Chapter 7 Toxic effects

Adverse effects faster (by 10-20%) after intervention experiment in which a single dose of with at a level of 0.15-100 mg of was given to 250 g/day (Pantuck et al., 1979, 1984). volunteers [number per group not The classic toxic effect of cruciferous Endemic goitre was common in given, a reduction in radioiodine vegetables is goitrogenicity, which has many countries in the past century and uptake was seen at doses ~ 50 mg. been described in cattle and humans in many European countries as late as Shortly after injection of 1311 and and is caused by goitrin and other 1985. Only the Nordic countries and determinations of baseline uptake, compounds derived from certain the United Kingdom had succeeded by groups of two men or women [sex not Brass/ca seeds. that time in decreasing the prevalence specified for each group] were given (European Thyroid Association, 1985). crystalline goitrin at one of six doses Humans Goitre can be controlled by compul- from 12.5 to 400 mg (Langer et al., Brassica vegetables sory addition of iodide to all household 1971). A dose-related decrease in Owing to the high content of vitamin K salt. Several studies were conducted radioiodine intake was observed, with in broccoli, drug interactions have been after 1947 on the goitrogenic effects of 50% inhibition at around 300 mg [value reported in patients receiving medica- dietary components and drugs by read from curve]. In two further experi- tion for hypoprothrombinaemia. Two injecting volunteers with radioiodine ments described in the same publica- such cases were reported in women and determining uptake into the thyroid tion, each individual served as his or who ate 230-450 g/day of broccoli by external counting (van Etten et al., her own control by undergoing two (Kempin, 1983). One survey showed 1969a). tests for radioiodine uptake 1 week that most health-care professionals A goitrogenic effect of cruciferous apart. After a baseline test for radioio- (> 87%) were aware of possible inter- vegetables was hypothesized on the dine uptake 1 week earlier, six volun- actions between warfarin medication basis of field observations of teers received 50 mg of crystalline and the high content of vitamin K in Tasmanian schoolchildren and the goitrin, and 13 received 25 mg 1 h certain Brass/ca vegetables, such as source of milk in their homes. The most before the second test. A third group of broccoli, whereas fewer [55%, read likely source of the goitrogenic com- 18 volunteers were given 10 mg of from figure] knew about the potential pounds in the diets of these children crystalline goitrin 12 h before and drug interaction with white was the milk of cows given Brass/ca for- another 10 mg 1 h before the second (Couris et al., 2000). age crops, particularly during the winter test. Radioiodine uptake was statisti- Drug interactions as a conse- months (Clements, 1957). cally significantly decreased in the two quence of effects of cruciferous veg- In Finland, when goitre was com- former groups, whereas no decline etables on xenobiotic metabolizing mon, it was suspected to be due to low was observed in the third group. In two enzymes are also well known from dietary iodine intake in combination individuals given 50 mg, uptake of controlled human intervention studies with consumption of milk from cows radioiodine was completely blocked. (see section 4 for more detail). In one grazing on fields rich in cruciferous Reduced iodine uptake by the thyroid study on the effects of eating 400 g/day vegetation. In an experiment with 22 gland was observed with the radioio- of Brussels sprouts for 2 weeks on healthy volunteers (7 women, 15 men) dine test after a daily intake of 500-600 warfarin clearance, a 29% increase aged 22-46 years, radioiodine uptake g of cabbage (raw, pickled, boiled or was observed (Ovesen et al., 1988). into the thyroid was determined while quick-steamed) for 2 weeks by a group The effect of eating Brussels sprouts they drank 1.5-2 I of milk containing of nine volunteers [previous work cited and cabbage on the clearance of 0-20.3 pg/I of goitrin, 2.1-8.0 mg/I of by Langer et ai. (1971)]. Each individ- antipyrine, oxazepam and aceta- SCN- and 8-142 pg/I of 1 (Vilkki et al., ual served as his or her own control by minophen has also been assessed in 1962). No effects were found on comparison with a baseline test per- controlled human intervention studies. radioiodine uptake, plasma-bound formed 6 months earlier. The drugs were excreted significantly iodine or total serum iodine. In an IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, and

In a parallel study of the effects of In a prospective open-label trial, of restorative growth were observed in ingestion of 150 g of Brussels sprouts -3-carbinol was given to 18 survivors. Abnormal thyroids were daily for 4 weeks by a group of three patients aged 1.2-66 years with recur- observed in non-pregnant females women and seven men, no effects rent respiratory papillomatosis; the given acutely toxic doses (150-650 were observed on serum concentra- children received a weight-related mg/kg bw) of epi-, 3-indolyl- tions of thyrotropic hormone, total or dose of 5-10 mg/kg bw per day, while acetonitrile, S-1 -cyan o-2-hydroxy-3- free thyroxine or total triiodothyronine the adults took 200 mg twice a day for butene or R-goitrin, in the form of (McMillan et al., 1986). The sprouts 8-24 months (average, 14.6 months). hyperplastic cells, smaller follicles, variety had been selected for its high Symptoms of disequilibrium developed foamy areas and scanty colloid, content of progoitrin and glucobras- in three persons who took excessive whereas the thyroids of pregnant sicin, and the daily doses of these com- doses, one of whom was a male vol- females were largely unaffected by pounds were confirmed by food analy- unteer given 800 mg/day for 10 days. these compounds. Slightly hyperplastic ses to be 99 mg and 105 mg, respec- Light tremor was also observed. These thyroids were observed in pregnant tively; however, the daily intakes were symptoms disappeared when the dose and non-pregnant rats after near-lethal decreased to 69 mg and 39 mg, was decreased to 400 mg/day. Two doses (100-700 mg/kg bw; see Table respectively, by cooking. The intake of girls aged 2.5 years and 12 years who 64) of 1 -cyano-3,4-epithiobutane, sink progoitrin in this study could have led to accidentally took triple doses both grin (including oral administration), an intake of 14 mg of the goitrogenic experienced unsteadiness for periods hydrate, para-hyd roxyphenyl- goitrin. The authors speculated that the of 8-24 h (Rosen et al., 1998). acetonitrile, iberin nitrile, 3-indolylaceto- low (2.4%) residual activity of myrosi- nitrile and indole-3-carbinol. Lberin at nase in the sprouts decreased the for- Experimental animals an acutely toxic dose (100 mg/ kg bw) mation of goitrogerlic derivatives and Acute toxicity did not affect thyroid tissue in rats. was indirectly the cause of the null and nitriles Sporadic changes in liver and adrenal effect observed in this study. The reported values for the median weights were found with the nitrites. 1- lethal dose (LD50) of Brass/ca com- Cyano-3,4-epithiobutano consistently Indoles pounds in animals are shown in Table increased adrenal weights of males Groups of 20 women at high risk for 64. and pregnant female rats by 25-50% breast cancer were assigned to 400 The acute toxic effects of subcuta- at a dose of 95 mg/kg bw. mg/day indole-3-carbinol or placebo neously injected Brass/ca glucosi no- In order to determine the lethal for 3 months. They were interviewed lates and nitrites were studied in dose, high single doses of the com- monthly about any adverse effects, groups of 5-8 Holtzman rats of each pounds were administered subcuta- and blood samples were collected for sex, maintained on Teklad 40% mouse neously to Sprague-Dawley rats. The extensive clinical chemistry. No sys- and rat diet and weighing 250-300 g rats were observed for 12 days after tematic adverse effects were reported, (Nishie & Daxenbichler 1980). Some of dosing, at which time they were killed. and there was no effect on plasma the rats were pregnant. lndole-3- The mean LD50 values were 90 mg/kg bilirubin and albumin or on serum glu- carbinol and 3-indolylacetonitrile bw for iberin, 109 mg/kg bw for 1- tamyl ornithine transaminase, suggest- induced sedation, ataxie, loss of right- cyano-3,4-epithiobutane, 200 mg/kg ing no effect on liver function. Also, ing reflex and sleep. In general, they bw for S-1 -cyano-2-hydroxy-3-butene plasma estrogen and the number of were less toxic in pregnant females and 255 mg/kg bw for 3-indolylacetoni- days between menses were unaf- and more toxic in the younger male trile; values could not be determined fected (Bradlow et al., 1994). rats. Several other compounds were for indole-3-carbinol or para-hydroxy- In a double-blind placebo-con- tested at only one or two doses, phenylacetonitrile, although their acute trolled dose-ranging study, 57 women administered by a single or by two toxicity was similar to that of 3-indoly- of an average age of 46.7 years repeated subcutaneous injections. The lacetonitrile. (range, 22-74 years) who were at kidneys, livers and adrenal and thyroid increased risk for breast cancer were glands were examined histologically at Isothiocyanates and derivatives randomized into groups of 7-10 and necropsy or 12 days after dosing. 1- In the study of Nishie and given indole-3-carbinol at a dose of Cyano-3,4-epithiobutane (total dose, DaxenbicNer (1980), the mean LD50 0-400 mg/day for 4 weeks (Wong et about 90 mg/kg bw) gave rise to value for allyl-ITC was reported to be al., 1997). No toxicity or consistent increased renal weights and renal 92 mg/kg bw (see Table 64). Mildly adverse effects were reported. necrosis in pregnant dams, and signs abnormal thyroid tissues were

192 Table 64. Median lethal doses (LD50) of Brassica compounds

Compound Species, strain Route of Duration of follow- LD5 Reference administration up {niglkg bw) 1-Cyano-3,4-epithio- Mouse Oral Not specified 178-240 van Etten et ai. (1969a) butane Rat, Sprague- Subcutaneous 12 days 109 Nishie & Daxenbichler (1980) Dawley Mouse Orai Not specified 170 van Etten et ai. (1 969a)

Rat, Sprague- Subcutaneous 12 days 200 Nishie & Daxenbichler (1980) Dawley

AlIyI-ITC Mouse (white) Subcutaneous 14 days 80 Klesse & Lukoschek (1955) Rat Oral 339 Jerrier et al. (1964) Rat, Sprague- Subcutaneous 12 days 92 Nishie & Daxenbichler (1980) Dawley

Phenethyl-ITC Mouse (white) Subcutaneous 14 days 250 Klesse & Lukoschek (1955) Mouse, Swiss- Subcutaneous 14 days 150 Lichtenstein et ai. (1962) Webster Oral 14 days 700 Intravenous 14 days 50

Goitri n Mouse Oral Not specified 1260-1415 van Etton eta?, (1969a)

Iberin Rat, Sprague- Subcutaneous 12 days 90 Nishie & Daxerbichler (1980) Dawley lndole-3-carbincl Mouse, ICR Oral 10 days cumulative 1670 Shertzer (1982) Intraperitoneal 10 days cumulative 500 Mouse, Cl Oral 1h <750 Shertzer & Sainsbury (1991a) Rat, Wistar Oral 10 days cumulative 1850 Shertzer (1982) Intraperitoreal 10 days cumulative 550 Rabbit, white Oral 10 days cumulative 1400 Intrapeiltoneal 10 days cumulative 420

3-Indolylacetonitrile Rat, Sprague- Subcutaneous 12 days 255 Nishie & Daxenbichler (1980)

('e IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles observed in pregnant and non-preg- Indoles > 100 mg/kg bw; the concentration had nant rats after near-lethal doses. The Preliminary LD50 values were deter- returned to background levels by 24 h LD50 of a 10% solution of allyl-ITC mined by Shertzer (1982) in male ICR at doses < 250 mg/kg bw. Leaking of given subcutaneously in corn oil to mice, Wistar rats and New Zealand hepatic enzymes into the bloodstream mice was reported to be 80 mg/kg bw white rabbits treated with indole-3- was seen at doses > 100 mg/kg bw. (IARC, 1985). carbinol for 10 days (Table 64). Male Neurological signs, including changes The acute toxicity of allyl-ITC in ICR mice, weighing about 30 g bw, in posture and activity, were seen at groups of five Fischer 344/N rats and were maintained on Purina animal doses ~ 100 mg/kg bw 2 h after gav- five B6C3F mice of each sex was chow and dosed orally or intraperi- age. A dose of 500 mg/kg bw induced examined in pilot studies before a long- toneally for 10 consecutive days. The coma. term cancer bioassay (National LD50 in male ICR mice was estimated A single oral administration of Toxicology Program, 1982). After 16 to be 500 mg/kg bw after iritraperi- indole-3-carbinol at a dose> 100 pmol days' administration of allyl-ITC in corn toneal administration and 1670 mg/kg per rat (15 mg/kg bw) to groups of four oil by gavage, growth retardation and bw when given by gavage. In male male Sprague-Dawley rats, 3-4 weeks dose-related signs of toxicity were Wistar rats (weighing 300 g), the LD50 old and maintained on a semi-purified seen at doses of 200 and 400 mg/kg values for intraperitoneal and oral diet for 7-8 days, led to toxic manifes- bw in rats and 100-800 mg/kg bw in administration were 550 mg/kg bw and tations in the small intestine (Bradfield mice, evaluated on the basis of com- 1850 mg/kg bw, respectively, whereas & Beldanes, 1987b). parisons with the lowest dose (no con- the LD50 values for rabbits (male New Groups of six male guinea-pigs, trols). No female mice and only one Zealand white, weighing about 2 kg) weighing 400-500 g, were given two male mouse survived the dose of 800 were 420 mg/kg bw when given oral doses of indole-3-carbinol at 0.3 mg/kg bw. intraperitoneally and 1400 mg/kg when mg/kg bw per day for 4 days. The ani- Groups of five male and five female administered orally. No further details mals were killed after treatment on day Osborne-Mendel rats were fasted for about the experiments were given. 4, and four tissue samples were col- 18h and then given a single high dose The acute toxicity of indole-3- lected from the liver and lung of each of allyl-ITC by gavage and observed carbinol was studied in groups of male animal and examined histopathologi- for 14 days. An LD., of 339 mg/kg bw CID-1 mice weighing 30-35 g, main- cally. Indole-3-carbinol caused hepatic (95% confidence interval, 318-361) tained on Teklad standard rodent diet steatosis and interstitial pneumonia was calculated (Jenner et al., 1964). for 7 days and then given indole-3- with septal hyperaemia (Gonzalez et The animals were described as carbinol orally at 50 mg/kg bw per day al., 1986). scrawny, with porphyrin-like deposits (dissolved in corn oil, 1.5 pug bw) for around the eyes and nose and rough 10 days (Shertzer & Sainsbury, Short-term effects fur. 1991a). Indole-3-carbinol did not Brassicas The LD50 values for phenethyl-ITC change the body weight or the Crambe oilseed meals processed to in mice were reported to be 700 mg/kg liver:body weight ratio. The activity of increase or decrease the levels of epi- bw after oral administration and 50 several plasma enzymes was deter- progoitrin, goitrin or total nitriles were mg/kg bw after intravenous injection. In mined 2 and 24 h after a single oral given at 10% in the feed to groups of the same species, the LD50 of dose of 50-500 mg/kg bw. Plasma cre- five weanling rats for 90 days, and the phenethyl-ITC administered subcuta- atine phospho-kinase activity was not weight and survival of the animals neously was reported to be 150 mg/kg changed at doses up to 500 mg/kg bw, were recorded as a measure of toxicity bw (Lichtenstein et al., 1962) or indicating a lack of severe cardiotoxic- (van Etten et al., 1969b). Fractions of 250 mg/kg bw (Klesse & Lukoschek, ity; however, indole-3-carbinol caused epi-progoitrin (0.85%), R-goitri n 1955). a dose-dependent increase in plasma (0.23%) and nitrile (0.1%) isolated In a study presented in an overview ornithine transcarbamylase activity (at from crambe seeds were mixed into (van Etten et al., 1969a), the LD50 val- doses > 100 mg/kg bw) and alanine the feed for additional groups. The ues in mice were 1260-1415 mg/kg transaminase activity (at doses nitrile-rich meals were the most toxic, bw for goitri n (R-5-vi ny!-2- > 250 mg/kg bw), indicating a hepato- leading to 100% mortality in a group thione), 169 mg/kg bw for S-1 -cyano-2- toxic effect, 24 h after treatment. fed 10% (w/w) meal treated to enrich hydroxy-3-butene and 178-240 mg/kg Indole-3-carbinol decreased the nitriles, and a 40% reduction in body bw for S-1 -cyano-2-hydroxy-3,4- hepatic GSH concentration in a dose- weight in a similar group in a second epithiobutane. dependent manner 2 h after oral doses experiment. The weight of animals fed

194 Toxic effects

0.1% nitrile fraction in the feed was 3-50 mg/kg bw, respectively, by gav- Benzyl-ITC at 200 mg/kg bw per day only 17% that of controls (60% sur- age for 14 days resulted in 'adhesion of caused histological changes in the vival). Animals fed meal containing the stomach wall to the peritoneum' in ductus choledochus, liver, ileum and active had only 40-600/. rats and a dose-dependent thickening mesenteric lymph nodes. The weights weight gain and reduced survival. of the stomach mucosa in both of the thymus (at 100 and 200 mg/kg These meals had high concentrations species. Furthermore, mice given the bw per day) and spleen (at 200 mg/kg of epi-progoitrin. The animals fed puri- highest dose developed thickening of bw per day) were decreased in relation fied epi-progoitrin or R-goitrin reached the urinary-bladder wall. [The Working to body weight, whereas the weights of 85% of the weight of controls. Group noted that histological details all other organs were increased. were not presented and that the signif- Even at a low dose of 7.5 mg/kg bw Isothiocyanates and derivatives icance of these findings is uncertain.] per day to ACI rats for 53 weeks, ben- WIST male rats weighing 70-90 g The doses of 200 and 400 mg/kg bw zyl-ITC resulted in a decrease in [number of animals per group not per day to rats and 50 mg/kg bw per weight gain of 8-9% as compared with reported] were given allyl-ITC at a day to mice were lethal. In a 13-week control animals (Sugie of al., 1991). dose of 10, 20 or 40 mg/kg bw (b study in groups of 10 male and female Morse et al. (1989a) examined the days/week) by gavage for up to 6 Fischer 344/N rats and B6C3F1 mice toxicity of phenethyl-ITC at 0, 0.75, 1 .5, weeks (Lewerenz et al., 1988). The given a dose of 1.5, 3, 6, 12 or 25 3 or 6 pmol/g of diet in male Fischer highest dose reduced body-weight mg/kg bw per day, no dose-related 344 rats (weighing 100-120 g) in a 13- gain and decreased blood glucose and effects were observed on growth or week study. Dietary administration of serum globulin levels. Examination of gross morphology (National Toxicology phenetliyl-ITC did not produce any the blood revealed an increased per- Program, 1982). deleterious effects on survival, body centage of neutrophils and a Administration of phenethyl-ITC at weight or food intake. Clinical evalua- decreased percentage of lymphocytes a dose of 41, 82 or 122 mg/kg bw per tion showed no adverse changes after treatment with the highest dose day for 6 days by gavage to groups of suggesting toxicity. In addition, no sig- for 2 weeks. After 1-3 weeks, 24 female and 24 male young NJ mice nificant differences from control ani- increased thymus, liver or adrenal resulted in significantly reduced body- mals were found on gross examina- weights (both relative and absolute) weight gains [absolute numbers not tion at necropsy or by determination were found in all treated groups. specified, hyperactivity, rough fur and of relative organ weights. Increasing the treatment period to 4 emaciation (Adam-Rodwell of al., Histopathological analyses, however, weeks resulted in no significant differ- 1993). A dose of 244 mg/kg bw per day revealed centribular and mid-zonal ences in thymus, liver or adrenal was lethal after two to four doses and fatty metamorphosis in the livers of weights between treated and control was discontinued. rats exposed to phenethyl-ITC at all animals. Renal dysfunction was indi- The toxicity of benzyl-ITC was doses tested. cated by increased urinary aspartate investigated in groups of 15 male rats ami notransferase activity, reduced weighing 85-110 g given the com- Indoles urine volume and changes in the spe- pound dissolved in sunflower oil at a Groups of eight male Sprague-Dawley cific gravity of the urine in the group of dose of 50, 100 or 200 mg/kg bw per rats weighing 70 g were given a control rats receiving the highest dose. day by gavage for 4 weeks (Lewerenz diet or control diet containing either Histopathological changes were et al., 1992). Control rats were given at 0.5 g/kg [30 mg/kg observed in the kidneys of animals the vehicle only Body-weight gain and bw per day], , gluconapin, glu- given 20 or 40 mg/kg bw per day and food consumption were decreased cosinalbin or each at in the livers of animals at 40 mg/kg bw with increasing doses of benzyl-ITC. 1.0 g/kg [60 mg/kg bw per day] or pro- per day. Haematological changes were goitrin at 3.0 mg/kg [160 mg/kg bw per The toxicity of aIlyl-ITC after observed at the highest dose, with day] for 29 days. Before treatment, the repeated doses was investigated in increased serum cholesterol concen- animals were held on control diet for 2 pilot studies before a long-term cancer trations in all treated groups and days, for 1 day on control diet contain- bioassay in rats and mice of each sex. decreased serum triglycerides at 200 ing 50% of the final level An experiment in which groups of five mg/kg bw per day. Renal dysfunction and for 1 day on control diet containing male and five female Fischer 344/N was indicated by reduced urine vol- 75% of the final glucosinolate level. rats and B6C3F1 mice received doses ume, proteinuria and enhanced urinary The dietary glucosinolates did not in corn oil of 25-400 mg/kg bw and lactate dehydrogenase activity. affect feed intake or body-weight gain

195 IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles throughout the period. Only progoitrin dose. Additionally, groups of six female diet (0.5-0.8 pg/day) with about 40 g of affected the relative weights of the thy- SW mice, 80-100 days of age, were cabbage or carrots (both vegetables roid, liver and kidney (Vermorel et al., maintained on Purina basal diet with contained negligible amounts of 1986). 10% corn oil for 3 days and then given iodine), or cabbage or carrots alone, The effect of indoles on body the same diet containing indole-3- for 60 days (Langer & Stole, 1965). The weight was investigated in groups of carbinol at 0,250, 1000 or 2500 mg/kg thyroid glands were significantly six male weanling Sprague-Dawley [30, 120 or 300 mg/kg bw per day] for enlarged and contained significantly rats fed A1N76 diet containing indole-3- 3 weeks. No difference in body weight less iodine per gram of organ. Plasma- carbinol at 50, 500, 5000 or 7500 was observed. A slight increase in the bound iodine was also significantly mg/kg ad Jib/turn for 3 weeks (Babish & liver:body weight ratio was seen in decreased. Stoewsand, 1978). Significantly lower treated animals, but there was no Pigs appear to be vulnerable to body-weight gain was observed with gross evidence of hepatic toxicity. Two meal glucosinolates. Groups 5000 mg/kg [1500 mg/kg bw per day] groups of 10 male and 10 female of 7-10 male and female pigs, initially and 7500 mg/kg [2250 mg/kg bw per C3H!OuJ mice, 21-28 days of age, weighing 20-25 kg, received their ordi- day], representing 85% and 59% of the were kept on control Purina semi-syn- nary diet containing 2-15% unrefined weight gain of untreated controls, thetic diet or the same diet containing rapeseed meal to replace skim milk respectively. The relative liver weights indole-3-carbinol at 2000 mg/kg [240 powder (Nordfeldt et al., 1954). were increased by 1.6- and 1.8-fold, mg/kg bw per day] for G months. The Thyroid and liver weights were respectively, in these two groups. In average body weights of the females in increased in all treated animals. another study, indole-3-carbinol was each group were recorded about every Adding an antibiotic (bacitracin), vita- given by gavage at 5, 25, 50, 100 or 10-15 days. No differences in body min B12 or wheat bran to the diet did 200 mg per day for 6 weeks to female weight were observed. not alleviate the effects. Water extrac- Sprague-Dawley rats from 43 days of Groups of seven male Sprague- tion of the rapeseed meal significantly age (Grubbs et al., 1995). The highest Dawley rats were fed diets containing attenuated the effect. dose caused a 10% depression in indole-3-carbinol at concentrations Studies in pigs were reviewed by body-weight gain and a 20% increase providing a dose of 0, 50, 100 or 150 Mawson et al. (1994). A generally lin- in liver weight. The modifying effect of mg/kg bw per day, 5 days/week for 7 ear relationship was observed indole-3-carbinol was investigated in a weeks (Exon et al., 2001). The anti- between glucosinolate intake from multi-organ carcinogenesis model in body response to an antigen challenge feed and thyroid weight at slaughter. groups of 20 male Fischer 344 rats, 6 was significantly decreased at the Significant increases in thyroid weights weeks of age, maintained on AIN-76A highest dose. No effects were found on were observed in pigs given feed with diet for 4 weeks and then switched to natural killer cell activity or delayed- a total glucosiriolate content of 2-3 the same diet containing 0.5% indole- type hypersensitivity. mg/kg, particularly when progoitrin 3-carbinol for 36 weeks [300 mg/kg bw was one of the main glucosinolates. per day]. An insignificant decrease in Goitrogenic effects body weight was observed (Jan g et al., Crucifers and unrefined fractions Isothiocyanates and derivatives 1991). A goitrogenic effect of crucifers was The spontaneous formation of goitrin The effect of dietary indole-3- first noted after feeding Hrassica seeds from 2-hydroxy-3-butenyl-ITC, result- carbinol on liver and body weight was to rabbits (Chesney et al., 1928). ing from degradation of progoitrogen investigated in groups of six female Several years later, the effect was catalysed by myrosinase, is one of the C3H/OuJ adult mice, 80-100 days old, found to be due mainly to the formation main causes of the goitrogenic action maintained on Purina basal diet with of organic and inorganic isothio- of rapeseed meal. Groups of eight 10% corn oil for 3 days and then given cyanates and goitrins (oxazo- female Sprague-Dawley rats given a the same diet containing indole-3- lidinethionos) (Astwood et al., 1949). diet containing about twice the minimal carbinol at 0, 1000, 3000 or 5000 Goitrogenic effects in various domestic required iodine content were given 5- mg/kg [120, 360 or 600 mg/kg bw per and laboratory animals have been vinyl-2-th iooxazolidone intraperito- day] for 3 weeks (Bradlow et al., 1991). reviewed (Fenwick et al., 1989; neally at a dose of 1-200 pg/day for 3 The average daily food consumption Mawson et al., 1994; Stoewsand, weeks (Elfving, 1980). Controls were decreased significantly only in mice at 1995). injected with saline. Significant the highest dose. The relative Groups of 4-9 male Wistar rats increases in thyroid weights were livecbody weight ratio increased with were fed 10-15 g/day of a low-iodine observed at doses > 5 pg/day. In the

196 Toxic effects thyroid, significantly increased ratios of were fed containing S-methylcys- of gestation, and the numbers of live 3 '-monoiodothyronine:3,5-diiodothyro- teine sulfoxide at 99 or 154 mg/kg. fetuses and resorptions were exam- nine and of 3,5,3'-triiodothyronine: Heinz bodies reached their peak level ined at necropsy or at termination 12 3,5,3,5-tetraiodothyronine were at 9 and 33 days of the feeding period, days later (Nishie & Daxenbichier, observed even at the lowest dose. respectively, and serum haemoglobin 1980). A significant 48% increase in Total 1251 uptake into the thyroid was reached minimal levels at 20 and 44 fetal resorption was observed after a unchanged at all doses, but iodine days, respectively. Additional groups of single dose of 1-cyano-3,4-epithiobu- excretion into urine was decreased at goats received different concentra- tane at 95 mg/kg bw given on day 8 of the highest dose. The authors con- tions: 10 animals received a fraction- gestation, whereas two doses of cluded that goitrin acts primarily on ated kale extract containing S-methyl- 44 mg/kg bw on days 8 and O did not thyroxin biosynthesis. cysteirie sulfoxide at 285 mg/kg, 97 affect fetal survival. A significant 19% Allyl-ITC has slight goitrogenic animals received S-methylcysteine increase in fetal resorption was activity in rodents. Studies in rats sulfoxide at 150 mg/kg, and 104 ani- observed after two doses of allyl-ITC at (weighing 150-320 g) given allyl-ITC mals received S-methylcystei ne sulfox- 100 mg/kg bw on days 8 and O of ges- as a single dose of 2-4 mg in water by ide at 195 mg/kg. The number of days tation, whereas one-half of this dose gavage showed 40-60% less iodine required to attain maximal formation of on the same days had no effect. uptake into the thyroid gland. Oral Heinz bodies was 7, 35 and 21, and Administration of iberin at 100 mg/kg administration of allyl-ITC at 2.5-5 the number of days to reach the lowest bw on day 8 of gestation increased mg/day for 60 days, however, did not haemoglobin level was 15, 42 and 35. fetal resorption by 29%. None of these decrease the total iodine content in the respectively. compounds resulted in malformations. thyroid (Langer & StoIc, 1965). The metabolite of S-methylcysteine Significant changes in mean fetal The degree of goitrogenicity sulfoxide, dimethyl disulfide, precipi- weights were recorded with epigoitrin, induced by allyI-ITC was found to be tates GSH to initiate the formation of S-1 -cyano-2-hydroxy-3-butene, R- weaker than that induced by better- Heinz—Ehrlich bodies in erythrocytes. goitrin, 1 -cyano-3,4-epithiobutane, sin- known goitrogens, such as thiouracil According to a recent review, rumi- grin, iberin nitrile, 3-indolylacetonitrile, (Duncan, 1991). nants, fowl and rats are also vulnerable indole-3-carbinol and para-hydroxy- to anaemia after being fed kale or phenylacetonitrile when given at near- Indoles dimethyl disulfide, but guinea-pigs and lethal doses to the pregnant dams. Groups of eight male Sprague-Dawley rabbits are refractory (Stoewsand, Sinalbin hydrate given at 300-600 rats, weighing 70 g, were fed control 1995). Dosing of rats with feed con- mg/kg bw to pregnant dams did not diet or control diet complemented by taining S-rnethylcysteine sulfoxide at lead to increased altered fetal weights, glucobrassicin at 0.5 g/kg [30 mg/kg 2-4% (w/w) elicited the effect, but it fetal resorption or malformations. bw per day] for 29 days. Before treat- was reversible after about 14 days. The teratogenic activity of allyl-ITC ment, the animals were given control was evaluated in mice, rats, hamsters diet for 2 days, then control diet con- Reproductive effects and rabbits in a study performed by the taining 50% of the final glucosinolate Decreased fertility has been described Food and Drug Research Laboratories level for 1 day and finally control diet repeatedly in domestic animals given in 1973 (IARC, 1985). Allyl-ITC dis- containing 75% of the final glucosino- rapeseed meal as part of their feed. solved in corn oil was administered by late level for 1 day. The animals were Mawson et al. (1994) summarized pub- gavage in all studies. Groups of 23-25 killed 29 days after the start of treat- lished and anecdotal evidence and CD-1 mice were given 0, 0.3, 1.3, 6 or ment. Neither thyroid weight nor thy- concluded that rats and pigs are the 28 mg/kg bw per day on days 6-15 of roid hormone concentrations in the most vulnerable species, whereas gestation, and fetuses were examined thyroid were affected (Vermorel et al., ruminants and chicken are less sensi- on day 17 for malformations. Groups of 1986). tive. Pigs may be affected by total rape- 25 Wistar rats received a dose of 0, seed gI ucosinolate concentrations 0.2, 0.85, 4 or 18.5 mg/kg bw per day Haemolytic effects exceeding 4 mg/kg of feed, whereas on days 6-15 of gestation, and fetuses Kale fed to ruminants in considerable rats appear to tolerate oven less. were examined for malformations on quantities for 1-3 weeks led to Groups of 3-12 pregnant Holzman day 20. Groups of 25-27 golden ham- haemolytic anaemia due to its content rats were given Brass/ca glucosino- sters received a dose of 0, 0.2, 1.1, 5.1 of S-methylcysteine sulfoxide (Smith, lates and degradation products at or 23.8 mg/kg bw per day on days 1980). Groups of 52 and 103 goats doses close to the LD,,) on day 8 or 9 6-10 of gestation, and fetuses were

197 IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles examined for malformations on day 14. doses > 0.5 mg/kg bw per day the direct mutagenic effects of nitrite- Groups of 11-14 Dutch-bolted rabbits decreased body-weight gain after four treated Chinese cabbage and indoles received a dose of 0, 0.123, 0.6, 2.8 or daily doses, whereas 3,3'-diindolyl- in Salmonella typliimurium. They found 123 mg/kg bw per day on days 6-18 methane had no effect on weight. The that the contributions of indoles to the of gestation, and fetuses were exam- latter compound slightly decreased total mutagenic activity of nitrite-treated ined for malformations on day 29. No ovarian weight gain after a challenge Chinese cabbage to evidence of maternal toxicity or treat- with 5 lU of equine chorionic S. typhimurivmTAloo were 0.2% from ment-related malformations was found gonadotropin and reduced the number indole-3-acetonitrile, 0.3% from in any species. In mice, there of ova shed after 72 h by about 30% in 4-methoxyindole-3-aceton itrile and appeared to be an increase in the all treated groups. [The Working Group 17% from 4-rnethoxyindole-3-aldehyde number of dead and resorbed fetuses noted that these changes were not sta- (Wakabayashi et al., 1986). Tiedink et at the highest dose (28 mg/kg bw per tistically significant]. I ndole-3-carbinol al. (1988) studied the potential of 30 day) (38/276 implantation sites had decreased both markers significantly vegetables to form biologically active dead or resorbed fetuses, compared and in a dose-dependent manner, N-n itroso compounds after treatment with 15/264 in the control group, and even at the lowest dose tested. with nitrite under acidic the average number of live pups per lit- Furthermore, the time courses of conditions. Although all the treated ter was 9.92 versus 11.3), although no serum luteinizing hormone, follicle- extracts contained N-nitroso com- statistical analysis of the data was pre- stimulating hormone and progesterone pounds, only half of the vegetables sented. were significantly decreased by indole- were mutagenic after nitrite treatment. Groups of pregnant Wistar rats 3-carbinol, but these end-points were Extracts of cru ciferous vegetables were given allyl-ITC in corn oil by gav- tested only at the highest dose. showed the highest mutagenic effect age at 0, 60 or 120 mg/kg bw per day Groups of juvenile (12-18-month- with respect to the number of on day 12 or 13 of gestation. Despite old) rainbow trout were given diets Salmonella revertants induced; how- severe maternal toxicity at the higher containing indole-3-carbinol (providing ever, there was no significant dose, no adverse effect on the fetuses five doses between 25 and 2000 correlation between the level of glu- was reported (Ruddick et al., 1976). mg/kg bw per day) or 3,3-diindolyl- cosinolates in these vegetables and Groups of pregnant Sprague- methane (2.5-250 mg/kg bw per day) the number of revertants. Subsequent Dawley rats were given indole-3- or control feed for 2 weeks (Shilling et detailed studies on the role of glucosi- carbinol at a dose of O (vehicle, corn al., 2001). 3,3'-Diindolylmethane signif- nolates in the formation of N-nitroso oil:acetone 19:1), 1 or 100 mg/kg bw icantly increased vitellogenin produc- compounds showed that only indole on day 15 of gestation (Wilker et al., tion in the liver in a dose-dependent glucosinolates are involved in the 1996). Each observation group con- manner, showing saturation at around formation of mutagenic agents (Tiedink sisted of three to five litters of male off- 25 mg/kg bw per day. In an additional et al., 1991). Nevertheless, the contri- spring, standardized at 10 per litter experiment, the effect was shown to be bution of these glucosinolates to the after whelping. Ano—genital distance additive to that of estradiol. mutagenicity of nitrite-treated crucifer- and crown—rump length were ous vegetables appears to be negligi- decreased significantly at the higher ble. For instance, although Brussels dose on day 1 after birth. Several Cytotoxicity, genotoxicity and sprouts are one of the richest sources markers of sperm count and abnor- mutagenic and related effects of indolyl glucosinolates (6.6 pmol/g of malities were affected on day 62 after dry weight) they induced only 61 ± 2 birth at either or both doses, including revertants of S. typhimurium TA1 00 per daily sperm production and epididymat Cruciferous vegetables 25 mg of dry matter, whereas radish, transit time. Plants are known to contain a variety with an i ndolyl-g lucosi no late concen- Groups of 6-8 weaned female of bioactive substances with toxic tration of 1 pmolig of dry weight, Sprague-Dawley rats, 23 days of age effects that may be comparable to induced 233 ± 63 revertants per 25 mg (weighing 55-60 g) were given indole- or greater than those of synthetic of dry matter (Tiedink et at, 1988). 3-carbinol (0.05, 0.5, 1 or 1.5 gfkg bw compounds (Ames et al., 1990a,b). Tiedink et al. (1990) found that the indi- per day), 3,3-diindolylmethane (0, Thus, some studies have shown vidual contribution of indole-3-acetoni- 100, 200 or 400 mg/kg bw per day) or that cruciferous vegetables are cyto- trile to the total mutagenicity of green vehicle (diniethyl sulfoxide) by gavage toxic, genotoxic and mutagenic. cabbage extracts treated with nitrite (Gao et al., 2002). lndole-3-carbinol at Wakabayashi et al. (1985) reported on was about 2%. The most abundant

198 Toxic effects

indole in the extract, indole-3-car- compounds, showed that 70-80% of amplitude swelling of the mitochondria, boxyaldehyde, was not mutagenic. the mutagenicity of the crude juice was disappearance of blobs with simultane- These studies therefore indicate that due to theisothiocyanate-containing ous appearance of microviili and con- the contribution of indole compounds portion. No correlation was found, centration of intermediate filaments in to the mutagenicity of cruciferous veg- however, between the mutagenicity of the juxtanuclear region were observed. etables treated with nitrite is negligible. the juices and their histidine or total These changes are characteristic fea- The mutagenic effect of nitrosated content. Charles et al. tures of apoptosis, and it has been indole compounds s not restricted to (2002) studied the cytotoxic effect of shown that isothiocyanates are potent certain groups of indoles. Eight indole Brussels sprouts, , red cab- inducers of apoptosis (see section 4). compounds, indole-3-acetonitrile, indolo- bage, broccoli and turnip towards Addition of excess GSH or cysteine 3-carbinol, indole-3-acetamide, indole- Chinese hamster ovary cells. The con- either abolished or diminished the 3-acetic acid, 3-methylindole, indole-3- centration of the crude extracts that cytotoxic effects of benzyl-ITC and aldehyde, indole-3-carboxylic acid and produced a 50% reduction in the num- allyl-ITC. The GSH and L-cysteine con- indole, tested in S. typhimurium strains ber of cells relative to the control was jugates of benzyl-ITC and allyl-ITC TA98 and TA100 and Escherichia coil < 10 td/ml of medium for broccoli and gave effects comparable to the parent WP2 uvrIPKM101 after nitrite treat- Brussels sprouts, 10-50 jil/mI of compounds, as a result of the ment at pH 3 were mutagenic in all medium for turnips and 50-100 gInil reversibility of the reaction between thi- three strains in the absence of meta- for cauliflower and red cabbage. In the oIs and isothiocyanates. In another bolic activation (at up to 0.5 mol/- same study, broccoli juice significantly study, in which the cytotoxic effect of plate), lndole-3-acetic acid had the induced chromosomal aberrations at a benzyl-ITC and phenethyl-ITC was strongest effect. Addition of a meta- concentration of 30 .il/ml of medium. investigated in NR50 BALB/c mouse bolic activation system decreased the Purified glucosnolates do not have 313 fibroblasts (0.016-0.02 mmol/ml), effect (Sasagawa & Matsushima, significant genotoxicity in vitro. heteropyknosis and vacuolization of 1991). None of the compounds was Chromosomal aberrations were the cytoplasm were observed (Babich mutagenic without nitrite. induced in Chinese hamster ovary et al., 1993). Intravesicular instillation As the effect of vegetable extracts cells by sinigrin and at of benzyl-ITC and allyl-ITC (2.8 mg/kg not treated with nitrite was rot investi- concentrations > 2 mg/ml of medium bw) or the same molar quantity (37 gated in these studies, it is not clear (Musk et al., 1995a). mol/kg bw) of benzyl-ITC metabolites whether other bioactive substances in conjugated either with GSH, cys- cruciferous vegetables contributed to Isoth iocyanates teine—glycine, cysteine or mercapturic the observed effect. The genotoxicity Isothiocyanates are biologically reac- acid to the urinary bladder of Fischer and mutagenicity of eight cruciferous tive compounds as a result of the 344 rats caused cytotoxicity. The effect vegetables (Brussels sprouts, white highly electrophilic central carbon of benzyl-ITC was greater than that of cabbage, cauliflower, green cabbage, atom of their —N=C=S group. They allyl- ITC. The benzyl- ITO metabolite , broccoli, turnip and black react with oxygen-, sulfur- or nitrogen- mercapturic acid, which is thought to radish) was tested in the Salmo- centred nucleophiles to give rise to be the main final metabolite, had less neila/microsome test, in the differential thiocarbamates, dithiocarbamates or effect than the other metabolites DNA repair assay with E. coli and in a thiourea, respectively. (Masutomi et al., 2001). test for chromosomal aberrations in Studies on the cytomorphological The genotoxic and mutagenic Chinese hamster ovary cells (Kassie changes induced by benzyl-ITC—GSH, effects of the predominant isothio- etaL, 1996). juice had (10 .tmol/ml of medium) and allyl- cyanates contained in cruciferous veg- the strongest effect in all three assays, ITC—GSH (25 .imol/ml) in RL-4 rat etables are presented in Table 65. and exogenous metabolic activation hepatocytes showed that these com- Ethyl-, n-butyl-, tert-butyl-, allyl-, ben- was not required for induction of the pounds cause considerable toxicity, zyl- and cyclohexyl-ITCs were muta- genotoxic and mutagenic effects. characterized by blebbing of the cells genie to S. typhimurium TA100 after Addition of Arochlor-induced rat liver (Bruggeman et al., 1986; Temmink et 1 In of preincubation at 37 C microsomes reduced the mutagenicity al., 1986). At higher concentrations, (Yamaguchi, 1980). AIlyl-ITC had the of the juices to S. typhimurium TA100 distinct patches of dense heterochro- strongest effect. Sinigrin, the parent by 20-50%. Tests carried out with two matin in the nucleus, complete degran- glucosinolate of aIlyI-ITC, was muta- fractions of the juices, one containing ulation of the endoplasmic reticulum, genic to the same degree as allyl- isothiocyanate and the other phenolic disappearance of polysomes, high- ITO. Addition of exogenous metabolic

199 Table 65. Genotoxic and mutagenic effects of isothiocyanates (ITC5)

lsothio- Concentration Genetic effect Remarks Reference cyanate or dose Allyl- 100 glplate Increase in number of his' Styphirnurlum TA1 00 S9 mix did not influence mutagenicity; weak Yamaguchi (1980) [[C revertants effect in TA98 but no effect in TA1 535, TA 1536, TA1 537 orTAt538

6.05-500 Not mutagenic in S. typhirnuriurn TA1 00 or TA98 No effects with or without S9 mix Kasarnaki at al. pg/p late (1982)

5 nmol/ml Significant increase in structural and numerical Colonies consisting of a pile of small cells medium chromosomal aberrations in Chinese hamster B241 cells were observed; SO mix did not influence the result 0.08-0.5 il/plate Fivefold increase in number of his S. typhirnuoum Mutagenic effect observed after proincubetion Neudecker & TAIOO revertants for 2 h; mutagenïcity only upon addition of S9 Henscbler (1985) mix

0.2-3 Itg/ml Did not induce chromosomal aberrations or sister Parent glucosinolate, sinigrin, induced Musk & Johnson medium chromatid exchange in Chinese hamster ovary cells; no chromosomal aberrations at 4.6 mg/mi (1993); Musk et at effect in SV40-transformed Indian muntjac cells medium (1995a)

12-200 .19/plate Marginal increase in number of S. typhim or/urn TAI 00 SO mix decreased effect Kassie & and TA98 revertants Knasm011er (2000)

1-25 kg/plate Dose-dependent increase in reparable DNA damage in Genctoxicity attenuated by rat liver S9 mix, rat E. coil 343/753 (uvrB/recA) liver homogenate, bovine serum albumin, human saliva, vitamins E and C, b-carotene and sodium benzoate

0.2-4 pg/nI Induction of micronucleus formation in human-derived Allyl-ITC less potent than phenethyl-ITC medium HepG2 cells

90-270 mg/kg Induction of reparable DNA damage in E. coli 343/753 Significant effect in liver, lung, colon, kidney bw (uvr31recA) in host-mediated assay in mice and stomach

0.5-5 mol/nil Increased formation of 8-oxo-78-dihydro-2 -deoxy- No effect in hydrogen peroxïde-resistant Murata et ai. medium guanosine in calf thymus DNA and human leukaemia leukaemic cells; genotoxicity of llyl-ITC (2800) cell line; increased DNA damage in P53 tumour stronger than that of benzylITC or phenethyl- suppressor gene and c-Ha-ras-1 proto-oncogene ITC

10-50 nmollml Induction of DNA strand breaks in human-derived Synergistic effect with bonzo[a]pyrene LJbI et ai, (2003) medium HepG2 cells

Benzyl- 100 pg/plate Increase in number of his* S. typh/muriurn TAI 00 S9 mix did not influence mutagenicity; weak Yamaguchi (1980) ITC revertants effect in TA98 but no effect in TA1 535, TA1 536, TA1 537 or TA1 538

0.22-0.88 pg/mI Dose-related increase in his* S. typh/muourn TAIOO Musk & Johnson medium revertants (1993) Table 65 (contd)

Isothio- Concentration Genetic effect Remarks Reference cyanate or dose 0.3-1.2 pg/mI Dose-dependent induction of chromosomal Frequency of sister chromatid exchange Musk et at (1995b) medium aberrations and sister chromatid exchange in much lower than that of chromosomal Chinese hamster ovary cells aberrations

1-25 pg/mI Dose-dependent increase in DNA strand Loss of comet heads at highest medium breaks in Chinese hamster ovary cells concentration

12-200 g/plate Marginal increase in number of S. typhimurium so mix decreased effect Kassie et at (1999) lAi DO and TA98 revertants

0.5-5.5 ig/ml Dose-dependent increase in reparable DNA Effect attenuated by rat liver S9 mix, rat medium damage in E. coil 343/753 (uvrB/recA) liver homogenate, bovine serum albumin, human saliva, vitamins E and C, )3-carotene and sodium benzoate

0.2-4 pg/mI Dose-dependent induction of micronuclei in medium human-derived HepG2 cells

0.8-10 pg/mI Dose-dependent induction of DNA strand Genotoxic effect in gastric mucosa cells medium breaks in primary rat hepatocytes and gastric completely attenuated upon incubation of mucosa cells benzyl-ITC with gastric mucus

90-270 mg/kg Moderate induction of reparable DNA damage Similar effects in liver, kidney, lung, bw in E. coli 343/753 (uvrB/recA) in host-mediated stomach and intestine assay in mice

110-220 mg/kg Moderate increase in DNA strand breaks in rat Strand break maximal 1 h after bw gastric mucosa and colon in vivo administration; after 4 h, it reached level in untreated animals

0.5-2 preol/mI Increased formation of 8-0x0-7,8-d ihyd ro-2 Benzyl-ITC more genotoxic than Murata et al. (2000) medium deoxyguanosine in calf thymus DNA and pbenethyl-ITC but less gerotoxic than human leukaemia cell line; increased DNA allyl-ITC damage in P53 tumour suppressor gene and c-Ha-ras-1 proto-oncogene

2.5-10 nmoL/ml Dose-related increase in DNA strand breaks in Synergistically genotoxic with Kassie et al. (2003b) medium human-derived HepG2 cells benzo[a]pyrene at low concentrations

Pheriethyl- 0.44-1.3 pg/mI Dose-related increase in chomatid gaps and Musk & Johnson (1993); ITC medium rearrangements in SV40-transformed Indian Musk et al. (1 995a) rnuntjac cell line and increased frequency of chromosomal aberrations and sister chromatid 3' exchange in Chinese hamster ovary cells

U) Table 65 (Gontd)

Isothio- Concentration or dose Genetic effect Remarks Reference cyanate

i-OU 19/mi meaiun Marginal mutagenicity to S. t yphimurium Effect diminished by SO mix Kassie & TA100 and TA98 Knasmuller (2000) 1 25 jig/plate Dose-dependent increase in reparable Genotoxicity attenuated by rat liver S9 mix; rat DNA damage in E. coil 343/753 liver homogenate, bovine serum albumin, (uvrB/recA) human saliva, vitamins E and C, n-carotene and sodium benzoate

02-4 119/mi medium Induction of micronucleus fornaton in Allyl-ITC less potent than phenethyl-ITC human-derived HepG2 cells

90-270 mg/kg bw Non-significant induction of reparable DNA damage in E. coli 3431753 (uvrBlrecA) in host-mediated assay in mice

0.5-2 ilmol/mI medium Increased formation of 8-oxo-7,8-dihydro- Less genotoxic than a lyl-ITC or benzyl-ITC Murata eta]. 2'-deoxyguanosine in calf thymus DNA (2000) and human leukaemia oeil line; increased DNA damage in P53 tumour suppressor gene and c-Ha-ras-1 proto-oncogene

2.5-10 nmol/mI medium Dose-related increase in DNA strand Synergistically genotoxic with benzoja]pyrene Kassie et al. breaks in human-derived HepG2 cells at low concentrations (2003b)

Methyl- 12-200 pg/rn plate Marginal mutagenicity to S. typhimur/um Mutagenicity diminished by 89 mix Kassie et a/. ITC TA 100 and TA98 (2001)

0.5-4 g/ml medium Dose-dependent increase in reparable Genutoxioity attenuated by l­at liver S9 mix; DNA damage in E. coil 343/753 human saliva, bovine serum albumin and (uvrB/recA) gastric juice

0.5-4 jig/ml medium Dose-related increase in micronucleus frequency in human-derived HepG2 cells 3.6-5.4 jig/mI medium Dose-related induction of DNA strand breaks in human-derived HepG2 cells 90 mg/kg bw Moderate induction of reparable DNA Significant effect in liver damage in E. coli 343/753 (uvr6/recA) in host-mediated assay in nice

SO mix, 9000 x g supernatant of rodent liver Toxic effects activation did not change the effect. assays with benzyl-ITC, which weakly (1999, 2001) reported that The effect of allyl-ITC in S. induced DNA damage at doses of 110 benzyl-ITC, allyl-ITC, phenethyl-ITC typhimurium TA98 was weak, and no and 220 mg/kg bw, with a maximum and methyl-ITC induced thiobarbitu nc effect was seen in TA1535, TA1536, effect 1 h after exposure; by 4 h after acid-reactive substances, indicators of TA1537 or TA1538. In another study exposure, the damage was reduced to lipid peroxidation, in HepG2 cells (Neudecker & Henschler, 1985), allyl- the level of that in untreated controls exposed to 0.5-4 .ig/ml of the com- ITC had clear mutagenic effects only (Kassie et al., 1999). The reduction in pounds for 1 h, the order of potency of after preincubation for 120 min. the effect of isothiocyanates in vivo induction being benzyl-ITC > allyl-ITC Although exogenous metabolic activa- could be due to non-specific binding to = methyl-ITC > phenethyl-ITC. In tion was a prerequisite for mutagenic- proteins, as witnessed by the pro- another study on the induction of reac- ity, an excess reduced the mutagenic nounced reduction in the mutagenicity tive oxygen intermediates, allyl-ITC potency. Other workers reported only and genotoxicity of the compounds induced a significantly higher level of borderline mutagenic effects of allyl- with addition of liver homogenate, an 8-oxodG in hydrogen peroxide-suscep- ITC (Eder et ai., 1980), benzyl-ITC, exogenous metabolic activation sys- tible human myelogenous leukaemia phenethyl-ITC and methyl- ITC (Kassie tem, bovine serum albumin or saliva. cells than their resistant counterparts et al., 1999; Kassie & Knasmüller, Moreover, radical scavengers might (Murata et al., 2000). in the same 2000; Kassie et at, 2001) in the stan- also contribute to the weak effect of the study, the authors compared induction dard test. In all these studies, addition compounds in vivo, as vitamin E and by allyl-ITC, benzyl-ITC and phenethyl- of an exogenous metabolic activation C, 13-carotene and sodium benzoate ITO of 8-oxodG in calf thymus DNA system abolished the effect almost reduced isothiocyanate-induced differ- and DNA damage in completely. Allyl-ITC was not muta- ential DNA damage in vitro. The effect 32P-labelled DNA fragments obtained genic in S. typhimuriumT498 orTAlOO of benzyl-ITC on gastric mucosa cells from the human P53 tumour suppres- in the presence or absence of meta- was drastically diminished by incuba- sor gene and the c-Ha-ras-1 proto- bolic activation (Kasamaki et al., tion of the compound with gastric oncogene. All the isothiocyanates 1982). mucus, corroborating the result caused Cu[ll]-mediated formation of Benzyl-ITC, allyl-ITC, phenethyl- observed in the differential DNA repair DNA damage and 8-oxodO, the order ITC and methyl-ITC induced reparable assay with E. co/i. of potency being allyl-ITC > benzyl-ITC DNA damage in E. coli in vitro and in The cytotoxic and genotoxic effects > phenethyl-ITC. Catalase and the vivo and DNA strand breaks and cyto- of isothiocyanates might be related to Cu[ll]-specific chelator bathocuproine, genetic effects in various mammalian their potential to induce formation of reduced 8-oxodG formation, suggest- cells (Kasamaki et al., 1982; Musk & reactive oxygen intermediates. ing the involvement of hydrogen perox- Johnson, 1993; Musk et al., 1995a,b; Treatment of rat liver epithelial (RL34) ide and Cu[l], respectively. Superoxide Kassie et al., 1999; Kassie & cells with benzyl-ITC or allyl-ITC (10 dismutase was also inhibitory, indicat- Knasm011er, 2000; Kassie et al., 2001, nmol/ml) resulted in an immediate ing participation of superoxide in the 2003b; Uhl et ai., 2003) (Table 65). increase in reactive oxygen intermedi- DNA damage. Benzyl-ITC was the most potent of all ates, which corresponded to induction Another possible mechanism of the the isothiocyanates. Al lyl-ITC was of class zGST pi (Nakamura et al., cytotoxicity and genotoxicity of isothio- more genotoxic than phenethyl-ITC in 2000b). Depletion of GSH by diethyl cyanates may be oxidative desulfura- the bacterial assay, but the latter was maleate significantly increased the tien of the compounds to reactive iso- more potent in cytogenetic tests. In an production of isothiocyanate-induced cyanates by cytochrome P450 enzymes. assay for differential DNA repair with reactive oxygen intermediates (hydro- Arochlor-inducible cytochrome P450 E. co//in vivo, the pattern of genotoxic- gen peroxide, lipid hydroperoxide and enzymes convert phenyl-ITC, benzyl- ity was similar to that found in vitro, the peroxynitrite) and accelerated isothio- ITC, phenethyl-ITC and methyl-ITC to effect of phenethyl-ITC being the cyanate-induced GST activity, while the respective isocyanates (Lee, 1992, weakest. The doses of isothiocyanates treatment of cells with GSH inhibited 1996). Phenyl-ITC underwent the most required to induce moderate genotoxic both reactions. Whereas benzyl-ITC metabolic conversion, followed by ben- effects in mice were much higher was the most potent inducer of zyl-ITC and phenethyl-ITC; methyl-ITC (90-270 mg/kg bw) than those reactive oxygen intermediates, the was only weakly metabolized. Iso- required for the highly cytotoxic and effect of allyl-ITC was intermediate, cyanates are reactive electrophilic genotoxic effects in vitro. A similar and phenyl-ITC did not induce these agents capable of modifying nucleic observation was made in comet compounds. Kassie and colleagues acids in vitro and in vivo and cause

203 IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles chromosome aberrations, sister chro- Reduction of ingested nitrate to nitrite genesis, the level of gap junction pro- matid exchange, mutations and cancer by bacteria in the oral cavity and reac- teins is generally reduced, and (Mason et a/., 1987; Bucher etal., 1989). tion of nitrite with ingested vegetables Herrmann et al. (2002) found inhibition Exposure of Chinese hamster at the acidic pH of the stomach may of gap-junction intracellular communi- B241 cells to allyl-ITC at 5 pmol/ml for lead to formation of mutagenic indole cation between primary rat hepato- 24 h and further cultivation for genera- compounds, which may be risk factors cytos co-cultured with a rat liver epithe- fions caused transformation of the for stomach cancer. To shed light on liai cell line, WB-F344, in response to cells (Kasamaki et al., 1987). Sub- the possible association between the indolo[3,2-b]carbazole at 0.1 nmol/ml sequent isolation and subcutaneous risk for stomach cancer and exposure for 8 or 12 h, with maximum inhibition injection of the anchorage-indepen- to nitrosated indoles, rats were given after 24-48 h. Both plasma membrane dent cell population resulted in tumour high doses of these compounds, and staining and the mRNA levels of con- formation after 3-8 months. In another effects on the stomach mucosa were nexin 32 were reduced. study, phenethyl-ITC failed to trans- examined. 1 -Nitrosoindole-3-acetoni- Prostaglandin E2 is believed to form BALB/c 3T3 cells but enhanced true, a nitrosated form of indole-3-ace- contribute to formation of tumeurs by the cell transforming potential of tonitrue, at 100 mg/kg bw caused the increasing cell proliferation, preventing benzo[ajpyrene (Perocco et aI., 2002). formation. of DNA adducts in the apoptosis and facilitating angiogene- forestomach and glandular stomach sis, particularly in the colon. In a study In do les (Yamashita et al., 1088), whereas of the regulation of prostaglandin E2 in With the exception of one report in indole-3-acetonitrile did not. 1-Nitro- a colon carcinoma cell line, HCA7, which indole-3-carbinol was found to soindole-3-acetonitrile also induced Sherratt et al. (2003) found that be cytotoxic to BALB/c 3T3 mouse replicative DNA synthesis and orni- indole[3,2-b]carbazole at 1 nmol/ml for fibroblasts at a concentration of 1 thine decarboxylase activity in the gas- 6 h increased COX-2 mRNA by 2.8- nmol/ml (Babich et a/., 1993), none of trie mucosa of rats treated at doses of fold. Co-treatment with interleukin-1 3 the studies of indole compounds con- 40-300 mg/kg bw (Furihata et al., increased the mRNA levels even fur- tained in cruciferous vegetables has 1987), suggesting that the compound ther. Subsequent increases in pros- shown them to be genotoxic or muta- has tumour promoting activity. taglandin E2 were also observed. genie. After treatment with nitrite, how- lndolo[3,2-b]carbazole has some The Working Group concluded that ever, most indole compounds become structural and functional properties in exposure to indole compounds as a mutagenic. The first report on the common with the tumour promoter result of consumption of cruciferous mutagenicity of nitrite-treated indole- 2,3,7,8-tet rac h lorodi ben zo-para-di oxi n vegetables does not appear to have compounds was that of Wakabayashi (TCDD), and some of the cellular adverse genetic effects. et al. (1985). changes induced by this indole in vitro Vegetables are the main sources of might favour tumour promotion rather dietary nitrate (Meah et aI., 1994). than chemoprevention. During carcino-

204