Phenethyl isotiocyanate modulates clastogenicity of mitomycin C and cyclophosphamide in vivo
Soumitra Sen 1, Anita Mukherjee *, Kalpana Agarwal, Archana Sharma Centre for Advanced Stuclv in Cell and Chromosome Research, Unicersity of Calcutta, 35 Ballygunge Circular Road, Calcutta 700 019,
India
Received 15 August 1995; revised 5 July 1996; accepted 24 July 1996
Abstract
Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is an effective chemopreventive agent against N-nitrosamine-induced carcinogenesis. We have investigated the extent to which PEITC modulates the clastogenicity of standard genotoxicants, mitomycin C and cyclophosphamide, using bone marrow cells of Swiss albino mice. PEITC, 1 txmol/kg body weight in corn oil was administered by gavage for 7 consecutive days to prime the animals. 24 h later, mice received a single dose of cyclophosphamide (10 or 20 mg/kg body weight)or mitomycin C (1 or 2 mg/kg body weight) intraperitoneally. Clastogenicity of the chemicals was compared using PEITC-primed and non-primed animals 24 h after clastogen treatment. As a single agent, PEITC is not clastogenic even after 7 days of priming. Oral priming with PEITC decreased the aberrations per cell values by 22-67% in all cases. PEITC could only alleviate the clastogenicity of I mg/kg body weight mitomycin C to near-control values ( p < 0.05). Although PEITC is reported to be effective against N-nitrosamine-induced tumorigenesis by preventing metabolic activation and by blocking the reactive species formed, it is virtually ineffective against the clastogenicity of cyclophosphamide. The results of inhibition by PIETC of the clastogenicity of mitomycin C suggest that the modulation of mitomycin C bio-activation contributes to, but may not be sufficient for, PIETC chemoprevention of clastogenicity by mitomycin C.
Keywords: Anticlastogen; Bone marrow chromosome; Chemoprevention; Isothiocyanate
Phenethyl isothiocyanate (PEITC) is a naturally- occurring aromatic isothiocyanate found in many cruciferous vegetables (Wattenberg, 1985; Morse and Stoner, 1993). It has been shown to inhibit N- nitrosamine (Guo et al., 1991, 1992; Morse et al.,
* Corresponding author. t Now at the Mario Negri Institute, Milan.
1991, 1992; Stoner et al,, 1991) and polycycl ic hydrocarbon-induced (Wattenberg, 1977) tumorigen- esis in vivo.
The purpose of the present study was to determine whether and to what extent orally administered PEITC would provide a broad spectrum of protection against two standard clastogens when administered intraperitoneally. Mice bone marrow cells were cho- sen as indicator cells for their high sensitivity to the clastogens.
160 S. Sen et al. / Mutation Research 371 (1996) 159-164
1. Materials and methods 1.3. Bone marrow chromosome preparation
1.1. Animals
Male Swiss albino mice, 8-10 weeks old and weighing 25-308 were obtained from the Depart- mental animal house, housed four/cage under stan- dard husbandry and feeding schedules, and provided with clean conventional colonies (temperature 25 _+ 2°C, relative humidity 60_+ 5%, 12 h light/dark photo-period). Animals were allowed ad libitum ac- cess to standard rodent pellet diet (Gold Mohar, Lipton India, Chandigarh, India) and water.
Ninety minutes before they were killed, the ani- mals received 5 mg colchicine/kg body weight (Sigma, St. Louis, MO) intraperitoneally as a chro- mosome pretreatment agent. Bone marrow chromo- some preparations were made by standard hypotonic (0.075 M KCI) - fixative (1:3 glacial acetic acid/methanol) - flame-drying schedule (World Health Organisation, 1985; Preston et al., 1987). All slides were coded and stained in dilute Giemsa solu- tion.
1.4. Scoring of aberrations
1.2. Chemicals and selection of dose
Based on our preliminary studies and the studies reported by Guo et al. (1992) and Morse et al. (1991, 1992)), the concentration of 1 txmol PEITC/kg body weight was chosen for the present study. This con- centration of PEITC did not affect either the food intake or the body weight gain of the animals during priming. PEITC (Aldrich, Milwaukee, WI) diluted in corn oil (Sigma, St. Louis, MO) as per the protocol suggested for mammalian in vivo cytogenetic assays (Preston et al., 1987) was administered by garage for 7 consecutive days for priming.
In separate experiments, we have tested the modu- latory effect of PEITC dissolved and diluted in olive oil (Bertolli Olio Extra Vergino, Bortolli, Lucca, Italy) and the chromosome aberration values have been compared with those obtained using corn oil.
Doses of 10 and 20 mg cyclophosphamide (CP, in saline)/kg body weight and 1 and 2 mg mitomycin C (MMC, in saline)/kg bodyweight were used in the study as standard clastogens. CP and MMC were purchased from Sigma, St. Louis, MO and Kwowa Hakko Kogyo, Tokyo, Japan, respectively. These were administered intraperitoneally as a single agent to PEITC-primed and non-primed mice 24 h after the last PEITC administration. These concentrations of CP and MMC caused a dose-dependent highly repro- ducible and significant increase in chromosome aber- rations in mice bone marrow cells (Tice et al., 1987a; Mukherjee et al., 1991). The animals were killed 24 h after clastogen administration.
Four animals were used per point. Fifty well- spread metaphase plates were scored per animal (200 metaphase plates per treatment set) at random. The types of aberration were scored and recorded strictly in accordance with the method of Tice et al. (1987b). All aberrations (chromatid gaps, isochromosome gaps, chromatid breaks, isochromosome breaks and rearrangements) were considered equal - regardless of the number of breakages involved. The percent- ages of damaged cells (%DC) and chromosomal aberrations cell (CA/cell) values were calculated excluding gaps.
1.5. Statistical analysis
For all statistical analyses, the level of signifi- cance was established at an alpha of 0.05. A one-way ANOVA followed by Duncan's new multiple range test (Duncan, 1955; Sokal and Rohlf, 1981) was carried out to detect significant differences in clasto- genicity, if any, amongst different treatment sets.
2. Results
The different parameters screened were the fre- quencies of chromosomal aberrations per cell (ex- cluding gaps) and the percentage of damaged cells (excluding gaps). Oral priming for 7 days with 1 pmol PEITC/kg body weight did not induce chro- mosomal aberrations in the animals. The values of
S. Sen et al . /Mutation Research 371 (1996) 159-164 161
Table 1 Total chromosomal aberrations recorded after administration of CP or MMC alone or when given to PEITC (1 ~mol /kg b.w.) primed mice
Treatment Chemical Dose Total chromosomal aberrations % DC CA/cell %Reduction in recorded per 200 cells (n = 4) CA/cell value
(days) (per kg b.w.) G' G" B' B" CR (mean _+ SEM) (mean _+ SEM)
7 Corn oil 10 ml 2 l 3 0 0 1.500 _+ 0.950 0.015 +_ 0.009 (negative control)
G',G", chromatid and isochromatid gaps; B',B", chromatid and isochromatid break; CR, chromosomal rearrangement; DC, damaged cells; CA, chromosomal aberration; mean, mean of four animals; SEM, standard error of the mean.
% D C and C A / c e l l w e r e ve ry s i m i l a r to n e g a t i v e
con t ro l va lues . A s s ing le agen t s , C P and M M C
i n d u c e d c l a s t o g e n i c i t y d i rec t ly p r o p o r t i o n a l to the
d o s a g e used . C h r o m a t i d t ype a b e r r a t i o n s w e r e pre-
d o m i n a n t . C P at d o s e s o f 10 and 20 m g / k g b o d y
w e i g h t i n d u c e d 10- and 13-fold i n c r e a s e s in % D C ,
r e spec t ive ly , wh i l e M M C at d o s e s o f 1 and 2 m g / k g
b o d y w e i g h t i n d u c e d 8- and 15-fold i n c r e a s e s r e spec -
t ive ly in % D C o v e r that i n d u c e d b y P E I T C alone .
S i m i l a r r e su l t s w e r e a l so o b s e r v e d w h e n C A / c e l l
va lue s w e r e c o n s i d e r e d ( T a b l e 1). T h e C A / c e l l de-
c r e a s e d by 27 and 3696, r e spec t ive ly , fo r P E I T C -
Table 2 Modulation of clastogenicity of CP or MMC by PEITC (1 Ixmol/kg b.w.) oral priming on bone marrow cells of mice
Treatment Chemical Dose Damaged cells (%)
(days) (per kg b.w.) Mean ± SEM
Chromosomal aberrations/cell
Observed Sum of Mean_+ SEM Observed Sum of increase individual increase individual (over increase (over increase corn oil) of the two corn oil) of the two
chemicals chemicals
7 Corn oil 10 ml 1.5 _+ 0.95 a * - - 0.015 _+ 0.009 a - - 7 PEITC 1 Ixmol 2.0 _+ 0.82 a 0.5 - 0.020 _+ 0.008 a 0.005 - 1 CP 10 mg 20.0 ± 1.63 b 18.5 - 0.505 _+ 0.045 b 0.490 - 7 + 1 PEITC + CP 1 + 10mg 16.5 +2.06 b 15.0 < 19.0 NS 0.370_+ 0.020 b 0.355 < 0.495 NS 1 CP 20 mg 25.0 _+ 4.30 c 24.5 - 0.920 _+ 0.230 c 0.906 - 7 + 1 PEITC + MMC 1 + 2 0 m g 17.0_+ 1.08b 15.5 < 25.0NS 0.590_+ 0.040 b 0.575 <0.910 NS 1 MMC I mg 17.5 _+ 3.77 m 16.0 - 0.270_+ 0.070 m 0.255 - 7 + 1 PEITC + MMC 1 + 1 mg 6.0_+0.81 n 4.5 < 16.5 * * 0.090___ 0.015 n 0.075 0.260 b I MMC 2 mg 31.5 _+ 1.70 m 30.0 - 0.790_+ 0.060 m 0.775 7 + 1 PEITC+MMC 1 + 2 m g 25.0_+4.20m 23.5 <30.5NS 0.620_+0.140m 0.605 <0.780NS
* Values in a vertical column followed by the same letter (a,a) or (b,b) or (m,m) are not significantly different at 5% level, whereas values with different letters (b,c) or (m,n) are significant at the 5% level as determined by Duncan's new multiple range test. Each value is the mean of four animals _+ standard error of the mean (SEM). * ~ Significantly less; NS, not significant.
162 S. Sen et al. / Mutation Research 371 (1996) 159-164
primed animals receiving l0 and 20 mg CP but the reduction was not significant statistically. Only in the group receiving MMC at 1 mg was the reduction (66%) in clastogenicity statistically ( p < 0.05) sig- nificant (Table 1). On the basis of Heddle (1974) method in the combination sets, PEITC and CP or PEITC and MMC, the increase in percentage of damaged cells and CA/cel l as compared to the control, was less than the sum of increase induced by individual compounds (PEITC, CP or MMC) over control. Therefore, PEITC was found to decrease the clastogenicity of C P and MMC (Table 2). Statistical analysis using one-way ANOVA followed by Dun- can's new multiple range test was used to compare the modulatory effect of PEITC on CP- and MMC- clastogenesis. Only in the last group was the reduc- tion in clastogenicity statistically (p < 0.05) signifi- cant (Table 2). Because of the potential interest of the data, we have repeated the entire set of experi- ments twice. The results were essentially the same as reported in this paper and deviations of the mean values never exceeded 10%.
In sharp contrast to the study reported by Raj and Katz (1984) that corn oil itself could inhibit DMBA- induced micronucleated polychromatic erythrocyte formation in the bone marrow cells, our studies failed to show any interference of corn oil with the clastogenic potential of MMC or CP. In separate sets, we have tested the modulatory effect of PR. ITC, dissolved and diluted in olive oil. Both the CA/cel l and %DC values have been found to be very similar to' those obtained by using corn oil (data not shown). The observation is in line with earlier reports that orally administered corn oil did not interfere with the clastogenicity of MMC or CP (Krishna et al., 1985) or that of DMBA (Tice et al., 1987a).
3. Discussion
An increasing number of dietary compounds have been shown to have significant chemopreventive ac- tions. Consumption of cabbage, cauliflower, broccoli and Brussels sprouts has been shown to be associ- ated with a decreased incidence of human cancers (Graham et al., 1978) as well as inhibition of experi- mental tumorigenesis in animals (Boyd et al., 1982;
Wattenberg, 1983). Investigations have focused on various organo-sulphur compounds in these vegeta- bles that appeared to possess the protective activity. Of these, isothiocyanates and alkyl polysulphides have demonstrable antitumorigenic activity (Miller and Stoewsand, 1983; Wargovich and Goldberg, 1985). PEITC or benzyl isothiocyanate (BITC) pre- treatments for extended periods, or concomitant ad- ministration, inhibited tumorigenicity of a large num- ber of compounds requiring metabolic activation (Guo et al., 1993). It was elegantly demonstrated by Guo et al. (1992) on rats and Smith et al. (1993) on mice that PEITC inhibited bio-activation of the xenobiotics by decreasing a cascade of enzyme activ- ities; and also facilitated detoxification of the reac- tive species and removal of DNA adducts by increas- ing activities of several enzymes involved in detoxi- fication/repair process. Differential modulation of cytochrome P450 enzymes has been shown to be one of the major mechanisms underlying chemopreven- tion of nitrosamine tumorigenesis by PEITC or BITC. The same cytochrome P450 enzymes have been shown to be involved in bioactivation of MMC in vivo (Bachur et al., 1979; Keyes et al., 1984; Pan et al., 1984).
To our knowledge, this is the first attempt to study PEITC modulation of clastogenicity. Single doses of 1 ~zmol PEITC/mouse are known to have no significant effect on activation of nitrosamines or other carcinogens. We have, therefore, primed ani- mals for 7 consecutive days before challenging with standard clastogens. Our results suggest that PEITC fails to protect animals completely against a single dose of CP, even when the mice were primed for 7 days with 1 ~zmol PEITC/kg body weight. Unfortu- nately higher doses of PEITC could not be used since it interfered with food intake and body weight gain of the experimental animals. The extent to which MMC activity was inhibited by PEITC prim- ing was not sufficient to elicit complete suppression of MMC-clastogenicity, particularly at low doses. Although a reduction of about one-fifth in abberation frequency may not be statistically significant, these are all reductions and the significance of this biologi- cal phenomenon should not be ignored. This means that altered P450 activity by PEITC is affecting the clastogens and aberrations were reduced in both cases. We agree that the best positive control would
s. Sen et al. / Mutation Research 371 (1996) 159-164 163
have been to look at the e f fec t o f our r e g i m e n of
P E I T C p r i m i n g on c l a s togenes i s in b o n e m a r r o w by
a m u t a g e n k n o w n to be ac t iva ted by p rocesses af-
fec ted b y PEITC. Unfo r tuna te ly , such an e x p e r i m e n t
was i m p o s s i b l e to pe r fo rm, s ince d i m e t h y l n i -
t r o s a m i n e is no t de tec tab le as a c l a s togen in the b o n e
m a r r o w ( p r e s u m a b l y b e c a u s e the ac t ive me tabo l i t e s
are largely p r o d u c e d in the l iver and do not r each the
b o n e m a r r o w in suf f ic ien t quant i t ies) . W e have ,
therefore , a t t emp ted to e luc ida te w h e t h e r and to wha t
ex ten t P E I T C cou ld m o d u l a t e c l a s togenes i s o f CP
and M M C . B o t h the c l a s togens are k n o w n to be
ac t iva ted by P450 e n z y m e s , p resen t ly k n o w n to be
mos t sens i t ive to the ef fec ts o f P E I T C ( M o r s e et al.,
1991; G u o et al., 1991, 1992).
Apa r t f rom m o d u l a t i o n o f b io -ac t iva t ion , a fu r the r
poss ib i l i ty exists , howeve r , for the m o d u l a t i o n of
ce l lu la r r e sponses to geno tox i c d a m a g e ( M a r m e l -
stein, 1993). Inc reased repa i r of d a m a g e d D N A or
de tox i f i ca t ion o f reac t ive species m ay p r e d o m i n a t e
ove r P E I T C - i n d u c e d inh ib i t i on of c l a s togen ac t iva-
t ion - par t i cu la r ly w h e n cel ls are t rea ted for sus-
t a ined per iods l onge r than 1 week. Th i s aspec t of
c h e m o p r e v e n t i o n m a y be re la ted to p ro tec t ion aga ins t
d i rec t ac t ing geno tox i can t s and shou ld be inves t i -
ga ted in detail .
Acknowledgements
This work was suppor ted in par t by the Counc i l
for Scient i f ic and Indus t r ia l Resea rch , India, Con-
t r a c t N o . 3 8 ( 8 3 0 ) y E M R - I I o f 1 / 9 1 a n d
9 / 2 8 ( 2 5 7 ) / 9 0 E M R - I of 6 / 9 0 .
References
Bachur, N.R., S.L. Gordon, M.V. and H. Hon (1979) NADPH-cy- tochrome P450 reductase activation of quinone anticancer agents to free radicals. Proc. Natl. Acad. Sci. USA, 76, 954-957.
Boyd, J.N., J.C. Babish and G.S. Stoewsand (1982) Modification by beet and cabbage diets of aliatoxin Bt-induced rat plas- mafoetoprotein elevation, hepatic tumorigenesis and muta- genicity or urine. Fd. Chem. Toxicol., 20, 47-52.
Duncan, B.D. (1955) Multiple range test. Biometrics, 11, 1-42. Graham, S., H. Dayal, M. Swanson, A. Mittleman and G. Wilkin-
son, (1978) Diet in the epidemiology of cancer of the colon and rectum. J. Natl. Cancer Inst., 61, 709-714.
Guo, Z., T.J. Smith, P.E. Thomas and C.S. Tang (1991) Metabolic activation of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone as measured by DNA alkylation in vitro and its inhibition by isothiocyanates. Cancer Res., 51, 4798-4803.
Guo, Z., T.J. Smith, E. Wang, N. Sadrieh, Q. Ma, P.E. Thomas and C.S. Yang (1992) Effect of phenethyl isothiocyanate, a carcinogen inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats. Carcinogenesis, 13, 2205- 2210.
Gun, Z., T.J. Smith, E. Wang, K.I. Eklind, F.L. Chung and C.S. Yang (1993) Structure-activity relationships of arylalkyl i soth- iocyanates for the inhibition of 4-(methylnitrosamine)-l-(3- pyridyl)-1-butanone metabolism and the modulation of xenobi- otic-metabolizing enzymes in rats and mice. Carcinogenesis, 14, 1167-1173.
Heddle, J.A. (1974) Combinations of mutagens: distinguishing possible outcomes. Mutation Res., 24, 77-79.
Keyes, S.R., P.M. Fracasso, D.C. Heimbrook, S. Rockwell, S.G. Sligar and A.C. Sartorelli (1984) Role of NADPH:cyto- chrome-c reductase and DT-diaphorase in the biotransforma- tion of mitomycin C. Cancer Res., 44, 5638-5643.
Krishna, G., J. Xu, J. Nath, M. Petersen and T. Ong, (1985) In vivo cytogenetic studies on mice exposed to ethylene dibro- mide. Mutation Res., 158, 81-87.
Marmelstein, N.H. (1993) Potential mechanism for food-related carcinogens and anticarcinogens. A scientific status summary by the Institute of Food Technologists" Expert Panel on Food Safety and Nutrition. Food Technol., 47, 105-118.
Miller, K.W. and G.S. Stoewsand, (1983) Dietary Brussels sprouts and glucosinolate influence on rat hepatic polysubstrat8 monoxygenases. J. Plant Foods, 5, 647-674.
Morse, M.A. and G.D. Stoner, (1993) Cancer chemoprevention: principles and prospects. Carcinogenesis, 14, 1737-1746.
Morse, M.A.K.T. Eklind, S.S. Hecht, K.G. Jordan, C.I. Choi, D.H. Desai, S. G. Amin, and F.L. Chung, (1991) Structure- activity relationship for inhibition of 4-methylnitrosamino)-l- (3-pyridyl)-l-butanone lung tumorigenesis by arylalkyl isoth- iocynates in A / J mice. Cancer Res., 5 l, 1846-1850.
Morse, M.A., K.I. Eklind, S.G. Amin and F.L. Chung (1992) Effect of frequency of isothiocyanate administration on inhibi- tion of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone-in- duced pulmonary adenoma formation in A /H mice. Cancer Lett., 62, 77-81.
Mukherjee, A., K. Agarwal, M.A. Aguilar and A. Sharma (1991) AnticlastoSenic activity of beta-carotene against cyclophos- phamide in mice in vivo. Mutation Res., 263, 41-46.
Pan, S., P.A. Andrews, C.J. Glover and N.R. Bachur (1984) Reductive activation of mitomycin C metabolites catalyzed by NADPH-cytochrome ]?450 reductase and xanthine oxidase. J. Biol. Chem., 259, 959-966.
Preston, R.J., B.J. Dean, S. Gallowway, H. Holden, A.F. McFee and M. Shelby (1987) Mammalian in vivo cytogenetic assays of chromosome aberrations in bone marrow cells. Mutation Res., 189, 157-165.
Raj, A.S. and M. Katz, (1984) Corn oil anod its minor con- stituents as inhibitors of DMBA-induced chromosomal breaks in vivo. Mutation Res., 136, 247-253.
164 S. Sen et al. / Mutation Research 371 (1996) 159-164
Smith, T.J., Z. Guo, C. Lt, S.M. Ning, P.E. Thomas and C.S. Yang (1993) Mechanism of inhibition of 4-(methylnitrosa- mine)-l-(3-pyridyl)-l-butanone bioactivation in mouse by di- etary pbenethyl isothiocyanate. Cancer Res., 53, 3276-3282.
Sokal. R.R. and F.J. Rohlf (1981) Biometry, 3rd Edn., Freeman, San Francisco.
Stoner, D.G., D.T. Morrissey, Y.H. Heur, E.M. Daniel, A.J. Galati and S.A. Wagner (1991) Inhibitory effects of phenethyl iso- thiocyanate on N-nitrosobenzylmethylamine carcinogenesis in the rat esophagus. Cancer Res., 51, 2063-2068.
Tice, R.R., J.L. Ivett and A.F. McFee (1987a) The effect of agent treatment time on the induction of sister-chromatid exchanges in mouse bone marrow cells in vivo. Mutation Res., 182, 15-29.
Tice, R.R., C.A. Luke and M.D. Shelby (1987b) Methyl isothio- cyanate: an evaluation of in vivo cytogenetic activity. Environ. Mutagen, 9, 37-58.
Wargovich, M.G. and M.T.D. Goldberg (1985) Diallyl sulfide: a naturally occurring thioether that inhibits carcinogen-induced nuclear damage to colon epithelial cells in vivo. Mutation Res., 143, 127-129.
Wattenberg, L.W. (1977) Inhibition of carcinogenic effect of polycyclic hydrocarbons by benzyl isothiocynate and related compounds. J. Natl. Cancer Inst., 58, 395-398.
Wattenberg, L.W. (1983) Inhibition of neoplasia by minor dietary constituents. Cancer Res., 43. 2448s-2453s.
Wattenberg, L.W. (1985) Cbemoprevent ion of cancer. Cancer Res.~ 45. 1-8.
World Health Organisation (1985) Environmental Health Criteria 51: Guide to Short Term Tests for Detecting Mutagenic and Carcinogenic Chemicals. WHO, Geneva, pp. 100-114.
