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Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes. I. Mutagenicity in Salmonella typhimurium.

Ptaquiloside, a potent carcinogen of an illudane-type sesquiterpene glycoside isolated from Pteridium aquilinum, and its related compounds, hypolosides having the same nucleus isolated from the Pteridaceae, exhibited marked mutagenicity in the modified Ames test with Salmonella typhimurium TA98 and TA100 using a preincubation at pH 8.5. Illudins M and S, sesquiterpenes of the same illudane type from basidiomycetes, also exhibited mutagenicity. The structural requirements for mutagenicity are discussed.

In Vitro Techniques↗

Widespread absence of outer dynein arms in the spermatozoids of lower plants.

Transverse sections of flagellar axonemes from a variety of lower plant spermatozoids were examined by electron microscopy. Motile sperm of four ferns (Marsilea, Pteridium, Lygodium and Aneimia), a horsetail (Equisetum) and a liverwort (Marchantia) were fixed in the presence of tannic acid to visualise the dynein arms. In all cases the inner dynein arms were clearly resolved but the outer arms were absent. Absence of outer arms therefore appears to be a common feature of the archegoniate plants. The implications of these findings to our understanding of the evolution of the land plants and the role of the dynein arms in flagellar beating is discussed.

Adenosine Triphosphatases↗

An evaluation of acetone extracts from six plants in the Ames mutagenicity test.

Acetone extracts from six plants were evaluated for mutagenic activity with the Salmonella/mammalian-microsome mutagenicity test (Ames) utilizing tester strains TA98 and TA100 and in the presence and absence of induced rat liver microsomes. Extracts from alfalfa (Medicago sativa), lettuce (Lactuca sativa) and thread-leaf groundsel (Senecio longilobus) produced only negative responses. Comfrey (Symphytum officinale) and tansy ragwort (Senecio jacobaea) extracts produced toxic responses that were abolished in the presence of the microsomal bioactivation system. Bracken (Pteridium aquilinum) and tansy ragwort extracts produced positive responses following bioactivation with the liver microsomal system. The results suggest that the Ames mutagenicity test may be of some value in initial evaluations for potential toxic effects of plants consumed by animals and man.

Acetone↗

The mutagenic and cell transforming properties of shikimic acid and some of its bacterial and mammalian metabolites.

Known and proposed metabolites of shikimic acid were synthesised, characterised and tested for genotoxic activity using the Salmonella/mammalian microsome mutagenicity test, the bacterial fluctuation mutagenicity test and the BHK 21 cell transformation test. Under the conditions used, none of these compounds showed any activity. However, shikimic acid itself was active in the BHK 21 cell transformation assay. It therefore seems unlikely that shikimic acid is a carcinogenic initiating agent, but it may act as a carcinogen-promoting agent in the bracken fern (Pteridium aquilinum).

Animals↗

Folk phytotherapeutical plants from Maratea area (Basilicata, Italy).

Field ethnobotanical survey was undertaken for the period of 2002-2003 in the Tyrrhenian part of the Basilicata region of southern Italy. Data of 56 species of plants belonging to 29 families where gathered through interviews; among the species, 47 are used in human therapy, 6 as insect repellents, 15 in veterinary medicine, 1 for its ichthyotoxic properties and 3 for magic therapeutic purposes. The most important findings in ethnomedicine relate to Nasturtium officinale (renal colic, liver diseases), Foeniculum vulgare subsp. piperitum (mouth ulcers), Leopoldia comosa (toothache, headache), Micromeria graeca subsp. graeca (coughs) and Ceterach officinarum (malaria), while in the ethnoveterinary field, we have Pteridium aquilinum (wolf bites) and Spartium junceum (fractures of animal limbs).

Abdominal Pain↗

Antifungal activity of some Tanzanian plants used traditionally for the treatment of fungal infections.

Using the ethnobotanical approach, some Tanzanian plants reported to be used by traditional healers for the treatment of oral candidiasis and fungal infections of the skin were collected and screened for their antifungal activity against Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei and Cryptococcus neoformans. A total of 65 crude methanol extracts belonging to 56 plant species and 38 families were screened using the broth microdilution method, according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI) (formerly, National Committee for Clinical and Laboratory Standards) [National Committee for Clinical Laboratory Standards, 2002. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard-2nd Edition M27-A2, National Committee for Clinical Laboratory Standards, Wayne, PA, USA]. Among the tested plant species, 45% (25 species) showed antifungal activity against one or more of the test fungi. The most susceptible yeasts were Cryptococcus neoformans, followed by Candida krusei, Candida tropicalis, and Candida parapsilosis. The least susceptible were Candida albicans and Candida glabrata. Strong antifungal activity was exhibited by extracts of Clausena anisata Oliv., Sclerocariya birrea Sond, Turraea holstii Gurk, Sterculia africana (Lour) Fiori, Acacia robusta subsp. Usambarensis (Taub) Brenan, Cyphosterma hildebrandti (Gilg), Desc, Elaeodendron buchannanii (Lows), Acacia nilotica (L.) Wild ex Del, Jatropha multifida L., and Pteridium aquilinum (L.) Kuhn.

Antifungal Agents↗

HPV-18 transformed cells fail to arrest in G1 in response to quercetin treatment.

Previous work with primary human keratinocytes demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 with concomitant elevation of the cyclin-dependent kinase inhibitor (cdki) p27Kip1. Expression of the human papillomavirus type 16 (HPV-16) E6 and E7 oncoproteins, under transcriptional control of a heterologous promoter, in transformed keratinocytes failed to abrogate this arrest [Beniston, R., Campo, M.S., 2003. Quercetin elevates p27Kip1 and arrests both primary and HPV-16 E6/E7 transformed human keratinocytes in G1. Oncogene 22, 5504-5514]. Given the link between papillomavirus infection, bracken fern in the diet and cancer of the oesophagus in humans, we wished to investigate further whether cells transformed by the whole genome of HPV-16 or HPV-18, with E6 and E7 under the transcriptional control of their respective homologous promoters, would be similarly arrested in G1 by quercetin. In agreement with earlier work, quercetin arrested HPV-16 transformed cells in G1 with an increase in the cyclin-dependent kinase inhibitor p27Kip1. However, HPV-18 transformed cells did not arrest after quercetin treatment. The failure of HPV-18 transformed cells to arrest in G1 was linked to the up-regulation of the HPV-18 long control region (LCR) by quercetin, maintaining high expression of the viral transforming proteins. Transcriptional up-regulation of the HPV-18 LCR was mediated by a "quercetin responsive element" homologous to the one identified previously in the bovine papillomavirus type 4 (BPV-4) LCR.

Cell Cycle Proteins↗

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability.

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.

Animals↗

Lateglacial and Early Holocene vegetation history of the northern Wetterau and the Amöneburger Basin (Hessen), central-west Germany.

The Lateglacial and Early Holocene vegetation history of the northern Wetterau and Amöneburger Basin, two intra-montane basins in Hessen, central-west Germany, is reconstructed by means of pollen and macrofossil analyses. Regional pollen assemblage zones are defined for the Lateglacial and Early Holocene. After calibration of the radiocarbon dates and establishment of age/depth relationships, the ages of the pollen zone boundaries are calculated. The regional vegetation changes correlate closely with the major fluctuations in the delta18O curve of the Greenland ice cores spanning the same time period. During the early Lateglacial, the open herbaceous vegetation with dwarf shrubs in the northern Wetterau was replaced by woodlands. Initially these woodlands consisted of birch, but after the immigration of pine, mixed forests of pine and birch developed. Soon after its immigration pine became dominant and pine woodlands largely replaced the birch forests. Only on the locally wetter substrates of the river floodplain did Betula stands persist. Gradually the importance of herbaceous communities declined and the pine woodlands lost their open character. During the Lateglacial two regressive phases in the vegetation succession are reflected in the data which are equated with the Older- and Younger Dryas biozones. At the beginning of the Younger Dryas, the forest-limit was lowered and the importance of herbaceous communities increased. Later, pine woodlands thinned and Ericales became part of the vegetation, indicating the development of more acid, nutrient-poor soils. A subdivision of the Younger Dryas biozone into a wetter, colder first part and a drier, warmer second part is suggested. At the beginning of the Early Holocene, pine woodlands became more closed and soils more stabilised. The transition between the Younger Dryas and Preboreal biozones is indicated by a lithological change to organic (-rich) deposits. Betula stands persisted on the locally wetter substrates of the fluvial plains. At the end of the early Preboreal, there was an extension of Betula stands on the river floodplains. This is interpreted as a phase in which climate was temporarily wetter, perhaps a reflection of a short-term climatic oscillation, the so-called Preboreal oscillation. During the Preboreal biozone, ferns became more important in the local vegetation of the mires, and later during the Boreal they formed the undergrowth of swamp forests. During the Boreal biozone, a major expansion of hazel took place in both areas. On the hills and slopes of the northern Wetterau and the Amöneburger Basin, open pine forests and hazel scrub developed with grasses and Pteridium in the field layer. In the river valleys, pine forests were replaced by deciduous mixed forests with oak, elm and later also with lime.

Journal Article↗

Occurrence of the carcinogenic Bracken constituent ptaquiloside in fronds, topsoils and organic soil layers in Denmark.

Bracken (Pteridium aquilinum (L.) Kuhn) is a common fern found on all continents except Antarctica. It is under suspicion of causing cancer among people who utilizes it as food. The main carcinogenic compound is thought to be the water-soluble compound ptaquiloside. Ptaquiloside-uptake may occur not only through food, but also via drinking water as ptaquiloside might leach from plant material. The purpose of the study was to identify environmental parameters that correlate with the ptaquiloside-content in fronds, and to quantify the amount of ptaquiloside in the soil environment. The ptaquiloside-content in fronds, Oi/Oe-, and Oa/A-horizons was quantified at end of the growth season at 20 sites in Denmark. The fronds had ptaquiloside-contents between 108 and 3795 microgg(-1). The Oi/Oe-horizons had contents between 0.09 and 7.70 microgg(-1), while Oa/A-horizons had contents between 0.01 and 0.09 microgg(-1). The ptaquiloside-content in the standing biomass, which could be transferred to the soil by the end of the growing season, ranged between 10 and 260 mgm(-2), with nine sites having ptaquiloside loads over 100 mgm(-2). The carbon-content in the O-horizon, the precipitation, the amount of Bracken-litter, the turnover rate and the size of Bracken-stands determined the ptaquiloside-content in the soil materials while the content in fronds was found to be a function of the frond-height and the light-exposure in the ecosystem.

Biomass↗

Bracken carcinogens in the human diet.

The ubiquitous bracken fern (genus Pteridium) is the only higher plant known to cause cancer naturally in animals. In addition to the well-recognized syndromes of thiamine deficiency, acute haemorrhage associated with myeloid aplasia and blindness due to retinal degeneration, it causes neoplasia of the urinary bladder and in some circumstances, of the upper gut. In addition, it has been shown to cause neoplasia in a wide range of tissues in many experimental species. The major carcinogen (and the cause of the retinal degeneration and the myeloid aplasia) has been shown to be ptaquiloside (PT), a norsesquiterpene glucoside that can be present in bracken in extraordinary concentrations, up to 13 000 ppm. The highest concentrations were found in the crosiers and young unfolding fronds. The mutagenicity, clastogenicity, teratogenicity and carcinogenicity have been convincingly demonstrated. Under alkaline conditions the loss of the glucose gives rise to the formation of a dienone intermediate which possesses a highly reactive cyclopropyl ring capable of reacting with cellular macromolecules. PT has been shown to alkylate DNA at N3 of adenines in the minor groove, preferentially in 5'-TAG and 3'-A in 5'-AA-3' sequences. It also alkylates N7 guanines in the major groove occurring in 5'-TG sequences. It is believed that these alkylations lead to mismatch repair and subsequent mutations in particular proto-oncogenes. Recently a rat model of carcinogenesis has been established using intravenously (iv) administered PT. Some epidemiological evidence has indicated higher risk of cancer in people who consume bracken crosiers, people who consume milk of cows feeding on bracken and those who live in bracken-infested areas. PT has been found in the milk of cows fed on bracken fern experimentally and the milk of bracken-fed cows has been shown to cause cancer in rats. PT carcinogenesis presents an excellent model of environmental and experimental carcinogenesis.

Animals↗

The spatial distribution of larvae of Culicoides impunctatus biting midges.

The spatial distribution of Culicoides impunctatus Goetghebuer (Diptera: Ceratopogonidae) larvae was sampled at a site in western Scotland, and geostatistical analyses were used to quantify spatial dependencies. Nested sampling and analysis indicated that most of the spatial variance occurred within distances of 160-640 cm, levelling off at distances >640 cm. Semivariograms for transformed data from three 100 m x 100 m grids showed similar, isotropic patterns for larval counts, soil organic content, soil water content and the distribution of Juncus spp. rushes, with the variance increasing with separation distance. High levels of significance were associated with power models used to describe the semivariograms, which was indicative of the absence of a plateau (or 'sill') in the respective data. Correlation analysis of transformed data revealed significantly positive relationships between larval counts and soil pH, soil percentage organic content, soil percentage water content and also the distribution of Sphagnum spp., Juncus spp. and Myrica gale. There were also significantly negative relationships between larval counts and the distribution of Pteridium aquilinum and all mosses other than Sphagnum spp. The results suggest a far more structured and predictable pattern of C. impunctatus larval sites than previous studies and are discussed in relation to their application in localized Culicoides control and to studies of the mechanisms determining the spatial distribution of C. impunctatus larvae.

Animals↗

Ultrasonographic evaluation of urinary bladder in normal, fern fed and enzootic bovine haematuria-affected cattle.

A total of 19 adult hill cattle of both sexes were subjected to trans-rectal ultrasound scanning of urinary bladder to evaluate bladder wall thickness and the presence of space-occupying lesions. The animals were divided into four groups. Eight apparently healthy hill cattle maintained under standard ration served as control (group I) and the remaining II animals were divided into three groups (II, III and IV). Group II animals (n = 8) were fed with different type of ferns which were further divided into subgroups II-P, -D and -B and fed with Polystichum squarrosom (n = 2). Dryopteris juxtaposita (n = 2) and Pteridium aquilinum (n = 4) ferns, respectively. The one animal in group III was a natural case of enzootic bovine haematuria (EBH) and the two animals in group IV were natural cases of microscopic EBH fed with Polystichum squarrosum fern. In group I animals, the average bladder wall thickness was 1.45 mm. The delineation of the bladder wall was uniformly smooth and the echo pattern of the bladder was homogeneously black, which was suggestive of clear urine content. In group II (P, D and B) the average bladder wall thickness of the six animals was 1.87 mm and the sonographic features were within normal limit when compared with controls. In two of the animals of group II-B, the bladder wall was apparently thick (4.36 mm) and there was no intraluminal mass except at one or two focal elevated points. Animals of groups III and IV showed the average bladder wall thickness of 4.86 mm and were characterized by the presence of irregular sessile masses extending into the bladder lumen. The homogeneous anechoic area was reduced centrally due to the presence of a hypoechoic soft tissue mass all around the bladder wall. Post-sonographic urinalysis, biopsy and necropsy of selected cases further confirmed the sonographic findings.

Animal Feed↗

Smooth muscle relaxant activity of pterosin Z and related compounds.

The natural products pterosin Z, acetylpterosin Z, originally from the fern Pteridium aquilinum, and their isopterosin analogues have been synthesized and their smooth muscle relaxation activity has been measured. All of the test compounds show activity with pterosin Z being the most active (EC50 = 1.3 +/- 0.1 x 10(-6) M), exhibiting 100 times the activity reported for the related fern metabolites onitin (EC50 = 1 x 10(-4) M), onotisin (EC50 = 2 x 10(-3) M) and otninoside (EC50 = 7 x 10(-4) M). The smooth muscle relaxant activity of pterosin Z is approximately equipotent with that of the related fungal pterosin (EC50 of 2.9 +/- 1.6 x 10(-6) M). These results suggest that smooth muscle relaxant activity is reduced when a phenol group is present in the pterosin nucleus and when one of the dimethyl groups is derivatized.

Animals↗

Biologic effects of plant toxins and aflatoxins in rats.

After weaning, groups of rats were fed a semi-purified diet containing fiddlehead greens (the unfuried frond of the ostrich fern Matteuccia struthiopteris), mature bracken fern (Pteridium aquilinum), or cycad meal (Cycas circinalis), or this diet plus aflatoxin B1. The groups fed 1-10% fiddlehead greens for 70-73 weeks developed no tumors, whereas those fed mature bracken fern for 67 weeks developed small-bowel adenocarcinomas (3/17). Those fed 3% cycad meal for 64 weeks developed tumors of the liver (4/18), kidney (2/18), small bowel (1/18), and large bowel (6/18). Those given aflatoxin B1 and observed for 73 weeks developed liver tumors (11/15). Thus this study indicates that fiddlehead greens from M. struthiopteris are not toxic or carcinogenic.

Adenocarcinoma↗

Interaction between Antheridogens and Fatty Acids in Fern Spore Germination.

Certain fatty acids in the C(5) to C(18) range, at concentrations as low as 10(-5) M, were found to inhibit the germinations of spores of the sensitive fern, Onoclea sensibilis L. The addition of gametophytic culture flltrates of the bracken fern, Pteridium aquilinum (L.) Kuhn, containing antheridogen A, was found to overcome this inhibition and allow the spores to germinate and the gametophytes to develop in a normal fashion. Some fatty acids were found to increase the antheridium-inducing potency of antheridogen A as much as 10-fold. An effect similar to this may promote the diecious reproduction of ferns.

Journal Article↗

Enhancement of protein synthesis in isolated chloroplasts by irradiation of fern gametophytes with blue light.

Irradiation of the gametophytes of Pteridium aquilinum with blue light led to a nearly 5-fold increase in the amino acid-incorporating activity of isolated chloroplasts. The blue light effect was not due to increased synthesis of ATP or other energy donors by the chloroplasts but was probably related to an increased production of chlorophyll and photosynthetic enzymes.

Journal Article↗

Bovine enzootic haematuria in Queensland.

Bovine enzootic haematuria was diagnosed in Queensland in coastal areas in association with bracken fern (Pteridium esculentum) on 3 properties or with mulga fern or rock fern (Cheilanthes sieberi) on 4 properties, and in inland areas in association with C. sieberi on 3 properties. In the absence of bracken fern, long-term ingestion of C. sieberi is suggested as a cause of bovine enzootic haematuria. Haemangiomas, haemangiosarcomas, transitional cell carcinomas, papillomas, fibromas and an adenoma were detected in the urinary bladders of 19 affected cattle and were accompanied by chronic cystitis.

Animals↗