Fd Cosmer. Tnrirol. Vol. 18. pp. 31 I to 316 Pergamon Press Ltd IYgO. Printed in Grcal Britain

Information Section

ARTICLES OF GENERAL INTEREST

MORE CHAPTERS IN THE BRACKEN SAGA

Bracken (Pteridium aquilinum) has been implicated in the induction of tumours in cattle (Cited in F.C.T. 1968, 6, 108; ibid 1970, 8, 85). Some workers have attributed its carcinogenicity to shikimic acid, which induces tumours of the gastric mucosa when fed to mice (ibid 1975, 13, 405), but the feeding of shikimic acid to rats has failed to induce tumours (ibid 1978, 16,630). Other carcinogenic principles have been sug- gested, including a more potent compound, pterosin B and its chemical relatives among the sesquiterpenes (ibid 1976, 14, 514), some of which appear capable of inducing tumours in the alimentary tract and the bladder of cattle and experimental animals.

Yoshihira et nl. (Chem. pharm. Bull., Tokyo 1978, 26, 2346) have described the fractionation of organic- solvent extracts of young bracken fronds (P. aquilinum var. latiusculum) and the characterization of their con- stituents. Successive partitioning of a methanolic extract of young bracken fronds with hexane, ben- zene, ethyl acetate and butanol demonstrated more than 20 sesquiterpenes with the 1-indanone nucleus (pterosins) and their glycosides (pterosides), together with benzoic, p-hydroxybenzoic, vanillic and p-cou- maroylquinic acids, vanillin, kaempferol and the steroids /I-sitosterol, stigma.+4-en-3-one and 5- a-stigmastane-3,6-dione. A later paper by the same team (Fukuoka et al. ibid 1978, 26, 2365) describes the chemical and physical characterization of 23 sesqui- terpenes derived from young bracken fronds. Cyto- toxicity testing against HeLa cells, acute SC and oral toxicity tests in rats, mice. and guinea-pigs, and feed- ing tests in rats were carried out on various fractions and on the dried fronds or rhizomes of the plant (Yoshihira et al. lot. cit.). Several fractions showed cytotoxicity for HeLa cells. Further separation of these led to the isolation of very small amounts of a number of indanone derivatives as well as several phenolic compounds and steroids. Some of the inda- none compounds were cytotoxic and all of these had a similar effect on the HeLa cells, which were reduced in size, showed a tendency to become spindle-shaped and were characterized by a clear, sometimes vacuo- lated, cytoplasm, a rounded nucleolus, and a decrease in mitotic figures. There was, however, no indication of the occurrence of any alkylation. A diet containing 30% dried bracken rhizome was fed to guinea-pigs for 5 days, and residues from extractions as well as dried samples of bracken frond and rhizome were fed to rats for 35-205 days again at levels of 30”/, in the diet. In addition, various fractions were mixed with the diet in ratios calculated to provide levels 16-3 times as high as did the dried-bracken diets. Ileal adenoma and papillary adenocarcinoma occurred in rats fed the dried frond or rhizome, but diets containing pter-

osin B and pteroside B failed to induce any significant incidence of tumours, and thus provided no support for their involvement in bracken carcinogenicity.

The approximate amounts of these two compounds administered in the diet were tabulated as 4 and 10 mg/day, respectively, in the report of the study (Yoshihira et al. lot. cit.) but were quoted as 4 and 10 mg/kg/day (for 120 days) in another paper by this group (Fukuoka et al. J. Pharmucobio-Dyn. 1978, 1, 324). The latter paper reports the mutagenicity testing of the bracken-derived compounds and extracts in Salmonella typhimurium, strains TAlOO and TA98. AlI 15 pterosins tested gave negative results in concen- trations up to 1 mg/plate. On the other hand, kaemp feral, one of the flavonoids isolated from bracken, showed marked mutagenicity in both strains of 5. typhimurium when tested in the presence of S-9 mix, but not in its absence. Since it was known that the glucosides of pterosin-sesquiterpenoids are hydro- lysed before being absorbed from the intestine of rats, some of the bracken extracts were pretreated with ‘hesperidinase’, as a model for the glycosidases of the intestinal flora, before being subjected to the Ames test. Using these procedures, the mutagenic activity of the boiling-water extract of bracken was traced to a fraction consisting principally of astragalin (kaemp feral 3+Dglucoside). This was only active, however, after the ‘hesperidinase’ treatment, which hydrolysed astragalin to kaempferol. Another compound, which was more water-soluble than astragalin, was also shown to have some mutagenic activity. The authors of this paper are extending their work on the muta- genic potential of flavonoids, which are common components of naturally-occurring materials. The group is also continuing its studies of the bracken sesquiterpenes, and earlier this year reported the characterization of more than 30 1-indanone deriva- tives from bracken and the demonstration that the sesquiterpene constituents of the rhizomes differ from those of the fronds, particularly in the relative amounts of ghtcosides and the stereochemistry of several compounds at the C-2 and C-3 positons (Kur- oyanagi et al. Chem. pharm. Bull., Tokyo 1979, 27, 592). The rhizomes of bracken have been shown to be more carcinogenic to rats and more toxic to cattle than are the young fronds (Cited in F.C.T 1974, 12, 285), but whether this is a reflection of any differences in their sesquiterpene chemistry has yet to be estab- lished.

The findings of Hirono et al. (Gann 1978, 69, 383). who compared, in small groups of rats, the carcinogen- icity of aqueous extracts of bracken with that of dried fronds, provided further evidence for the solu- bility of a bracken carcinogen in boiling water. One

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312 Articles of general interest--Fd Cosmet. Toxicol. Vol. 18, no. 3

group of animals (group 1) received a diet containing 33% dried bracken powder for 3 months. A second (II) received for 16 months as drinking-water an in- fusion prepared from 60 g dried bracken in 2 litres boiling water. A third (III) received a 72-hr cold-water infusion of bracken (60 g/2 litres) for 16 months, while a fourth (IV) was fed for up to 12 months a basal diet containing, at a level of 33%, a boiling-water extract of bracken that had been concentrated by evapor- ation to a tarry liquid. In a second experiment, rats received a basal diet containing a concentrated triple hot-water extract of bracken for up to 16 months. Surviving animals in all these groups were killed 480-500 days after the start of the treatment. In group I, 13 of the 14 rats, all of which survived for longer than 10 months, developed multiple ileal tumours, comprising adenomas and adenocarcinomas, and one also had a bladder tumour. In group II, six of the ten rats surviving longer than 7 months developed ileal or bladder tumours or both. There were no tumours in group III, but in group IV and in the treated rats in the second experiment, all but one rat had bladder tumours and most also had ileal tumours. These results suggest that the carcinogen present in bracken is water-soluble and extractable by boiling water, but is not appreciably leached out by cold water.

A somewhat different approach to the question of bracken-induced carcinoma has been put forward by

Jarrett et al. (Nature, Lond. 1978, 274, 215). Virus- induced papillomas of the alimentary tract are com- mon in British cattle and occur in animals raised on farms with and without areas of bracken. Cattle with alimentary carcinomas show a much more restricted geographical distribution than those with papillomas, but it has been noted that in the areas of high cancer incidence, the papillomas occur in greater numbers in individual animals and are found at multiple sites. In the bracken-infested upland areas of Scotland and the north of England there is a high incidence of alimen- tary cancer in beef cows, in contrast to the neighbour- ing, relatively bracken-free, lowland areas. In a detailed study of these cattle Jarrett et al. (lot. cit.) found that papillomas and carcinomas occurred at the same sites, and in some cases there was clear evi- dence of the progression of the one to the other. On the basis of this work and of epidemiological studies reported elsewhere, they suggest that the long-term effect of consuming bracken at levels too low to cause the radiomimetic lesions of acute bracken poisoning may promote persistent papillomatosis and progres- sion of the papillomas to malignant tumours, possibly by inhibiting normal immunological reactions to the papilloma virus.

[P. Cooper-BIBRA]

ALCOHOL AND PREGNANCY-PART 1: ANIMAL DATA

Early this year the US Department of the Treasury launched an educational programme to warn the pub- lic of the dangers of drinking during pregnancy. How- ever a clear picture of the various effects of different levels of drinking has not yet emerged from the con- siderable animal and human data on this topic (Cited in F.C.T. 1978, 16, 290). We now report the results of some more recent studies in animals.

The effects of chronic and acute maternal ethanol intake on foetal development were compared by Hen- derson et al. (Alcoholism c/in. expl Res. 1979, 3, 99). Groups of 15 female rats were given a liquid diet con- taining 6% ethanol or its isocalorific equivalent for at least 30 days before mating and for 20 days of ges- tation. The acute exposure groups, of 11-13 dams, were given two doses/day of 5 g/kg ethanol by gastric intubation on days 11-13 or 14-16 of gestation. The acute dose was selected to give roughly the same 24hr intake as in the chronic-exposure group. Blood levels were about 70-200 mg/lOO ml in chronic-expo- sure groups and reached peaks of 100-300 mg/lOO ml in acute-exposure groups. Resorption rates in all the ethanol-exposed rats were significantly higher than for controls and highest in groups exposed chronic- ally or acutely on days 11-13. There was also an increase in the number of dead foetuses among ani- mals exposed chronically. Body weights of all the treated groups were 20-25% less than those of the controls and organ weights were also reduced in the treated animals although there were few significant differences when relative organ weights were com- pared. Tissue protein tended to be higher in the

groups exposed to ethanol. Both acute and chronic exposure had adverse effects on foetal viability and growth.

In a study using mice, Randall & Taylor (Terar- ology 1979,19,305) investigated the effects of different doses of alcohol. They administered the ethanol in a liquid diet at doses providing 17, 25 or 30% of the total calories as ethanol from days 5 to 10 of ges- tation. The dams were killed and the foetuses were examined on day 19. Results are given for groups of 14 to 21 dams. The number of implantation sites was significantly greater in the group given the diet pro- viding 25% of the calories as ethanol than in the con- trols. There were significantly more resorptions and malformations in the two groups given the highest doses but not in the low-dose group. The malfor- mations produced were of a wide variety of types. Blood alcohol levels were negligible in the low-dose group, 36261 pg/lOO ml in the mid-dose group and 124-384 mg/lOO ml for the high-dose group.

Yanai & Ginsburg (Alcoholism clin. expl Res. 1977, 1, 325) investigated the effects of chronic ethanol ex- posure in two species of mice. One group was given 10% ethanol in water sweetened with dextrose and saccharin as its sole liquid supply (the ethanol pro- vided up to 20% of the total calories), while control groups were given sweetened water or tap water ad lib. until 60 days of age. At this age they were mated to animals of the same strain (C57 or DBA) given the same pretreatment. Pregnant females continued to receive ethanol or control drinking water until day 14 after parturition. Their progeny were never directly