Cytotoxic and clastogenic effects of benzyl isothiocyanate towards cultured mammalian cells

FdChem. Toxic. Vol. 33, No. I, pp. 31-37, 1995

Pergamon

0278-6915(94)00109-X

Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0278-6915/95 $9.50 + 0.00

Cytotoxic and Clastogenic Effects of Benzyl Isothiocyanate Towards Cultured Mammalian Cells S. R. R. M U S K * ,

S. B. A S T L E Y , S. M. E D W A R D S t , P. S T E P H E N S O N : ~ , R. B. H U B E R T a n d I. T. J O H N S O N *Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich, NR4 7UA and %Radiotherapy Research Unit, Institute of Cancer Research, Downs Road, Belmont, Sutton, Surrey, UK (Accepted 27 July 1994) Abstract--Benzyl isothiocyanate (BITC), a compound found in cruciferous vegetables present in the human diet, has previously been shown to induce chromosome aberrations in an Indian muntjac cell line. The results of this study show that it also induces both chromosome aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells in the absence of an exogenous metabolic activation system and induces DNA strand breaks as measured by the single-cell gel electrophoresis assay. However, whereas it increased the number of aberrations four-fold, it was not able to raise SCE levels by more than 50% and there was a levelling-off in the dose-response curve. Whereas the survival curve of CHO cells exposed to BITC was linear in shape, that of the human colorectal adenocarcinoma cell line HT29 was found to fit the exponential model (with an ct equivalent of 0.28 and a fl equivalent of 2.80, where the concentration of BITC is measured in p g/ml). This pattern of clastogenic and cytotoxic activities is reminiscent o1' that generated by ionizing radiation and certain radiomimetic chemotherapeutic agents.

et al., 1978; Kune et al., 1987) might be due to a high intake of these organic isothiocyanates. One such isothiocyanate, benzyl isothiocyanate (BITC) has also been used as an antibiotic for the treatment of infections of the respiratory and urinary tracts (Mennicke et al., 1988). However, we have previously shown thai BITC and the closely related compound phenethyl isothiocyanate (PEITC), are both capable of inducing chromosome aberrations in an SV40-transformed Indian muntjac cell line (SVM), even in the absence of any metabolic activation, at doses below 1 pg/ml (Musk and Johnson, 1992 and 1993a). BITC has also been reported to be mutagenic to Salmonella typhimurium (Yamaguchi, 1980). These results raise the suspicion that isothiocyanates might be carcinogenic in their own right. The related compound allyl isothiocyanate has been shown to induce tumours in the bladder of the male F344 rat (Dunnick et al., 1982) but this compound showed no genotoxic activity in our studies (Musk and Johnson, 1992 and 1993a,b; Musk et al., 1993). Those workers who have described the antitumorigenic effects of isothiocyanates state that these compounds do not themselves induce a significant number of tumours in laboratory animals (Morse et al., 1989b; Stoner et al., 1991) but careful reading of one paper does reveal an increase in tumours of male F344 rats fed on a diet containing 3 / t m o l

INTRODUCTION

Cruciferous vegetables contain relatively large amounts of isothiocyanates, either as such or as the precursor glucosinolates from which isothiocyanates are liberated by action of the enzyme myrosinase (Fenwick and Heaney, 1983; Van Etten et al., 1980; Van Etten and Tookey, 1979). This enzyme is spatially separated from the glucosinolates in the intact vegetables but, on disruption of the tissue, is able to break down the: parent compounds to liberate the isothiocyanates. Certain of these isothiocyanates have been shown to protect laboratory animals from the tumorigenic effects of a variety of model carcinogens when taken as a dietary supplement (Morse et al., 1989a,b,c; Stoner et al., 1991; Wattenberg, 1981 and 1987). It has been proposed that the protective effect of cruciferous vegetables ~.gainst cancer identified by epidemiological evidence (Benito et al., 1990; Graham ~Present address: John Innes Centre, Institute of Plant Science Research, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Abbreviations: BITC = benzyl isothiocyanate; CHO = Chinese hamster ovary; D O= slope of survival curve; Dq = threshold concentration below which no cells were killed; EDTA = ethylendiaminetetraacetic acid; PEITC = phenethyl isothiocyanate; SCEs = sister chromatid exchanges; SVM = SV40-transformed Indian muntjac. FCT 33/1--C

31

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S.R.R.

PEITC/g (Morse et al., 1989c). Although the induction of tumours at any one site did not appear to be significant, the total number of tumorous organs (excluding testes which have a high background level of tumours) in 20 rats fed PEITC was 17 as opposed to six in the rats not exposed to PEITC. In view of these findings we decided to conduct some further investigations to determine whether BITC was also able to induce chromosome aberrations and sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells, which have been used as the test system to construct a large part of the database on the clastogenicity of substances in vitro (Ishidate et al., 1988). We also assayed BITC for the ability to induce DNA breaks in CHO cells using the single-cell gel electrophoresis ('comet') assay. In view of the results obtained in the cytogenetic assays which, as discussed below, suggested a radiomimetic mode of action, we also examined the cytotoxic effects of BITC on the human colorectal adrenocarcinoma line HT29. As a human tumour cell line this exhibits a bending survival curve when treated with ionizing radiation (Steel and Peacock, 1989) and we determined whether BITC induced a similar pattern of cell killing. Given the high chromosome number and inherent chromosomal instability of HT29 (Rutzky and Moyer, 1990) this cell line was not used to assay BITC for the capacity to induce chromosomal aberrations or SCEs. MATERIALS AND M E T H O D S

Cell culture CHO and HT29 cells were obtained as gifts from Dr R. T. Johnson (Cambridge) and Dr I. Gibson (University of East Anglia), respectively. CHO were grown in Eagle's minimum essential medium (ICN Flow, High Wycombe, UK) supplemented with 2 mM glutamine, 50 U penicillin/ml, 50,ug streptomycin/ml and 5% foetal calf serum (all from Imperial, Andover, UK). Cultures were incubated in 5% CO, at 37°C in a humidified incubator and split 1: 8 every 4 days, conditions under which the cell cycle is about 12 hr. Culture conditions were similar for HT29 except that they received a 10% serum supplement and were split 1:3 every 8 days as their cell cycle was about 24 hr (data not shown). Test compound BITC (purity 98%) was obtained from Aldrich (Gillingham, UK). Ethanolic solutions were prepared at a concentration of 1 mg/ml immediately before use, with further dilutions being made in medium before addition to the cultures. The final concentration of ethanol in the medium was never greater than 1%; this concentration had no effect on the survival or mitotic index of either cell line and induced no chromosome aberrations in CHO (data not shown). All exposures to BITC were for I hr. Little or no data exist on the stability of BITC in

Musk et al.

aqueous solution, but it is a highly reactive compound (Hasegawa et al., 1993) and we found that longer exposures did not markedly increase the number of aberrations of SCEs induced (data not shown). Thus, it would appear that the initial interaction of BITC and cellular components is essentially complete within I hr. Determination q[" clonal survival Cells were harvested from log-phase cultures and plated out in tissue culture flasks at the required low densities 4 h r before exposure to BITC. Following addition of BITC each flask was sealed airtight to prevent the escape of volatilized compound. After 1 hr of incubation, the cells were given fresh medium and incubated for 10-12 days until colonies were visible to the naked eye. At this point flasks were fixed, stained with crystal violet and scored for colonies over 50 cells (over 95% of colonies consisted of more than 200 cells at this time: data not shown). Each experiment was carried out in triplicate. Untreated CHO and HT29 cells both show a cloning efficiency of between 60 and 80% (data not shown). Induction o f chromosome aberrations CHO cells were plated out at 105 per culture flask and left for 24 hr in order to achieve logarithmic growth, following which they were exposed to BITC for 1 hr. The medium was then removed and replaced with a fresh supply. 15 hr later colcemid was added to a final concentration of 0.1/~g/ml. After incubation in colcemid for l hr, chromosome preparations were made, stained with crystal violet and scored 'blind' for the induction of all forms of aberration, both of chromatid and chromosome types, by a trained and experienced observer. 100 cells per treatment were scored; only well-spread and clearly-stained cells with at least 20 centromeres were considered acceptable for analysis. The effect of BITC on the mitotic indices was also investigated. Induction o f sister chromatid exchanges In a procedure similar to that described above, CHO cells were plated out 24 hr prior to treatment, exposed to BITC for 1 hr and then cultured in the dark for 32hr in the presence of 10-sM BrdU. Chromosome preparations were made and SCEs visualized using the direct staining technique of Alves & Jonasson (1978). SCE frequencies were scored in 25 well-spread clearly-stained cells per sample. Single-cell gel electrophoresis ('comet') assay DNA strand breaks were measured using the singlecell gel electrophoresis assay essentially as described by Green et al. (1992) and by McKelvey-Martin et al. (1993). Log-phase cultures of CHO were exposed to BITC for 1 hr following which they were harvested, spun down and resuspended in liquefied I% low melting point agarose (buffered with 40 mM Trizma

Clastogenicity of benzyl isothiocyanate base, 40 mM acetic acid a n d 1 mM ethylenediaminetetraacetic acid (EDTA), p H 8.0). They were then layered onto frosted, cleaned slides and the agarose was allowed to set over ice, following which they were incubated for 1 hr in lysis buffer (2.5 M NaCI, 100 mM E D T A , 10mM Tris base, p H 10.0 plus 1% sodium sarcosinate, 1% Triton X-100, I % dimethyl sulfoxide) a n d then for 40 rain in alkaline unwinding buffer (0.5 M N a O H , l mM EDTA). The slides were then electrophoresed for 1 0 m i n at 1 0 V (3 A) at room temperature, neutralized a n d stained in 0.4 M Tris base plus ethidium hromide. The comets were then examined under a fluorescence microscope and scored on an arbitrary scale of 0 - 5 for the p r o p o r t i o n o f fluorescence in the tail where 0 represents no tail and 5 represents all tail.

Statistical method

100%

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0.3

0.6

0.9

1.2

1.5

10%

1%

The statistical significance of the relationship between induction of c h r o m o s o m e aberrations, mitotic index and the level o f BITC exposure was determined by one-way analysis o f variance followed by t-tests for linear trend across ordered groups (Altman, 1991). The linear quadral:ic model was fitted to the cell survival data for HT29 by least squares after linear transformations.

Fig. 1. Survival curves of CHO cells exposed to BITC for 1 hr. Points represent mean_+ SE values from four experiments. The parameters of the curve are as follows: Dq = 0.17 + 0.01 pg/ml; D O= 0.36 + 0.04 pg/ml; D37 = 0.53 + 0.04 ,ug/ml.

(albeit for a 24-hr exposure: M u s k and J o h n s o n , 1993a). RESULTS

Cell killing of CHO Figure 1 shows the survival curve o f C H O exposed to BITC for 1 hr. It could be expressed in terms of an initial shoulder (the 'I)q ': the threshold concentration below which no cell killing was seen) followed by a linear decrease in survival (defined as the slope or 'Do': the c o n c e n t r a t i o n required to lower survival from any point on the slope to 37% of that value). The Dq a n d D O values of 0.17 ,ug/ml a n d 0.36 pg/ml, respectively, compare with those of 0.11 # g / m l and 0.23 p g / m l that we have previously reported for SVM

Chromosome aberrations The effects of BITC on the levels of c h r o m o s o m e aberrations and the mitotic indices of C H O cultures are presented in Table 1. BITC was found to induce a b e r r a t i o n s in a dose-dependent fashion a n d was active down to a concentration of 0.3/~g/ml, a dose that was only marginally toxic in the colony-forming assay. A l t h o u g h the majority of the a b e r r a t i o n s were o f the c h r o m a t i d type, approximately 2 5 % were c h r o m o s o m e type aberrations; details of the specific types of lesion generated are given in the footnote to Table 1. Unusually, the mitotic index o f cultures

Table 1 Induction of chromosome aberrations in CHO cells following exposure to BITC for I hr, and mitotic indices of the cultures Aberrations* (no. per 100 cells) Percentage of BITC c o n c n Chromatid Chromosome cells with Mitotic index (k~g/ml) type type aberrations (%) 0.0 1.3 0.7 2.0 6.1 0.3 4.7 1.3 5.3 5.1 0.6 5.7 1.3 6.0 8.7 0.9 7.7 3.0 9.7 6.6 1.2 8.3 3.3 10.7 2.0 PooledSDvalues 1.77 0.93 2.30 Signifcance P<0.001 P<0.01 n.s. *The data include all types of aberration including gaps and breaks. Pooling all the concentrations to v,hich the cells were exposed, 36/79 of the chromatid type aberrations were exchange events while, of the 27 chromosome type aberrations seen. 11 were dicentrics, 12 were deletions and four were rings. Values are means from three experiments. Standard deviations (SD) were obtained from pooled datasets and the significancevalues derived from /-tests for linear trend across ordered groups.

34

S . R . R . Musk et al. [BITC] (gg/ml)

0.6 100%

0.3 \ ,

0.6 ,

0.9 ,

1.2 ,

1.5 ,

0.4

0.3

~(

"\\\

0.2 10%

'~"Xx x

0.1.

i 00

i 0.3

i 0.6

i 0.9

[BITe] (gg/ml)

Q "d

Fig. 2. Induction of SCEs in CHO following exposure to BITC for 1 hr. Values represent mean _+ SE values from three experiments.

exposed to BITC was observed to drop only at a concentration (1.2/~g/ml) that was highly cytotoxic in the clonal survival assay and that led to the induction of as many as 10 chromosome aberrations per 100 cells. This may be due to the ability of a number of isothiocyanates, inducing BITC, to arrest cells in metaphase (Hasegawa et al., 1993, and our own unpublished observations)--an effect that would tend to counteract any reduction in mitotic index induced by the directly genotoxic effects of isothiocyanates. Clearly, the use of reduced mitotic indices to assess cytotoxicity will be inappropriate in the case of agents that arrest cells in metaphase. Although accepting that the difference in recovery times between a clonal survival assay and an aberration study makes it difficult to make direct comparisons between the two endpoints, we consider the former assay to be the most useful measure of survival available when assaying metaphase arrestants for clastogenic activity. Sister chromatid exchanges

The effect of BITC on the levels of SCEs in C H O cells is shown in Fig. 2. As with the induction of chromosome aberrations, induction of SCEs was seen at a concentration of 0.3 Itg/ml. This dose raised the SCE level by about 40% of the control value. However, little further significant induction was seen up to a concentration of 0.9 itg/ml, which was the maximum compatible with assay. That the cells were

\X\x 1%

0.1%

Fig. 3. Survival curves of HT29 cells exposed to BITC for 1 hr. Points represent mean + SE values from four experiments. The parameters of the curve are as follows: c~ =0.28 (#g/mt)-t; /~ =2.80 (~g/ml)--'. The dashed line recapitulates the survival data for CHO. competent to exhibit higher levels of SCEs was shown by simultaneously treating cultures with benzpyrene and rat liver S-9 activation fraction, which led to a three- or four-fold increase in exchanges (data not shown). D N A strand breaks

Table 2 shows the effect of BITC on the levels of D N A strand breaks in C H O cells. BITC was found to induce D N A strand breaks in a dose-dependent fashion and was active at the lowest concentration tested, 1/ag/ml. At the higher concentrations BITC was such an effective inducer of D N A strand breaks that all the nuclei were highly damaged and, in many cases, holes were observed in the 'comet heads' (not shown). This was presumably due to BITC-induced breaks bringing about the loss of large areas of D N A from the in situ preparations.

Table 2. Induction of D N A strand breaks in C H O following exposure to BITC for I hr as measured by the single-cell gel electrophoresis ('comet') assay BITC concn (,ug/ml) 0.0 1.0 5.0 25.0

Percentage o f cells in each scoring category* ............................................................ 0 I 2 3 4 19.0 12.0 2.0 0.0

61.0 54.5 14.0 0.5

17.0 23.0 22.0 7.5

1.5 7.5 35.0 12.0

0.5 2.5 25.0 50.0

5 1.0 0.5 2.0 30.0

*A cell in categotry 0 is undamaged; category 5 indicates that all the D N A is in the tail o f the comet. Values are means from two experiments.

Clastogenicity of benzyl isothiocyanate Cell killing o f H T 2 9

Figure 3 shows the survival curve of HT29 cells exposed to BITC for 1 hr. Unlike that for CHO, this, being continuously curving, could not be satisfactorily expressed in terms of a shoulder and linear slope. It could, however, be modelled in terms of two components, ct and fl, related to the concentration of BITC and to the square of the concentration respectively, such that percentage survival = 100 x e ~c l~c2where c is the concentration of BITC in pg/ml. DISCUSSION

The data presented here confirm our previous results (Musk and Johnson, 1992 and 1993a) in showing the clastog:enic nature of BITC towards a mammalian cell line in vitro in the absence of any exogenous metabolic activation system. We have now extended our observations to include the induction of SCEs and find that, in contrast to its ability to raise the level of aberrations about four-fold above baseline levels, BITC was not able to induce large numbers of SCEs in that in no experiment did it so much as double that level of SCEs observed in control cells. This contrasts markedly with the effects of many chemical agents, such as mitomycin C, quinacrine mustard, N - m e t h y l - N ' - n i t r o - N - n i t r o s o guanidine and ethyl methane sulfonate, which are all able to increase levels of SCEs in CHO by eight- to 10-fold at concentrations which induce few or no chromosome aberrations (Perry and Evans, 1975). This pattern of activity, wherein the ratio of aberrations to SCEs is very high, is reminiscent of that generated by ionizing radiation and radiomimetic agents such as bleomycin (Garner and Martin, 1979; Perry and Evans, 1975; Vig, 1979). One of us has tested both bleomycin and BITC for the ability to induce SCEs in SVM and it is of interest that both agents show a saturation effect, whereby no more than four or five SCEs can be induced per cell, even at the maximum cortcentrations of compounds compatible with assay (Musk, 1987; Musk and Johnson, 1993b). This plateau effect contrasts with the response of this cell line to a wide variety of other DNA-damaging agents such as UV light, monofunctional alkylating agents and mitomycin C, which is similar to that of CHO in intensity (Musk, 1987). The results obtained with the HT29 cell line indicate that BITC does not only mimic ionizing radiation in its pal:tern of inducing chromosome damage. It is a characteristic of human tumour cells when grown in vitro that they exhibit a bending survival curve, when treated with ionizing radiation, which can be defined in terms of two components, one proportional to the dose and the other to the square of the dose (lhe linear quadratic model: Steel and Peacock, 1989). Here we have shown that the survival curve of HT29 cells exposed to BITC is also

35

bending and can be similarly described in terms of ~t and/~ values. This contrasts with the survival curves for CHO and SVM exposed to both BITC and ionizing radiation, which are all exponential with an initial shoulder (Musk, 1987; Musk and Johnson, 1993a; Musk et al. 1993; Thompson et al., 1982). Unfortunately, the continuing obscurity of the molecular basis for the shape of the survival curves of tumour cells (Fertil et al., 1994) means that a mechanistic discussion of the different shapes would be highly speculative and, at present, we feel unable to do more than draw attention to the differences. Thus we have an overall picture in which BITC exhibits radiomimetric behaviour both in terms of its clastogenic and its cytotoxic effects towards mammalian cells in vitro. Similar results have been obtained with PEITC (unpublished data) but it is of interest that allyl isothiocyanate, which induces neither chromosome aberrations nor SCEs in CHO or SVM (Musk and Johnson, 1992 and 1993a,b; Musk et al., 1993), does not produce an exponential survival curve when applied to HT29 cells (Musk and Johnson, 1993c). Thus the clastogenic and cytotoxic aspects of the radiomimesis of isothiocyanates would appear to be linked. That BITC exhibits a radiomimetic pattern of behaviour has certain implications for the type of lesions that one might predict it to generate within DNA. Ionizing radiation and radiomimetic agents are thought to generate directly relatively high numbers of DNA strand breaks, which are proposed as efficient substrates for the formation of chromosome aberrations but not for SCEs (Speit et al., 1984). Conversely, certain damaging agents which form adducts within the DNA, such as UV light and alkylating and cross-linking agents are capable of inducing large numbers of SCEs in exposed cells (Nishi et al., 1984). The fact that restriction endonucleases have been shown to be efficient inducers of SCEs (Natarajan et al., 1985) is not incompatible with this overall picture, given that the repair of the breaks induced by these enzymes is postulated to resemble that of those induced by enzymes involved in the repair of adducts rather than that of those introduced directly into the DNA by damaging agents (Balajee and Natarajan, 1993). Thus, one might predict from the results of the cytogenetic assays alone that the type of lesion generated by BITC would tend to be a strand break rather than an adduct. The results of the comet assay provide more evidence for this hypothesis in that they demonstrate that BITC is a powerful inducer of DNA strand breaks. In order to investigate further the radiomimesis of BITC we plan to investigate the spectrum of mutations induced by this compound in mammalian cells in vitro and to expand our studies on its ability to induce DNA strand breaks. Both the epidemiological evidence and the fact that the concentrations of BITC and its precursor glucotropaeolin in cruciferous vegetables are very low

36

S . R . R . Musk et al.

(values of 0.03-1.4 p p m a n d 0.4-1.0 ppm, respectively, being reported in fresh cabbage: Van Etten et al., 1976 a n d 1980) would seem to indicate t h a t there is no risk to h u m a n health in the c o n s u m p t i o n of ' n o r m a l ' levels of BITC. However, in view of its genotoxic nature in vitro, it would clearly be unwise to r e c o m m e n d an increase in dietary intake of BITC beyond n o r m a l levels as a putatively antitumorigenic measure without further investigation. It is also interesting to note that, whereas the ripe fruit of the p a p a y a (Carica papaya) has been reported to contain no more t h a n 4 p p m BITC, unripe fruit and seeds can have levels of BITC as high as 290 and 2 9 0 0 p p m , respectively (Tang, 1971). F u r t h e r m o r e , glucotropaeolin is present in the latex of unripe fruit at levels as high as 11.6% of the dry weight (Tang, 1973). Given that L e M a r c h a n d e t a l . (1991) have f o u n d a positive association between c o n s u m p t i o n of p a p a y a a n d the risk o f prostate cancer in H a w a i i a n males over 70, it is not entirely clear that the c o n s u m p t i o n of papaya fruit or juice is without hazard.

REFERENCES

Altman D. G. (1991) Practical Statistics for Medical Research. pp. 218-220. Chapman & Hall, London. Alves P. and Jonasson J. (1978) New staining method for the detection of sister chromatid exchanges in BrdU-labelled chromosomes. Journal of Cell Science 32, 185-195. Balajee A. S. and Natarajan A. T. (1993) Restriction endonucleases do induce sister chromatid exchanges in Chinese hamster ovary cells. Mutation Research 302, 25-31. Benito E., Obrador A., Stiggelbout A., Bosch F-X., Mulet M., Mufioz N. and Kaldor J. (1990) A population-based case-control study of colorectal cancer in Majorca. International Journal of Cancer 45, 69-76. Dunnick J. K., Prejean J. D., Haseman J., Thompson R. B., Giles H. D. and McConnell E. E. (1982) Carcinogenesis bioassay of allyl isothiocyanate. Fundamemal and Applied Toxicology 2, 114-120. Fenwick G. R. and Heaney R. K. (1983) Glucosinolates and their breakdown products in cruciferous crops, foods and feedingstuffs. Food Chemistry 11, 249 271. Fertil B., Reydellet I. and Deschavanne P. J. (1994) A benchmark of cell survival models using survival curves for human cells after completion of repair of potentially lethal damage. Radiation Research 138, 61 69. Garner R. C. and Martin C. N. (1979) Fungal toxins, aflatoxins and nucleic acids. In Chemical Carcinogenesis and DNA. Vol. 1. Edited by P. L. Grover. pp. 187-225. CRC Press, Boca Raton, FL. Graham S., Dayal H., Swanson M., Mittelman A. and Wilkinson G. (1978) Diet in the epidemiology of cancer of the colon and rectum. Journal of the National Cancer Institute 61, 709-714. Green M. H. L., Lowe J. E., Harcourt S. A., Akinluyi P., Rowe T., Cole J., Anstey A. V, and Arlett C. F. (1992) UV-C sensitivity of unstimulated and stimulated human lymphocytes from normal and xeroderma pigmentosum donors in the comet assay: a potential diagnostic technique. Mutation Research 273, 137-144. Hasegawa T., Nishino H. and lwashima A. (1993) Isothiocyanates inhibit cell cycle progression of HeLa Cells at G2/M phase. Anti-Cancer Drugs 4, 273-279. Ishidate M., Harnois M. C. and Sofuni T. (1988) A comparative analysis of data on the clastogenicity of 951

chemical substances tested in mammalian cell cultures. Mutation Research 195, 151-213. Kune G. A., Kune S., Read A., MacGowan K., Penfold C. and Watson L. F. (1991) Colorectal polyps, diet, alcohol and family history of colorectal cancer: a case control study. Nutrition and Cancer 16, 25-30. Kune S., Kune G. A. and Watson L. F. (1987) Case~:ontrol study of dietary etiological factors: the Melbourne colorectal cancer study. Nutrition and Cancer 9, 21-42. LeMarchand L., Hankin J. H., Kolonel L. N. and Wilkens L. R. (1991) Vegetable and fruit consumption in relation to prostate cancer risk in Hawaii: a re-evaluation of the effect of dietary beta-carotene. American Journal of Epidemiology 133, 215-219. McKelvey-Martin V. J., Green M. H. L., Schmerzer P., Pool-Zobel B. L., DeM6o M. P. and Collins A. (1993) The single cell gel electrophoresis assay (comet assay): a European review. Mutation Research 288, 47-63. Mennicke W. H., G6rler K., Krumbiegel G., Lorenz D. and Rittmann N. (1988) Studies on the metabolism and excretion of benzyl isothiocyanate in man. Xenobiotica 4, 441 447. Morse M. A., Amin S. G., Hecht S. S. and Chung F. L. (1989a) Effects of aromatic isothiocyanates on tumorigenicity, 06-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)l-(3-pyridyl)-l-butanone in A/J mouse lung. Cancer Research 49, 2894-2897. Morse M. A., Eklind K. I., Amin S. G., Hecht S. S. and Chung F. L. (1989b) Effects of alkyl chain length on the inhibition of NNK-induced neoplasia in A/J mice by arylalkyl isothiocyanates. Carcinogenesis 10, 1757-1759. Morse M. A., Wang C. X., Stoner G. D., Mandal S., Conran P. B., Amin S. G., Hecht S. S. and Chung F. L. (1989c) Inhibition of 4-(methylnitrosamino)-l-(pyridyl)l-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate. Cancer Research 49, 549 553. Musk S. R. R. (1987) Defective responses of a Simian virus 40-transformed Indian muntjac cell line to DNA damaging agents. PhD thesis, University of Cambridge. Musk S. R. R. and Johnson I. T. (1992) In vitro genetic toxicity testing of isothiocyanates in the human diet. Mutagenesis 7, 383. Musk S. R. R. and Johnson I. T. (1993a) The clastogenic effects of isothiocyanates. Mutation Research 300, 111-117. Musk S. R. R. and Johnson I. T. (1993b) In vitro genetic toxicology testing of isothiocyanates in the human diet. In Food and Cancer: Chemical and Biological Aspects. Edited by K. W. Waldron, 1. T. Johnson and G. R. Fenwick. pp. 58-61. Royal Society of Chemistry, Cambridge. Musk S. R. R. and Johnson I. T. (1993c) In vitro studies into the selective toxicity of allyl isothiocyanate towards transformed human cells. In Food and Cancer: Chemical and Biological Aspects. Edited by K. W. Waldron, I. T. Johnson and G. R. Fenwick. pp. 285 289. Royal Society of Chemistry, Cambridge. Musk S. R. R., Stephenson P. and Johnson I. T. (1993) Clastogenic activity of secondary metabolites from food plants. Mutagenesis 8, 475-476. Natarajan A. T., Mullenders L. H. F., Meijers M. and Mukherjee U. (1985) Induction of sister chromatid exchanges by restriction endonucleases. Mutation Research 144, 33-39. Nishi Y., Hasegawa M. M., Taketomi M., Ohkawa Y. and Inui N. (1984) Interrelationships of SCEs, mutation at the HGPRT locus and toxicity in Chinese hamster V79 cells. In Sister Chromatid Exchanges: 25 Years of Experimental Research. Edited by R. R. Tice and A. Hollaender. pp. 361-384. Plenum, New York.

Clastogenicity of benzyl isothiocyanate Perry P. and Evans Iq. J. (1975) Cytological detection of mutagen carcinogen exposure by sister chromatid exchange. Nature 258, 121-125. Rutzky L. P. and Moyer M. P. (1990) Human cell lines in colon cancer research. In Colon Cancer Cells. Edited by M. P. Moyer and G. H. Poste. pp. 155-202. Academic Press, London. Speit G., Hochsattel R. and Vogel W. (1984) The contribution of DNA single strand breaks to the formation of chromosome aberrat:ions and SCEs. In Sister Chromatid Exchanges: 25 Years of Experimental Research. Edited by R. R. Tice and A. Hollaender. pp. 229-244. Plenum, New York. Steel G. G. and Peakcock J. (1989) Why are some human tumours more radio:~ensitive than others? Radiotherapy and Oncology 15, 63--72. Stoner G. D., Morrissey D. T., Heur, Y. H., Daniel E. M., Galati A. J. and Wagner S. A. (1991) Inhibitory effects of phenthyl isothiocyanate on N-nitrosobenzylmethylamine carcinogenesis of the rat oesophagus. Cancer Research 51, 2063 2068. Tang C. S. (1971) Benzyl isothiocyanate in papaya fruit. Phytochemistry 10, 117-121. Tang C. S. (1973) Localisation of benzyl glucosinolate and thioglucosidase in CaHca papaya fruit. Phytochemistry 12, 769-773. Thompson L. H., Brookman K. W., Dillehay L. E., Carrano A. V., Mazrimas J. A., Mooney C. L. and Minkler J. L. (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange. Mutation Research 95, 427 440.

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Van Etten C. H., Daxenbichler M. E., Tookey H. L., Kwolek W. F., Williams P. H. and Yoder O. C. (1980) Glucosinolates: potential toxicants in cabbage cultivars. Journal of the American Society for Horticultural Science 105, 710 714. Van Etten C. H., Daxenbichler M. E., Williams P. H. and Kwolek W. F. (1976) Glucosinolates and derived products in cruciferous vegetables. Analysis of the edible part from twenty two varieties of cabbage. Journal of Agricultural and Food Chemistry 24, 425-455. Van Etten C. H. and Tookey H. L. (1979) Chemistry and biological effects of glucosinolates. In Herbivores: Their Interaction with Secondary Plant Metabolites. Edited by G. A. Rosenthal and D. H. Janzen. pp. 471 500. Academic Press, New York. Vig B. K. (1979) Hyperthermic enhancement of chromosome damage and lack of effect on sister chromatid exchanges induced by belomycin in Chinese hamster cells in vitro. Mutation Research 61, 309-317. Wattenberg L. W. (1981) Inhibition of carcinogeninduced neoplasia by sodium cyanate, tert-butyl isocyanate, and benzyl isothiocyanate administered subsequent to carcinogen exposure. Cancer Research 41, 2991 2994. Wattenberg L. W. (1987) Inhibitory effects of benzyl isothiocyanate administered shortly before diethylnitrosamine of benz[a]pyrene on pulmonary and forestomach neoplasia in A/J mice. Carcinogenesis 8, 1971-1973. Yamaguchi T. (1980) Mutagenicity of isothiocyanates, thiocyanates and thioureas on Salmonella typhimurium. Agricultural and Biological Chemistry 44, 3017-3018.