CHEMOPREVENTION BY ISOTHIOCYANATE: THE IN VIVO EVIDENCE

VI. CHEMOPREVENTION BY ISOTHIOCYANATE: THE IN VIVO EVIDENCE

One of the major characteristics of chemical carcinogens is their ability to act as DNA alkylating agents inducing DNA aberrations that have the potential to lead to genetic mutations. Several well-characterized carcinogens, such as

benzo[a]pyrenes, nitrosamines, and aflatoxin B 1 , can form DNA adducts with exposed bases in double-stranded DNA (Fig. 2). The overall consequence of this is the possible impairment of DNA replication and the induction of genetic mutation. This can be especially significant if these occur in tumor suppressor genes such as p53. A possible mechanism to prevent these deleterious effects is to use dietary agents that can alter the metabolism of carcinogens and thus reduce or prevent DNA damage. Of the many dietary compounds studied, low-dose exposure to ITCs has been proven to be highly effective in preventing DNA damage and the development of cancers in animal models (reviewed in Refs. 2,7 and summarized in Table 2). Recent experiments have focused on the inhibition of tumor formation induced by several nitrosamines and polycyclic aromatic hydrocarbons found in tobacco smoke. Of the approximately by 4000 compounds identified 43 have the potential to induce tumor formation in animal models (reviewed in Ref. 63). Two of the most effective carcinogens identified were the polycyclic aromatic hydrocarbons represented by benzo[a]pyrene and the nitrosamine 4-(meth- ylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Both compounds promote tumor formation in animal models. B[a]P and NNK are also suggested to be potent initiating factors in the induction of tobacco-related human cancers. Dose-dependent inhibition by arylalkyl ITCs and their N-acetylcysteine conjugates on NNK-induced tumor formation in rodents has been widely addressed; several of these studies are summarized in Table 2 (64–74). Common to many of these studies is a reduction in the deposition and an increase in Phase II detoxification metabolites of many of these carcinogens.

Cruciferous Vegetables and Chemoprevention 375

F IGURE 2 Metabolism, DNA adduct formation, and detoxification of the car- cinogenic agents aflatoxin B1 (AFB 1 ) and 4-(methylnitrosamino)-1-(3 pyridyl)-

1-butanone (NNK) in mammalian cells. (This figure is by no means compre- hensive. See Refs. 90,95.)

376 Rose et al. T ABLE 2 Summary of the In Vivo Chemopreventive Properties of Isothiocyanates

in Rodents Animal model

Isothiocyanate Effect Ref. A/J mice

Tissue

Carcinogen

Inhibition 64 forestomach A/J mice

Pulmonary,

B[a]P

Aromatic Inhibition 65

ITCs

Inhibition 66 ACI/N rats

Fisher rats Esophagus

NBMA

PEITC

Inhibition 67 Fischer rats

BITC/PBITC No effect 68 A/J mice

Esophagus

NBMA

Inhibition 69 Sprague-

Inhibition 70 Dawley rats

tissue Fischer rats

Inhibition 71 Fischer rats

Sulforaphane Inhibition 85 Rats

BITC/PEITC Promotion 51

BBN

Promotion 73 liver A/J mice

Fischer rats Bladder and

B[a]P and

BITC/PEITC Inhibition 74

NNK

ICN mice Stomach

Sulforaphane Inhibition 46 A/J mice

B[a]P

Inhibition 83 BITC, benzyl isothiocyanate; PEITC, phenylethyl isothiocyanate; PPITC, phenylpropyl isothio-

Lung

B[a]P

2BITC

cyanate; PBITC, phenylbutyl isothiocyanate; sulforaphane, 4-methylsulfinylbutyl isothiocyanate; AOM, azoxymethane; B[a]P, benzo[a]pyrene; DEN, 1, 2-diethylnitrosamine; DMBA, 7,12-di- methylbenzanthracene; MAM, methylazomethanol acetate; NNK, 4-(methylnitroamino)-1-(3- pyridyl)-1-butanone; NBMA, N-nitrosobenzylmethylamine.

PEITC and BITC, both prominent isothiocyanates in watercress, garden cress, and papaya, have been the focus of much of this attention. PEITC can inhibit tumor formation induced by NNK in both rat and mouse models but it is ineffective at inhibiting tumor formation induced by B[a]P (75–79). In contrast, BITC can inhibit tumor formation induced by B[a]P while having no inhibitory effect on NNK (79–83). These contrasting differ- ences are currently under investigation in several laboratories, and suggest that for effective chemoprevention a combination of ITCs or other phyto- chemicals may be necessary in preventing tumor formation. Additional research also shows both PEITC and BITC can inhibit DMBA-induced mammary tumors in rats, with PEITC being additionally effective in the inhibition of N-nitrosbenzylmethylamine (NBMA)-induced esophageal tumors (66,68–72). Likewise, sulforaphane, a very potent Phase II detoxfica-

Cruciferous Vegetables and Chemoprevention 377

tion enzyme inducer from broccoli, can inhibit DMBA-induced tumor formation in rat mammary tissues while also showing a protective effect against azoxymethane (AZO)-induced aberrant crypt foci in rats (84,85). However, it is ineffective against B[a]P-induced lung tumor formation in A/J mice (74).

Of all the in vivo studies only a few have been conducted on human subjects, many of these being coordinated by Hecht and colleagues. In humans, it is hypothesized that PEITC prevented the metabolic activation of carcinogens to more toxic forms. During the early investigation it was demonstrated that consumption of watercress in smokers significantly in- creased the levels of the detoxification products of the NNK metabolite NNAL and NNAL-Gluc in the urine (86). It was suggested that this effect was either due to the inhibition of NNK metabolism by CYP450 isoenzymes or due to the induction of Phase II detoxification enzymes involved in their excretion. Follow-up studies determined the latter to be the influencing factor with data suggesting that components in watercress were inducing the phase

II detoxification enzyme UDP-glucuronosyltransferase (87). Many of the in vivo traits of synthetic isothiocyanates are also observed for the respective vegetables from which they are derived. Cabbage, brussels sprouts, and broccoli have all been shown to reduce mammary tumor formation in rats exposed to DMBA (82). More recent investigations have shown that selenium-enriched broccoli is also effective at reducing intestinal tumor formation in mice exposed to dimethylhydrazine (88). Additional studies using multiple intestinal neoplasia mice as a rodent model in which the rodents are predisposed to the development of tumors in the small and large intestine show a significant reduction in the numbers of tumors when the rodents are fed selenium-enriched broccoli (89). Whether an interaction between GSL components and methylselenocysteine (the predominant seleno- compound in plants) can occur in vivo has not been determined; however, both agents do have chemopreventive properties and such effects are feasible. Nevertheless this requires further investigation.

Aflatoxin B 1 (AFB 1 ), a potent hepatocarcinogen, is recognized by the International Agency for Research on Cancer (IARC) as a group 1carcin- ogen in humans. It is generally accepted that the formation of AFB 1 -8,9- epoxide generated by the metabolism of AFB 1 by cytochrome P450 can cause the covalent binding of the epoxide to guanine bases present in DNA. More important is the finding that AFB 1 epoxide is able to induce mutations in the tumor suppressor gene p53 and K-ras oncogenes. These factors are suggested to contribute to AFB 1 -induced carcinogenesis (reviewed in Ref. 90). The first study showing the modulation of AFB 1 -DNA adduct formation by Brassica vegetables was conducted by Godlewski et al. In feeding studies GSL- containing fractions of brussels sprouts were shown to diminish the number

378 Rose et al.

of g-glutamyl transpeptidase foci induced by AFB1in hepatic tissues, a marker of hepatocarcinoma (91). Additional research indicates that the consumption of brussels sprouts had a pronounced inhibitory effect on aflatoxin B

1 DNA binding in rats. Rodents administered H-AFB 1 either intraperitoneally or intragastrically, showed a 50–60% reduction in AFB 1 DNA binding in hepatic tissues (92). Likewise, consumption of cabbage

induced an 87% reduction of AFB 1 DNA binding in weaning Fischer F344 rats (93). Associated with all three studies was a significant increase in de- toxification enzyme activity in hepatic tissues. These findings could be at- tributed to the presence of unidentified GSL hydrolytic products in the vegetable extracts. Supporting this hypothesis is the study by Hayes et al. showing that ITCs can induce GSTA5-5, a major GST involved in the de- toxification of AFB1-epoxide (94,95).