Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells

Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells

Mutation Research, 158 (1985) 89-95 89 Elsevier MTR 01007 Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells Lynnette R. Fe...

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Mutation Research, 158 (1985) 89-95


Elsevier MTR 01007

Condensed tannins induce micronuclei in cultured V79 Chinese hamster cells Lynnette R. Ferguson


P i e r r e v a n Zijl


W a r r e n D . H o l l o w a y b a n d W i l l i a m T. J o n e s

aCancer Research Laboratory, Universityof Auckland School of Medicine, Private Bag, Auckland (New Zealand) and bApplied Biochemistry Division, Department of Scientific and Industrial Research, Private Bag, PalmerstonNorth (New Zealand)

(Received11 January 1984) (Revision received29 May 1985) (Accepted 31 May 1985)

Summary The tannins, delphinidin and procyanidin were isolated from flowers of white clover (Trifolium repens) and the leaves of Arnot Bristly Locust (Robina fertilis) respectively, and tested for mutagenic properties in a range of systems. There was no evidence for either compound causing significant levels of frameshift or base-pair mutagenesis in bacterial mutagenicity assays, although both were weakly positive in a bacterial DNA-repair test. Both compounds very slightly increased the frequency of petite mutagenesis in Saccharomyces cerevisiae strain D5. In V79 Chinese hamster cells, both were efficient inducers of micronuclei. In each of these test systems, increasing the potential of the compound for metabolic activation by addition of '$9' mix had little effect on toxicity or mutagenicity of either tannin. It would seem that potential chromosome-breaking activity of condensed tannins could represent a carcinogenic hazard for animals grazing on pastures of white clover in flower. It may also have wider implications for human carcinogenesis by some, if not all, condensed tannins.

Tannins are polyphenols that occur mainly in a few families of the dicotyledons such a s the Leguminosae. The function of tannins in the plant is not clear. However, it is possible that tannins inhibit microbiological attack of vulnerable plant tissue (McLeod, 1974). There are two classes of tannins, the hydrolysable, with a central carbohydrate which serves as a polyalcohol to which a number of phenolic carboxylic acids are bound by ester linkages, and the condensed tannins (for example, see Fig. 1) which are derived from condensation of flavonoid precursors. The condensed tannins are the principle tannins in foods and forage and occur in such fruits as bananas, persimmons, apples, pears, peaches and grapes, particu-

lady in the unripe fruit (Bate-Smith, 1959). In forage legumes they occur at high levels in flowers of white clover (Trifolium repens) (Jones et al., 1976). It has been suggested that some condensed tannins may be a contributory factor in the aetiology of oesophageal carcinoma. Subcutaneous injection of a range of condensed tannins into rats or mice produced both local sarcomas and liver tumours (Kirby, 1960). There is a high incidence of small intestine carcinoma in New Zealand sheep (Ross, 1982). To investigate the hypothesis that this may be related to condensed tannin ingestion, two types of condensed tannin, a pure delphinidin and a mixed polymer of procyanidin and delphinidin

0165-1218/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)


o(V° i R-R1-OH,R2-H- Procyanidin R-R1-R2-OH- Delphinidin Fig. l. Structures of the two tannins.

were isolated (Fig. 1). A range of in vitro test systems were used to study the genetic toxicology of these compounds. Materials and methods

Tannins Condensed tannins were isolated from the flowers of white clover, Trifolium repens and from the leaves of Arnot Bristly Locust Robina fertilis by acetone/water extraction and purified by chromatography on Sephadex LH-20. The tannin from white clover was shown to be a pure delphinidin, and from Robina a mixed polymer of 40% delphinidin and 60% procyanidin (Jones et al., 1976). The structures are illustrated in Fig. 1. All tannins were dissolved in dimethyl sulphoxide (DMSO) for the experiments, and fresh solutions made up immediately before use. Microbial strains The histidine auxotroph strains of Salmonella typhimurium, TA1535, TA1537, TA1538, TA98 and TA100, were kindly provided by Prof. B.N. Ames (Berkeley, CA, U.S.A.). For the DNA-repair assays, the Escherichia coli strains WP2 (trp ochre, UV-resistant), WP67, (trp ochre uvrA polA) and CM 871 (trp ochre uvrA recA lexA; Tweats et al., 1981) were obtained from Dr. J.M. Parry, University College of Swansea, Wales. Saccharomyces cereoisiae strain D5 (Zimmerman, 1973) was obtained from Dr. B.S. Cox (Oxford University, U.K) Upon receipt, all strains were grown to stationary phase, DMSO added to a final concentration of 10%, and 1-ml aliquots stored at -80°C. Before each experiment, an aliquot was thawed and grown to early stationary phase, as confirmed by optical density measurements.

Chinese hamster cells The Chinese Hamster cell line V79-5, was originally obtained from Dr. W.R. Inch (London, Ont., Canada) and has been maintained in the Cancer Research Laboratory since 1979. It is maintained in Alpha MEM without ribo- and deoxyribonucleosides containing 10% v / v heat-inactivated foetal calf serum, by trypsinization and subculture to 10 4 cells per T-25 flask twice weekly. Bacterial mutagenicity assays Preliminary experiments followed the standard protocol for the Salmonella/microsome mutagenicity test (Maron and Ames, 1983). However, it became apparent that the tannins were precipitating with top agar, and toxic doses of the compounds could not be reached in this manner. Therefore, following Yahagi et al. (1976), bacteria were pre-incubated with tannin in 2 ml of bacterial minimal medium with or without 100/~1 of $9 mix for 2 h. 1 ml triple strength soft agar containing 0.5 mM histidine/biotin was added, and the mixture poured onto minimal plates containing 20 ml of Vogel-Bonner medium E with 2% glucose (Vogel and Bonner, 1956). Subsequent incubation and scoring followed standard procedures (Maron and Ames, 1983). $9 mix This was prepared as described in Maron and Ames (1983), using a post-mitochondrial supernatant of the livers from male Sprague-Dawley rats, induced using aroclor 1254. Bacterial DNA-repair assays These followed closely the protocol of Tweats et al. (1981). Bacterial cultures were diluted in Davis Mingioli Salts (Boyle and Symonds, 1969) to approximately 2 × 103 bacteria/ml, and dispersed in small test tubes in 0.25-ml aliquots. Three separate doses (5, 12.5 and 25/xl) of tannin solution, DMSO (as negative control) or 1 #g/ml methyl methanesulphonate solution (as positive control) were added. '$9' mix (1/20) was also added in some experiments. The tubes were incubated at 37°C for 2 h, and for each strain, beginning with the highest concentration of extract, three 10-~1 drops were absorbed onto a nutrient agar plate. Plates were incubated overnight at 37°C and counted the following day.


DMSO and 200 ~1 of '$9' mix added to some dishes. (This gave a 2% concentration of liver homogenate in the dishes.) After a further hour incubation, 2 ml of alpha medium containing 30% foetal calf serum was added, and the cells incubated a further 44 h before harvest. Cells were trypsinised and a sample counted. The remainder of the cells were swollen in hypotonic KC1 (0.075 M), fixed in 3 : 1 v / v methanol/acetic acid, dropped 20 cm onto clean glass slides and Giemsa-stained. A s previously described (Wilson et al., 1984), structures were identified as micronuclei only if stained the same colour as the main nucleus, although staining intensity was not necessarily the same. All scored micronuclei were clearly distinguishable from protrusions from the face of the nucleus, with a diameter in the range 2.5-10 ~m. The range of nuclear diameters in these slides was 12.5-27.5 /~m for control cells. It was considered important that the micronuclei differ in diameter from the main nucleus by at least 2.5 /~m, to

Yeast 'petite' mutagenesis assay As described in Ferguson and Baguley (1981), a concentration range of each compounds was incubated with S. cereoisiae Ds, (Zimmermann, 1973) in growing conditions, for 24 h at 30°C. Samples of the culture were diluted 1 in 1 0 4, plated onto yeast complete medium (Cox and Bevan, 1962) and scored for 'petite' mutants using a tetrazolium overlay (Nagai, 1959). Yeast strain D 5 is known to have high levels of endogenous metabolic activation (Callen and Philpott, 1977), and we have previously found that addition of '$9' mix is not necessary for activation of many potential mutagens (Ferguson, 1985). Micronucleus assay V79 cells were dispersed at I × 1 0 4 cells/60-mm petri dishes, in 5 ml of alpha medium containing 10% foetal calf serum. 20 h later, cells were washed with phosphate buffered saline and 4 ml of Alpha medium with no foetal calf serum added. Test substances were added in no more than 10 #l

TABLE 1 M U T A G E N I C ACTIVITY (AS REVERSION TO H I S T I D I N E I N D E P E N D E N C E ) OF T A N N I N S IN Salmonella typhimurium Tannin

Revertants per plate (Average of 3 plates a )

concentration (mg per plate)

TA1535 - $9

D M S O (negative control)



+ $9



532 0.4 0.8 1.2 1.6 2.0 2.4 0.4 0.8 1.2 1.6 2.0

Positive control b Pro-delphinidin (white clover)

TA1537 - $9

TA1538 + $9

- $9

TA98 + $9


- $9

TA100 + $9








> 1 000

15 12 18 15 15 tox

23 15 22 21 14 tox

5 8 8 11 9 tox

9 5 6 7 14 tox

23 21 30 22 22 tox

23 22 28 30 29 30

27 31 42 48 41 tox

14 18 18 15 tox

16 18 18 24 19

7 4 9 7 tox

8 7 9 11 15

24 34 38 25 tox

25 27 28 29 27

26 33 39 35 tox

> 1 000


- $9





> 1000


30 31 37 40 39 40

106 140 114 132 144 133

136 141 152 143 155 150

36 35 40 37 36

132 148 104 100 tox

148 147 139 133 145


a A m e s test data, using preincubation protocol. b Positive controls were: TA1535, sodium azide (2 #g/plate); TA1537, 9-aminoacridine (50 #g/plate); TA1538, 4 nitro-o-phenylenediamine (20 #g/plate); TA98, 4 nitro-o-phenylenediamine (20 ug/plate); TA100, ( - $ 9 ) sodium azide (2 #g/plate); '$9' mix TA100, 4-aminobiphenyl (50 #/plate).

92 distinguish micronucleated cells from binucleate cells with small nuclei. Either 100 cells with micronuclei or 2000 cells in total were scored for each data point. Results

S. typhimurium mutagenesis A dose range of both compounds was tested in all S. typhimurium strains without $9 activation, and in the presence of a single (100 ~1) dose of $9 mix. Results are presented in Table 1. There was no .evidence for significant mutagenic activity in any test, although toxic levels were reached. E. coli DNA-repair test In a DNA-repair test, preferential killing of the repair-deficient strain implies that the chemical being tested is reacting with cellular DNA and may be mutagenic. In the present experiments, CM871 and WP67 are both DNA-repair-deficient. Results for the differential killing test, are presented in Table 2. There was a slight increase in toxicity of both tannins towards CM871, less towards WP67 as compared with WP2. Addition of '$9' mix made little difference to this pattern. Analysis of the slopes of the curves of the repairproficient as compared with the repair-deficient strains (as described in Tweats et al., 1981), showed these differences did not reach statistical significance.

S. cerevisiae "petite" mutagenesis assay Although the yeast could be killed by the compounds, there was little evidence for petite mutagenesis (Table 3) nor did any treated cultures have increased numbers of pink or red colonies which would have implied mitotic recombination or gene conversion. Although the frequency of petites did increase with dose, plate counts did not increase, and it was impossible to distinguish between a slight increase in petite mutagenesis, and a preferential killing for respiratory-proficient as compared with respiratory-deficient cells. V79 micronucleus assay Both tannins decreased the growth of V79 Chinese hamster cells (Fig. 2) and both showed ability to induce micronuclei (Fig. 3). Although delphinidin is slightly less toxic than procyanidin in microbial cells, it appears marginally more toxic towards V79 cells. We have illustrated data for experiments in which the tannins were depressing growth of the V79 cells, but the remaining cells still had high clonogenic ability. At higher dose levels the tannins were cytotoxic, and higher levels of micronuclei (up to 14%) were seen. Values reached were similar to those for ethyl methanesulfonate, and benzo[a]pyrene used as positive controls in these experiments, as well as published data for ionising radiation (Bettaga et al., 1980). The tannin induced micronuclei are considerably smaller than the main nucleus and in the size


Concentration (#g/ml) 20

% survival(average) Strain:WP2 WP67 100 100

CM871 100






400 1000 2000

83.9 64.6 36.2

82.8 63.7 31.6

81.8 65.2 21.7


400 1000 2000

94.9 87.2 57.62

78.7 65.4 30.6

85.8 80.3 26.7






"6 tO

Concentration (/~g/ml)

Survival a.c (%)

'petite b,c mutants



DMSO (negative control)




Ethidium bromide (positive control)




15.625 31.25 62.5 125 250 500 1000

97 99 98 82 46 14 3

2.15 2.05 1.69 2,04 2.51 3.64 4,08

15.625 31.25 62.5 125 250 500 1000

100 100 97 75 55 16 1

1.69 1.46 2.03 1.83 2.72 2.58 3.71




== ¢D

a Survival is expressed as colony numbers from tannin treated yeast as a percentage of the control value. b 'Petite' mutants scored by tetrazolium overlay (Nagai, 1959). c Each point represents an average of counts from 3 plates.


o t














Fig. 3. The proportion of V79 Chinese hamster cells containing micronuclei, following 45-h exposure to procyanidin (©) and pro-delphinidin (zx), with (closed symbols) or without (open symbols) '$9' mix. An average value for the benzo[a]pyrene control is also shown, for experiments with (11) and without (13) '$9' activation.

range (Yamamoto and Kikuchi, 1980) likely to result from chromosome breakage. It was possible in these experiments that the tannins were reacting with components of the foetal calf serum and thereby removing important growth factors. However, the V79 cells will survive and grow without micronucleus induction at very low serum levels, and we consider the present effects to be real. Discussion



O &


a~ O





Concentration (~uglml) Fig. 2. Numbers of V79 Chinese hamster cells grown for 45 h in the presence of procyanidin (C)) and pro-delphinidin (zx) with (closed symbols) and without (open symbols) '$9' mix.

Microbial mutagenicity (Ames and McCann, 1981), DNA-repair effects (Liefer et al., 1981), 'petite' mutagenesis in S. cerevisiae (Wilkie and Evans, 1982) and micronucleus induction (Heddle and Bruce, 1977) have all been found to correlate with carcinogenisis. The present results show that neither tannin causes microbial mutagenesis. Micronucleus induction data, however, differ significantly from control values, are dose-related and reproducible. The data suggest that both tannins tested are potential carcinogens. Although previous carcinogenicity studies on tannins have pointed to a tumour hazard most reports (Kirby, 1960; Kapodia et al., 1976; Pradhan et al., 1974; Pamukau et al., 1980) are based on subcutaneous injection of the material and many tumours are at the injection site. When the tannin fraction of bracken ferns was administered

94 orally, it did not induce tumours, although subcutaneous injection did so (Pamukau et al., 1980). Tannins are rather reactive molecules, and it may be that subcutaneous injection is the only feasible method of ensuring a depot of the compound, placed so that it is unlikely to react with other materials before it reaches the cells. In the alimentary stream there is a chance that a tannin will bind to proteins and carbohydrates or react with cell walls before it reaches any cells. However, in their summary statement on tannins, the I A R C working party (IARC, 1976) concluded that oral administration data were, at least to that date, inadequate on which to base any conclusions. Tannins represent a heterogeneous group of substances, and to date, no animal tests have been conducted on these particular forms. In view of the fact that tannins are often ingested as part of the human diet or in the diet of a grazing animal, tests of a wide range of condensed tannins, orally administered, might be appropriate. It is of importance, that the main genetic activity of both compounds is in inducing micronuclei. There is some evidence (Barret et al., 1983) that chromosome mutations are only one stage of the neoplastic transformation process. It might be useful to test the selected tannins as either initiators or promotors, in two-stage carcinogenesis experiments, rather than conventional carcinogenesis tests. Heddle and Carrano (1977) provided evidence that micronuclei in mouse bone marrow after Xirradiation arise from acentric chromosome fragments, and Y a m a m o t o and Kikuchi (1980) have distinguished these types of micronuclei from those arising by chromosome non-disjunction on the basis of size. We believe that the micronuclei seen in the present experiments are of a size compatable with chromosome-fragment origin. Joshi et al. (1982) looked at the colony-forming ability of cells with micronuclei and showed that, although most cells died, 21% produced a slow growth colony. Such colonies may well be showing one stage in the carcinogenesis process (Heddle and Bruce, 1977; Jenssen and Ramel, 1979). On the basis of the present results, it would seem that the micronucleus assay would provide a useful screen to decide which tannins should undergo more extensive testing.

In the local context, this study suggests that a tannin common in a New Zealand forage legume may have carcinogenic potential. If a range of condensed tannins have this effect, it might imply that tannins are a hazard to humans. It is interesting to note that one index of high colon cancer risk currently being investigated is the production of micronuclei in colon tissue (Heddle et al., 1982). It is possible that some types of tannin could be causing this effect. In view of the high incidence of colon cancer in New Zealand, both in humans and sheep, rigorous testing of selected local tannins, especially in combination with known tumour initiators or promotors, could well be appropriate.

Acknowledgments This work was supported by the Auckland and Hawkes Bay Divisions of the Cancer Society of New Zealand. We are grateful to Dr. P.B. Roberts, D.S.I.R., Lower Hutt, for preparing the '$9' mix used in these experiments. We also wish to thank Dr. B.C. Baguley for helpful discussions, Ms. Rosalee Nash for preparing the diagrams and Mrs. Sally Hill for typing the manuscript.

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