) was present in both developmental stages. In the larvae, 1,8-dihydroxyanthraquinone (=chrysazin) and 1,8,9-trihydroxyanthracene (dithranol) were also detected. Neither hydroxylated anthraquinones nor dithranol were found in bark and leaf extracts of the hostplant. After assessing the total amounts of these compounds in a single larva, their ecological significance was studied in feeding bioassays with M. sabuleti. Both P. viburni larvae and equivalent amounts of anthraquinones and dithranol deterred feeding by the ants. The role of anthraquinones and triterpenes in P. viburni is discussed.     

Calcium-binding protein regucalcin inhibits deoxyribonucleic acid synthesis in the nuclei of regenerating rat liver

The effect of regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, on deoxyribonucleic acid (DNA) synthesis in the nuclei of regenerating rat liver was investigated. At 1 day after partial hepatectomy, the liver weight was increased about 50% of that of sham-operated rats, and it reached to the same levels as sham operation at 3 days after hepatectomy. Nuclear DNA synthesis was markedly increased at 1 day after hepatectomy, and this increase was also seen at 3 days. Nuclear DNA synthesis was clearly enhanced in the presence of EGTA (0.4 mM) in the incubation mixture. The presence of Ca²⁺ ( 1.0–25 M) caused a significant decrease in the nuclear DNA synthesis of normal rat liver. Regucalcin (0.25 and 0.5 M) clearly inhibited the nuclear DNA synthesis of normal rat liver. This inhibition was also seen in the presence of Ca²⁺ (1.0 M). Moreover, in the liver nuclei obtained at 1 day after partial hepatectomy, the presence of regucalcin (0.05–0.5 M) caused a remarkable inhibition of nuclear DNA synthesis. This effect was also revealed in the presence of EGTA (0.4 mM). Thus, the inhibitory effect of regucalcin was remarkable in regenerating rat liver nuclei in comparison with that of normal rat liver. The present results demonstrate that regucalcin can suppress nuclear DNA synthesis in regenerating rat liver. We suppose that regucalcin may have a role in the regulation of nuclear DNA synthesis in liver cell proliferation.     

Escherichia coli mutY-dependent mismatch repair involves DNA polymerase I and a short repair tract

Repair of heteroduplex DNA containing an A/G mismatch in a mutL background requires the Escherichia coli mutY gene function. The mutY-dependent in vitro repair of A/G mismatches is accompanied by repair DNA synthesis on the DNA strand bearing mispaired adenines. The size of the mufY-dependent repair tract was measured by the specific incorporation of -[³²P]dCTP into different restriction fragments of the repaired DNA. The repair tract is shorter than 12 nucleotides and longer than 5 nucleotides and is localized to the 3 side of the mismatched adenine. This repair synthesis is carried out by DNA polymerase I.     

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.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HA hydroxyanthraquinones - LUE lucidinethylether - PRH primary rat hepatocytes - UDS unscheduled DNA synthesis     

Effect of zearalenone (F-2) on pea stem,maize root,and rat liver mitochondria

At 5 and 10 g ml^-1 concentration, zearalenone (F-2), a mycotoxin produced by a number of species of the genus Fusarium, causes an inhibition of the oxidative phosphorylation of isolated plant mitochondria, while at 20 and 40 g ml^-1 it causes uncoupling. However, when the mitochondria are pre-incubated for 20 min with F-2, the uncoupling appears to be the prevailing effect. F-2 is also able to inhibit the mitochondrial ATPase activity (Mg²⁺-dependent). Conversely, F-2 (40 g ml^-1) does not alter the ATP level of maize roots and only slightly affects the ATPase activity of pea stem and maize root microsomal fractions. In addition, F-2 (10–40 g ml^-1) inhibits ATP synthesis catalyzed by rat liver mitochondria. It is suggested that the phytotoxicity of F-2, also known for its ability to collapse the transmembrane electric potential of maize roots, may be mainly linked to its ability to increase the proton permeability of the cell, similar to the common uncouplers.Abbreviations F-2 zearalenone - DCCD N,N-dicyclohexylcarbodiimide - PCCP carbonyl cyanide, p-trifluoromethoxiphenylhydrazone - CBT Cerospora beticola toxin     

Induction by mycotoxins of somatic mosaicism in Drosophila and DNA repair in mammalian liver cell cultures

The genotoxic activity of four mycotoxins has been studied. High level of somatic mutagenesis in imaginal discs of Drosophila melanogaster larvae and DNA repair synthesis in human embryo and adult rat liver cell cultures were inducible only by highly carcinogenic aflatoxin B1. Patulin, a weak direct-action carcinogenic substance, slightly elevated the mutagenesis in somatic cells of Drosophila but did not induce DNA repair synthesis in liver cell cultures. Citrinin that did not exhibit any carcinogenic properties when used alone and stachybotrotoxin with non-reported carcinogenic activity appeared inactive in the test-systems applied. The possibilities of rapid recognition of carcinogenic mycotoxins by detecting their genotoxic properties are discussed.