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     

Characterization of lucidin formation in Rubia tinctorum L.

In order to approach lucidin formation (a strong mutagen or a carcinogen) from a physiological standpoint, hairy roots of Rubia tinctorum L. were established by a transformation of Agrobacterium rhizogenes strain 15834 and cultured in a liquid woody plant medium without plant hormones. The anthraquinone pigment composition of the intact hairy roots was essentially the same as that of the intact non-transformed (normal) roots, in which lucidin O-beta-D-primeveroside (LuP) was one of the major pigments. Lucidin was scarcely detected in the intact hairy roots, but was a main pigment after the squash treatment. The crude protein extract of intact hairy roots exhibited LuP-glycosidase activity (an activity converting LuP to lucidin). This activity was also detected in the roots of the normal plants at a high level, but slightly in the stems and not in the leaves. Methyl jasmonate enhanced the LuP production and LuP-glycosidase activity in the hairy roots. On the other hand, ethephon or salicylic acid had either no effect or rather an inhibitory effect on them. After partial purification of LuP glycosidase, the resultant active fraction producing a major band with an apparent Mr of 68 kDa exhibited the substrate specificity for both aglycon and sugar-moiety. The sugar released from LuP by this fraction was neither D-glucose nor D-xylose and was hydrolyzed into them. These results suggest that LuP specific beta-primeverosidase (EC 3.2.1.149) exists in the roots of R. tinctorum and is involved in the systematic defense system.     

Identification of a mutagenic substance, in Rubia tinctorum L. (madder) root, as lucidin

Mutagenicity of the extract from Rubia tinctorum L. (madder) root was demonstrated on Salmonella typhimurium TA100 and TA98. The active substance wa purified and characterized by TLC, UV spectrum, IR spectrum, mass spectrum and [1H]NMR spectrum. All the mutagenic activity of the extract from the root of Rubia tinctorum L. was due to lucidin (1,3-dihydroxy,2-hydroxymethyl-9, 10-anthraquinone).     

Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone

3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK+/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.     

An assessment of the in vivo rat hepatocyte DNA-repair assay

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.     

The mutagenic potential of madder root in dyeing processes in the textile industry

The roots of Rubia tinctorum L. have a long tradition of being used in dyeing processes of textiles from centuries ago until the present time. The colouring principles belong to the class of hydroxyanthraquinones. Concern arose because several of these compounds were recognised as mutagenic in vitro and even carcinogenic in rodents. To assess the possible risk to humans caused by coloured textiles, mutagenicity was investigated with two madder root samples of different origin (Iran and Bhutan) along the entire dyeing process from root extracts to the dyed wool. The Salmonella/microsome test (Ames assay) with the strains TA98, TA100 and TA1537 was used. Significant mutagenic effects could be detected in madder root extracts and also in the final product, the dyed wool. Madder root from Iran showed considerably higher mutagenic responses than samples from Bhutan. Analytical investigations of the extracts by HPLC showed the presence of a spectrum of anthraquinones typical for madder root. Three mutagenic compounds, lucidine, rubiadine and purpuroxanthine, together with the non-mutagenic alizarine could be detected. The mutagenic response of the different samples was positively correlated with the concentration of the mutagenic anthraquinones, and with lucidine in particular. Based on these investigations a risk to dye-house workers and users of textiles dyed with R. tinctorum must be anticipated.     

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.     

Effect of salt stress on the genes expression of the vacuolar H+ -pyrophosphatase and Na+/H+ antiporter in Rubia tinctorum

Molecular Biology Reports - Salinity which covers vast areas of the world is increasing every year. But some plants like madder can grow in these areas. Madder (Rubia tinctorum) is a perennial...     

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.     

Genotoxicity of aloeemodin in vitro and in vivo

The present in vitro and in vivo experiments were undertaken to clarify the genotoxic potential of the hydroxyanthrachinone aloeemodin which can be found in different plant derived products for therapy of constipation. The results demonstrate that aloeemodin is able to induce mutagenic effects in vitro. Positive results were obtained in the chromosomal aberration assay with CHO cells, as well as in the Salmonella reverse mutation assay (frameshift mutations in strains TA 1537, TA 1538 and TA 98). No mutagenic potential of aloeemodin, however, was observed in the gene mutation assay with mammalian cells in vitro (HPRT assay in V79 cells). Each assay was performed in the presence and absence of an extrinsic metabolic activation system (S9-mix). In in vivo studies (micronucleus assay in bone marrow cells of NMRI mice; chromosome aberration assay in bone marrow cells of Wistar rats; mouse spot test [DBA/2J × NMRI]) no indication of a mutagenic activity of aloeemodin was found. Information about a possible reaction of aloeemodin with DNA was derived from an in vivo UDS assay. Hepatocytes of aloeemodin-treated male Wistar rats did not show DNA damage via repair synthesis. All these data suggest that aloeemodin is able to interact with DNA under certain in vitro conditions. However, in vivo the results that were negative did not indicate a genotoxic potential. Therefore, it may be assumed that a genotoxic risk for man might be unlikely.     

Comparative mutagenicity testing of a drug candidate, U-48753E: mechanism of induction of gene mutations in mammalian cells and quantitation of potential hazard

U-48753E is a potential human drug which was subjected to a battery of short-term assays for genetic activity. The compound was negative in the Salmonella (Ames) test, the in vitro UDS assay, the mouse bone-marrow micronucleus test and the Drosophila sex-linked recessive lethal assay. However, it was weakly positive in the CHO/HPRT assay in the presence of metabolic activation (S9). The weak positive response might easily have been labeled artifactual since there was no dose response and the dose level producing positive findings varied from experiment to experiment. In addition, the weak positive response was not confirmed in V79 cells. However, a reproducible dose-related increase in mutants was observed in the AS52/XPRT assay in the presence of S9. Metabolism of this drug proceeds through conversion of aliphatic N-methyl groups to formaldehyde. Addition of formaldehyde dehydrogenase to the S9 resulted in elimination of the mutagenicity of the compound in AS52 cells. Thus, the mutants were probably induced by formaldehyde. From the endogenous levels of formaldehyde in human blood, and the limiting potential therapeutic dose levels, the genotoxic hazard associated with U-48753E is marginal. This assessment of risk and its quantitation depend upon an understanding metabolism and exposure limits imposed by known side effects of the drug. This study can serve as a model for quantitative genetic risk assessment when mutagenicity is due to N-demethylation and formation of formaldehyde in situ.     

Chemical and enzymatic hydrolysis of anthraquinone glycosides from madder roots

For the production of a commercially useful dye extract from madder, the glycoside ruberythric acid has to be hydrolysed to the aglycone alizarin which is the main dye component. An intrinsic problem is the simultaneous hydrolysis of the glycoside lucidin primeveroside to the unwanted mutagenic aglycone lucidin. Madder root was treated with strong acid, strong base or enzymes to convert ruberythric acid into alizarin and the anthraquinone compositions of the suspensions were analysed by HPLC. A cheap and easy method to hydrolyse ruberythric acid in madder root to alizarin without the formation of lucidin turned out to be the stirring of dried madder roots in water at room temperature for 90 min: this gave a suspension containing pseudopurpurin, munjistin, alizarin and nordamnacanthal. Native enzymes are responsible for the hydrolysis, after which lucidin is converted to nordamnacanthal by an endogenous oxidase.     

D and C Red No. 9: genotoxic or non-genotoxic carcinogen?

The azo-compound, D and C Red No. 9 was assayed for genotoxicity in vivo using the rat micronucleus test and the rat ex vivo liver UDS assay. Uniformly negative results were obtained in both assays, even though large oral doses were used (2 g/kg). These results suggest that the tumorigenic effects of this compound in rats are mediated through non-genotoxic rather than a genotoxic mechanism. Further experiments using additional end-points such as 32P-post-labelling would further substantiate this conclusion.     

D and C Red No. 9: Genotoxic or non-genotoxic carcinogen?

The azo-compound, D and C Red No. 9 was assayed for genotoxicity in vivo using the rat micronucleus test and the rat ex vivo liver UDS assay. Uniformly negative results were obtained in both assays, even though large oral doses were used (2 g/kg). These results suggest that the tumorigenic effects of this compound in rats are mediated through a non-genotoxic rather than a genotoxic mechanism. Further experiments using additional end-points such as ³²ap-post-labelling would further substantiate this conclusion     

Inability of chrysotile asbestos fibers to modulate the 2-acetylaminofluorene-induced UDS in primary cultures of rat hepatocytes

There is now growing evidence that asbestos fibers could act in association with genotoxic compounds, either as cocarcinogens or promoters, in the process of carcinogenesis. The hepatocyte/UDS assay system has been taken to advantage to investigate the capacity of fibers to modulate the effects of genotoxic compounds on the cell, as we previously demonstrated the hepatocytes can engage in phagocytosis of chrysotile fibers. Measurement of UDS was performed by a biochemical procedure involving liquid scintillation counting (LSC) of a purified DNA fraction as well as by radioautography. Both LSC and radioautography revealed that chrysotile asbestos fibers UICC B at concentrations up to 100 micrograms/ml do not elicit UDS, whereas 2-acetylaminofluorene (2-AAF) at low concentrations (0.05-0.625 micrograms/ml) significantly induces it in parallel positive controls. In an attempt to test the cocarcinogen hypothesis, cultures of hepatocytes were simultaneously exposed for 20 h to 2-AAF (0.05 and 0.25 micrograms/ml) and asbestos fibers (1 and 10 micrograms/ml) given as simple mixtures. It was found that the 2-AAF-induced UDS activity was the same whether fibers were present or not. This was observed with both UDS evaluation procedures at all concentration combinations selected. An analysis of variance applied to the data collected from several experiments confirmed that there was no significant 2-AAF-fiber interaction. Our data suggest the absence of intrinsic genotoxic properties for chrysotile fibers. They also indicate that the modulation of the cellular response to genotoxic agents by asbestos fibers is not detected under our test conditions and may require longer-term exposures to be expressed.     

Characterization of the in vitro unscheduled DNA synthesis assay in primary lung cells of the rat

The in vitro unscheduled DNA synthesis (UDS) assay has been evaluated in rat primary lung cells with known genotoxicants. The autoradiographic method was employed to detect UDS in both alveolar macrophages and primary pulmonary cells. Data of a time course study revealed that a high radioactive labeling of DNA repair was achieved after a 16-h incubation with [3H]thymidine. Coupled with low serum (1%), hydroxyurea at the concentration of 20 mM inhibited regular DNA synthesis in primary lung cells in a satisfactory manner (81-88% inhibition). With this protocol, a dose-related increase in UDS was induced by N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminoanthracene in both rat alveolar macrophages and primary lung cells. The results suggest that primary rat lung cells in culture possess DNA-repair ability and that the UDS assay may be useful for assessing the pulmonary genotoxic effect of chemicals in this cell system.     

Investigation of the mutagenic activity of tobacco smoke

The genotoxic effect of whole tobacco smoke was studied employing the Salmonella/microsome mutagenicity assay, the micronucleus test in mouse bone marrow and UDS in peripheral human lymphocytes. It was established that tobacco smoke (120-480 cm3 in a 16-1 glass chamber, at 1-10 min exposure time) induced a 3-9-fold increase of spontaneous his+ reversion mutation rate in S. typhimurium TA98, but not in strains TA97a, TA100 and TA102. Addition of S9 mix obtained from the liver of Aroclor 1254-treated rats was necessary to reveal the mutagenic activity of tobacco smoke. Treatment of BDF1 mice placed in a 14-1 glass chamber with tobacco smoke (600 cm3 smoke, 2 exposures of 30 min each, with a 1-min interval between them) caused a 2-fold dose-dependent elevation of the number of micronucleated PCE in bone marrow. No cumulative effect was detected when mice were treated with tobacco smoke during 2-28 consecutive days. The effect observed 24 h after tobacco-smoke exposure was abolished 48 h later. Tobacco smoke (180 or 360 cm3) passed through the culture medium (with or without S9 mix) of human peripheral lymphocytes (the cells were then incubated for 60 min at 37 degrees C) did not increase the spontaneous rate of UDS. Both the Salmonella/microsome mutagenicity assay employing S. typhimurium TA98 strain and the micronucleus test in mouse bone marrow might be useful in studying tobacco smoke-induced mutagenesis.     

Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo

In the first international guideline addressing the unscheduled DNA synthesis (UDS) assay in vivo (OECD guideline no. 486, adopted July 1997) only the genotoxic liver carcinogen N-nitrosodimethylamine (NDMA) is proposed as positive control for the short sampling time. Since NDMA is extremely volatile, alternative positive controls should be identified to facilitate handling and reduce exposure risk during routine testing. At Bayer AG and at RCC-CCR GmbH, the genotoxic but non-volatile dimethylhydrazine (DMH; as dihydrochloride) was used instead as positive control in livers of Wistar rats and to a limited extent of NRMI mice after 2–4 h exposure. As shown by the data presented in this paper DMH induced a positive result in a total of 21 UDS in vivo studies over a period of 7 years. A negative result was never seen for DMH. Due to these results DMH was proven to be a suitable and reliable positive control in the UDS assay in vivo. Consequently, DMH should be considered as positive control for the short sampling time in the next issue of OECD guideline no. 486.     

Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

Anthraquinones in cell suspension cultures of Morinda citrifolia

From cell suspension cultures of Morinda citrifolia five known anthraquinones, rubiadin, lucidin, morindone, lucidin-3-β-primeveroside and morindone-6-β-primeveroside, and seven new anthraquinones were isolated. Six of the seven new quinones were characterized as 2-methyl-3,5,6-trihydroxyanthraquinone, 3-hydroxymorindone, 5,6-dihydroxylucidin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, 3-hydroxymorindone-6-β-primeveroside and 5,6-dihydroxylucidin-3-β-primeveroside, respectively.