Some new methodological aspects of the hen's egg test for micronucleus induction (HET-MN)

In a previous publication we introduced the hen's egg test for micronucleus induction (HET-MN) as an extremely simple, inexpensive and rapid animal free genotoxicity assay which is positioned between pure in vitro and in vivo assays, strictly in line with animal protection regulations and ethical aspects. The HET-MN combines the use of the commonly accepted genetic endpoint "formation of micronuclei" with the well characterized and complex model of the chick embryo. The high metabolic competency provided by this model enables metabolic activation, elimination and excretion of xenobiotics including mutagens and promutagens.In this paper we present some new methodological aspects, which are important for improving the experimental protocol. We used cyclophosphamide (CP) and 7,12-dimethyl-benz[a]anthracene (DMBA) as model substances. Dose-response-relationship for both chemicals and cytotoxic effects for CP are described. In addition to the standard proliferation marker PCE/NCE-ratio we found an increased frequency of primitive erythrocytes (E I) and the appearance of proerythroblasts and erythroblasts as further alerting signals for cytotoxic or erythrosuppressive effects. From the total cell population we could further qualify the group of target cells. We found that all definite erythrocytes (E II), observed at day 11 (d11), are relevant target cells, independent from their stage of maturity (polychromatic as well as normochromatic definite erythrocytes). E I cells do not belong to the group of target cells, however. An additional important methodological aspect is the optimal time frame. We found the time period from d8 of incubation (administration of the test substance) up to d11 (time point of blood sampling) as most favorable. In this way an exposure period of up to 72h is covered. Further results indicate that the air cell route provides a higher response to the test substances than the albumen route. The consideration of the described methodological aspects will contribute to the improvement of the experimental protocol of the HET-MN.     

Applicability and robustness of the hen's egg test for analysis of micronucleus induction (HET-MN): results from an inter-laboratory trial

The hen's egg test for analysis of micronucleus formation (HET-MN) was developed several years ago to provide an alternative test system to the in vivo micronucleus test. In order to assess its applicability and robustness, a study was carried out at the University of Osnabrueck (lab A) and at the laboratories of Henkel AG & Co. KGaA (lab B). Following transfer of the method to lab B, a range of test substances that had been pre-tested at lab A, were tested at Henkel: the genotoxins cyclophosphamide, dimethylbenz(a)anthracene, methotrexate, acrylamide, azorubin, N-nitroso-dimethylamine and the non-genotoxins, orange G and isopropyl myristate. In a second phase, additional compounds with known in vivo properties were examined in both labs: the non-genotoxin, ampicillin, the "irrelevant" positives, isophorone and 2,4-dichlorophenol ("irrelevant" means positive in standard in vitro tests, but negative in vivo), the clastogen p-chloroaniline, and the aneugens carbendazim and vinorelbine. All substances were correctly predicted in both labs with respect to their in vivo genotoxic properties, indicating that the HET-MN may have an improved predictivity compared with current standard in vitro test systems. The results support the promising role of the HET-MN assay as a supplement to existing test batteries.     

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.     

Evidence for the persistence of wild Ginkgo biloba (Ginkgoaceae) populations in the Dalou Mountains, southwestern China

?Premise of the study: The possible persistence of wild Ginkgo biloba populations in China has long been debated but never scientifically confirmed. We test our hypothesis that the extant Ginkgo populations in the Dalou Mountains (SW China) represent fragments of the original natural Ginkgo range and offer a range of pertinent perspectives on the living fossil Ginkgo's history, prehistory, ecology, and place in human culture-all important aspects of this highly valued species. ?Methods: We analyzed the vegetation of the study area, determined the population age structure of Ginkgo, and compared it to existing fossil records. For supporting material, we also examined records of the lack of human presence before the mid-17th century in the area, the local people's beliefs regarding preservation of the forests and existing genetic studies. ?Key results: Current species composition of Ginkgo forests in the Dalou Mountains agrees closely with floristic assemblages from fossil records bearing G. biloba. Current populations are found in habitats similar to those of fossil Ginkgo, which, as today, favored rock crevices. Female to male ratios are 3:2. Estimated ages for many of the trees show that Ginkgo was present in this area prior to human settlement and indigenous peoples of this area are unlikely to have planted Ginkgo because of traditional beliefs. Our results agree with existing genetic studies that show that these mountains were glacial refugia for G. biloba. ?Conclusions: The corroborative evidence confirms the finding that these populations represent fragments of the original natural Ginkgo in the valley and lower mountain slopes of the Dalou Mountains.     

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.     

Isolation of an ATPase from the membrane complex of the hen's egg

The ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of the vitelline-plasma membrane complex markedly increases at the time of ovulation. A fraction, high in ATPase activity, that remains in the supernatant after centrifugation at 100000 × g for 60 min, can be isolated from this membrane complex. The method for solubilizing this fraction utilizes repeated extraction in buffered KCl and differential centrifugation, a procedure analogous to that used to solubilize muscle. ATPase. The solubilized fraction provides three bands on gel electrophoresis and demonstrates a 30-fold increase in activity over the bound fraction. It is stable during storage at 4° for many days. The fraction can be isolated from the unfertilized egg and appears to differ from the cleavage-membrane ATPase of the fertilized egg.     

Deterrents extracted from the leaves of Ginkgo biloba: effects on feeding and contact chemoreceptors

Powdered dried ginkgo tree leaves were subjected to various chemical extractions and successive extracts were monitored for antifeedant activity against larvae of Pieris brassicae. Many fractions moderately inhibited food intake, and some were deterrent at levels as low as 25–50 ppm. Some behaviourally highly active fractions were tested electrophysiologically for neural responses in the maxillary taste sensilla. These extracts appeared to stimulate deterrent receptors. There were distinct differences in responses between Pieris brassicae and P. rapae. Ginkgolide A, B, and C each strongly stimulated deterrent receptors in P. rapae, which corresponds with the observation (Matsumoto & Sei, 1987) that these compounds are effective antifeedants for this species. No toxic effects were observed in insects after feeding for 24 h on diets containing ginkgo extracts.

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Résumé Des feuilles de Gingko séchées et broyées ont été soumises à différentes extractions. On a testé l'activité déterrante (antiapprétant) des extraits successifs sur les larves de Pieris brassicae. Un grand nombre de fractions inhibent modérément la consommation, et certaines sont déterrantes à des concentrations aussi faibles que 25–50 ppm. Par électrophysiologie, on a testé la réponse nerveuse des sensilles gustatives maxillaires à certaines fractions qui montraient une forte action sur le comportement. Il s'avère que ces extraits stimulent les récepteurs déterrants. Les réponses diffèrent pour P. brassicae et P. rapae. Les gingkolides A, B et C stimulent chacun fortement les récepteurs déterrants chez P. rapae, ce qui correspond aux observations (Matsumoto & Sei, 1987) selon lesquelles ces composés sont des antiappétants efficaces pour cette espèce. On n'a pas observé d'effet toxique sur les insectes nourris durant 24 heures par des diètes contenant des extraits de Gingko.

     

银杏外种皮提取物对病原菌Cylindrocladium colhounii的抑制作用

采用菌丝生长抑制法,研究了银杏外种皮3种不同提取物对病原菌(Cylindroc ladium col-hounii)的抑制作用并测定了这些提取物的MIC/MBC。结果表明,在相同实验天数内,抑菌效果(最小抑菌率)由高到低依次为银杏外种皮乙醇提取物(37.4%)、石油醚提取物(23.7%)和新鲜外种皮汁液(18.4%)。当提取物的浓度提升到一定程度时,三者的抑菌率均可达到100%;三种提取物的MIC/MBC分别为:86.25/86.25,172.5/276,293.25/345mg·mL-1。可见银杏外种皮具有明显抑制该病原菌生长的作用,且抑菌成分在乙醇中的溶解度较大。这为进一步研究该抑菌成分乃至生物农药的开发奠定了基础。     

Long-chain betulaprenol-type polyprenols from the leaves of Ginkgo biloba.

A long-chain betulaprenol-type polyprenol mixture was isolated from the leaves of Ginkgo biloba mainly as acetate. The structure was determined by mass spectroscopy, 1H-n.m.r. spectroscopy and 13C-n.m.r. spectroscopy. The mixture contained polyprenols-14-22, predominantly polyprenols-17, -18 and -19, and consisted of the dimethylallyl terminal unit (omega-terminal), two trans-isoprene residues, a sequence of 11-19 cis-isoprene residues and a terminal hydroxylated isoprene unit (alpha-terminal) aligned in that order. The concentration of these polyprenols in leaves increased from 0.04 to 2.0% of dry wt. with maturing of the leaves, though the content of total lipids was constant. The distribution of chain length in these polyprenols showed little variation throughout the whole life of the leaves.     

Coumaroyl flavonol glycosides from the leaves of Ginkgo biloba.

Two coumaroyl flavonol glycosides, isorhamnetin 3-O-alpha-L-[6"'-p-coumaroyl-(beta-D)-glucopyranosyl-(1,2)-rhamnopyranoside], and kaempferol 3-O-alpha-L-[6"'-p-coumaroyl-(beta-D)-glucopyranosyl-(1,2)-rhamnopyranoside]-7-O-beta-D-glucopyranoside, were isolated from the n-BuOH extract of Ginkgo biloba leaves. These two, together with six other flavonol glycosides, kaempferol 3-O-alpha-L-[6"'-p-coumaroyl-(beta-D)-glucopyranosyl-(1,2)-rhamnopyranoside], quercetin 3-O-alpha-L-[6"'-p-coumaroyl-(beta-D)-glucopyranosyl-(1,2)-rhamnopyranoside], quercetin 3-O-alpha-L-[6"'-p-coumaroyl-(beta-D)-glucopyranosyl-(1,2)-rhamnopyranoside]-7-O-beta-D-glucopyranoside, quercetin 3-O-beta-D-glucopyranosyl-(1-2)-alpha-L-rhamnopyranoside, quercetin 3-O-beta-rutinoside, and quercetin 3-O-beta-D-glucopyranoside, showed profound antioxidant activities in DPPH and cytochrome-c reduction assays using the HL-60 cell culture system.     

The hen's egg test for micronucleus induction (HET-MN): Novel analyses with a series of well-characterized substances support the further evaluation of the test system

The hen's egg test for micronucleus induction (HET-MN) combines the use of the commonly accepted genetic endpoint “formation of micronuclei” with the well-characterized and complex model of the incubated hen's egg, which enables metabolic activation, elimination and excretion of xenobiotics—including those that are mutagens or promutagens. This assay procedure is in line with demands for animal protection. In three previous publications we presented the scientific rationale and methodological aspects for this assay as well as results for some well-characterized mutagens and promutagens. Here we present the results of new experiments involving further genotoxic and non-genotoxic model substances. Making a comparison with published data we have to date not found any false negatives or false positives in the experiments presented here and in trials published before, thus demonstrating a promising predictivity of genotoxic effects with this assay.We could confirm relevant genotoxicity for the following substances in the HET-MN: acetylamino-fluorene (2-AAF), acrylamide (ACM), cytarabine (AraC), methotrexate (MTX), cadmium chloride (CD), dipotassium monochromate (DPC), and epirubicine (EPI). Negative results were obtained for azorubin (E122), orange G (OG) and starch (STRC).The micronucleus frequencies (MNE II) of the concurrent negative controls were in agreement with the values of the historical negative control (0.87‰ ± 0.87; average ± s.d.). This value is based upon the scoring of 556,500 erythrocytes from 445 eggs. In historical positive controls the administration of 0.05 mg cyclophosphamide/egg at d8 resulted in an MNE II-frequency of 12.4‰ ± 6.8 (average ± s.d.) at d 10.5. This value is based upon the scoring of 249,250 erythrocytes from 223 eggs.