Waikato Medical Research Foundation
Humans are continually exposed to carcinogens, which are present in the atmosphere
and in our food, and which are produced in our bodies by the natural processes of
metabolism. But we do not all develop cancer, because we are protected against
carcinogens by a family of enzymes that convert them to unreactive metabolites, which
are readily excreted from the body. These are the Phase II detoxification enzymes,
and there is evidence from animal experiments that, after exposure to carcinogens,
cancer occurs only when these enzymatic defences are overwhelmed. If we could
increase the strength of our defences against carcinogens by increasing the levels of
these beneficial enzymes in our tissues, we could decrease the likelihood of
Bladder cancer is a major human health problem. In Western countries, it is the fourth
commonest cancer in men and the eighth commonest in women. Epidemiological
studies have shown that the incidence of bladder cancer is lower in individuals who
consume large amounts of Brassica vegetables, such as cabbage, cauliflower, broccoli
and Brussels sprouts. The protection against bladder cancer given by Brassica
vegetables is attributable to their ability to form compounds called isothiocyanates
when cut or chewed. These compounds are very effective inducers of Phase 2
enzymes in the urinary bladder, and we have shown that sulforaphane, an
isothiocyanate derived from broccoli, gives excellent protection against chemicallyinduced
bladder cancer in rats.
Isothiocyanates may thus be useful in protecting against bladder cancer in humans.
These compounds vary greatly in their inductive activity, however, and we need to
know which would be most effective. More than 120 isothiocyanates occur in nature,
and it would be very laborious to test all of these. An understanding of structure-activity
relationships would be valuable for selecting isothiocyanates most likely to be effective
for cancer chemoprevention.
We have previously shown that a methyl group adjacent to the isothiocyanate moiety
increases inductive activity, and that the way in which substituents are arranged
around the isothiocyanate group may also have a significant effect. In the present
study, we have compared the activities of a number of methylated and isomeric
isothiocyanates in inducing Phase 2 enzymes in rat tissues, and have confirmed the
effects of methylation and stereochemistry on inductive activity in certain tissues. This
work will help in understanding how isothiocyanates interact with the signalling
systems that trigger Phase 2 enzyme induction, and may lead to the development of
substances with exceptionally high activity, which would be of great value in cancer
There is evidence that the incidence of cancer in humans could be decreased by
changes in lifestyle, with dietary modifications being particularly important (1). The
recent report (2) by the World Cancer Research Fund/American Institute for Cancer
Research “Food, Nutrition, Physical Activity and the Prevention of Cancer: a Global
Perspective” emphasises the fact that individuals consuming diets high in fruit and
vegetables are less at risk of developing cancer than those with a relatively low intake
of these foodstuffs.
Most human cancers result from the irreversible interaction of cancer-causing
chemicals (carcinogens) with DNA. We are continually exposed to carcinogens in our
food and in the atmosphere, while others are produced within our bodies as normal
products of cellular metabolism. We do not all develop cancer, however, since we
have efficient mechanisms for detoxifying carcinogens, a process that involves the so called Phase II detoxification enzymes. These enzymes, which include NAD(P)Hquinone reductase (NQO1), glutathione S-transferase (GST), epoxide hydrolase and glucuronosyl transferase, convert carcinogens to non-toxic water-soluble materials that
are readily eliminated from the body (3, 4). If carcinogen detoxification is rapid,
irreversible damage to DNA is avoided, and cancer development prevented.
The efficacy of carcinogen detoxification is proportional to the activities of the Phase II
enzymes in tissues. In animal models, an inverse relationship between tissue levels of
Phase II enzymes and susceptibility to cancer has been observed (5). Furthermore,
genetic changes in humans, leading to deficiencies in Phase II enzyme activity, are
associated with increased risk of bladder cancer (6), lung cancer (7) colorectal cancer
(8) and leukaemia (9).
If Phase II enzyme activity in humans could be increased by practicable dietary
modifications, cancer incidence may be decreased. In work previously funded by the
Foundation, we have shown that isothiocyanates, which are compounds derived from
Brassica vegetables, increase tissue activities of Phase II enzymes in rats. The
greatest effect of isothiocyanates was observed in the urinary bladder (10-12),
suggesting that these substances, and the vegetables from which they are derived,
would be especially effective in protecting against bladder cancer. This is in accord with
epidemiological evidence (13) and we have shown that an isothiocyanate from broccoli
gave excellent protection against chemically-induced bladder cancer in rats (14).
Bladder cancer is an important problem.
In Western countries, it is the fourth commonest cancer in men and the eighth commonest in women (15). Treatment for bladder cancer continues to improve, but recurrence occurs in 60-75% of patients (16), so that repeated diagnostic evaluation and therapy are required, resulting in considerable patient discomfort and economic burden. The median age of first diagnosis of bladder cancer is 69 for men and 71 for women (17). The life expectancy of men in New Zealand is 78 years, and that of women 82 years (18) so that if the onset of disease could be delayed by 10 years by use of chemopreventative agents,
the incidence of bladder cancer would be greatly diminished. Isothiocyanates from
vegetables could play a major role in this process.
More than 120 different isothiocyanates have been shown to be present in Brassica
vegetables (19), but only a small number have been tested for their ability to increase
tissue activities of Phase 2 enzymes. Previous studies have shown that there are very
large variations in the inductive activity of different isothiocyanates, and that certain
aspects of the chemical structure of these compounds have a major influence on their
We have previously shown (20) that a methyl group on the carbon atom adjacent to
the isothiocyanate group increases inductive activity. For example, allyl isothiocyanate
[I] at a dose of 250 moles/kg/day for 5 days caused a 5.5-fold increase in NQO1 in
the urinary bladder, while the methylated compound 1-methylallyl isothiocyanate (II)
increased the activity of this enzyme by a factor of 7.8. We have also shown that cyclohexylmethyl isothiocyanate (III) is a very good inducer
of Phase 2 enzymes in the urinary bladder, and it would be expected that the
compound substituted with a methyl group on the carbon next to the isothiocyanate
group would be even better. This compound is 1-cyclohexylethyl isothiocyanate, which
exists in 2 isomeric forms, the R-(-)-enantiomer (IV), in which the methyl group lies
below the plane of the ring and the S-(+) enantiomer (V), in which it lies above the ring.
The results of a preliminary experiment with these two enantiomers were rather
surprising. The R-(-)-isomer was a better inducer than cyclohexylmethyl
isothiocyanate, as expected. But the S-(+) isomer was a much weaker inducer, and
less effective than the un-methylated compound.
In the present study, we have examined several more substituted isothiocyanates for
their effects on Phase 2 enzyme activity in various tissues of the rat.
Six female Sprague-Dawley rats, 10-12 weeks old at the start of the experiment, were
dosed by gavage with the isothiocyanates listed below at 250 moles/kg/day for 5
days. On the sixth day, the animals were euthanased and tissues harvested for
determination of levels of NQO1 and GST.
Experiment 1. The two enantiomers of 1-phenylethyl isothiocyanate, compared with
benzyl isothiocyanate, which lacks the methyl group next to the isothiocyanate moiety.
Experiment 2. The two enantiomers of 1-phenylpropyl isothiocyanate, compared with
2-phenylethyl isothiocyanate, which lacks the methyl group next to the isothiocyanate
Experiment 3. The two enantiomers of 2-hexyl isothiocyanate, compared with 1-pentyl
isothiocyanate, which lacks the methyl group next to the isothiocyanate moiety.
Experiment 4. The two enantiomers of 2-heptyl isothiocyanate, compared with 1-hexyl
isothiocyanate, which lacks the methyl group next to the isothiocyanate moiety.
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