THE METABOLISM OF DIETARY ISOTHIOCYANATES IN VIVO

VII. THE METABOLISM OF DIETARY ISOTHIOCYANATES IN VIVO

The first step in the metabolism of dietary or synthetic ITCs involves their conjugation with the nucleophilic sulfhydryl group of glutathione in the mercapturic acid pathway (Fig. 3) (96). After GSH conjugation the resultant ITC conjugates pass through a series of catabolic steps mediated by the enzymes g-glutamyltranspepsidase, cysteinylglycinase, aminopeptidase, and N -acetyltransferase to the final mercapturic acid metabolite excreted in urine, the N-acetylcysteine-S or, alternatively, S-N-(thiocarbomylisothiocyanate)- L -N-acetylcysteine conjugates. Several in vivo studies in A/J mice and rats have identified N-acetylcysteine conjugates of AITC, BITC, sulforaphane, and PEITC in urine, after feeding with either synthetic or plant-derived ITCs (these data are summarized in Table 3). Common to all these studies is the rapid rate at which ITCs are detoxified and excreted in the urine; typically between 30 and 80% of the total ingested ITCs is excreted within 24 hr.

Disposition and pharmacokinetic studies using 14 C PEITC and 14 CPHITC in male Fischer F344 rats addressed the effects of tissue distribution on the excretion of ITCs. High localization of PEITC within the liver, lungs, and blood was observed with 88.7% of the administered dose being excreted in the urine and feces within 48 hr. In contrast, PHITC, a structural analog of PEITC (increased chain length), showed greater retention within the liver, lungs, and blood with only 7% of the dose being excreted in the urine and 47% in feces during 48 hr. The observed retention of PHITC may explain its greater efficacy at inhibiting NNK-induced lung tumor development in rodents. The data also suggest that, unlike PEITC, PHITC may also be me- tabolized via a different route, as large quantities are eliminated in the feces (105).

Cruciferous Vegetables and Chemoprevention 379

F IGURE 3 Detoxification of isothiocyanates occurs through the mercapturic acid pathway. The initial conjugation reaction mediated by GSTs allows for the subsequent catabolic degradation of S-(N-thiocarbomylisothiocyanate)-L-glu- tathione intermediate to its N-acetylcysteine derivative. NAC conjugates are routinely used for measuring isothiocyanates in urine during epidemiological and/or feeding experiments (see text).

In humans the principal urinary metabolites of ITCs are the N-acetyl- cysteine derivatives (100,102). Quantification of these has been aided by the development of the 1,2-benzenedithiol derivatization assay developed by Zhang et al. (110). Indeed, this method has often been adopted for use in feeding and epidemiological studies (60,61). In determining the role of myr- osinase in the bioavailability of ITCs several studies have addressed the microbial metabolism of GSLs in humans. Getahun and Chung fed human subjects watercress with active and inactivated myrosinase and measured the urinary excretion of the N-acetylcysteine conjugates in urine (111). Con- sumption of 150 g of fresh watercress with active myrosinase resulted in the excretion of 17–77% of the administered dose of ITCs in the urine. In contrast, individuals consuming 350 g of watercress with inactivated myr- osinase showed a significant reduction to only 7% of the administered dose at

24 hr. In separate experiments these investigators demonstrated the ability of human fecal samples to hydrolyze GSLs to their respective ITCs, with 18% of

380 Rose et al.

T ABLE 3 Summary of the Metabolic Studies Conducted Using Dietary-Derived and Synthetic Isothiocyanates In Vivo

Glucosinolate or Species

Site of investigated

isothiocyanate

Metabolites

detection Ref. Rat, dog

studied

identified

BITC and its

Urine 97 mercapturic

NAC conjugate

Feces acids Rat

Urine 98 BTITC, AITC Rat

MITC, EITC,

NAC conjugate

BITC, AITC,

Urine 99 MITC, EITC, BTITC

NAC conjugate

Human BITC from

Urine 100 garden cress A/J mice

NAC conjugate

PEITC

4-Hydroxy-4-carboxyl-3-

Urine and 101 phenylethylthiazolidine-

tissues

2-thione and NAC conjugate

Human PEITC from

Urine 102 watercress Human

NAC conjugate

Urine 103 brown mustard Fischer F344

AITC from

NAC conjugate

Urine, feces, 104 rats, B6C3F1

AITC

Rat; NAC conjugate

expired air mice Fischer

Mouse; -SCN ions

Urine 106 F344 rats

Dose of

NAC conjugate

cauliflower, sinigrin, or AITC

Human PEITC from

Urine 111 watercress Fischer

NAC conjugate

Urine, tissues, 105 F344 rats

PEITC and PHITC

N/D

and expired air

Human Sulforaphane

Urine 106 from broccoli Human

NAC conjugates

Urine 107 containing ITCs Human

Broccoli sprouts

NAC conjugate

Plasma 108 PEITC Human

Single dose of

N/D

Broccoli sprouts

Urine, plasma, 109 containing ITCs

N/D

serum, erythrocytes

AITC, allyl isothiocyanate; BITC, benzyl isothiocyanate; BTITC, butyl isothiocyanate; EITC, ethyl isothiocyanate; MITC, methyl isothiocyanate; PEITC, phenylethyl isothiocyanate; sinigrin, 2-propenyl

Cruciferous Vegetables and Chemoprevention 381

the total GSLs being degraded with 2 hr. These data implicate colonic bacteria in the bioavailability of ITCs in the human diet. Further investiga- tions by Shapiro et al. also demonstrated that heat inactivation can reduce the levels of ITCs metabolites in urine. In subjects consuming cooked broccoli only 10–20% of the ITCs were excreted compared to those consuming 47% in myrosinase-treated broccoli in which most of the GSLs had been converted to their respective ITCs. Furthermore, removal of colonic bacteria in subjects using antibiotic treatments almost eliminated the detection of these urinary metabolites (107). These data highlight the complex nature of the bioavail- ability of ITCs in the diet with a reliance on both endogeneous plant and microbial myrosinase.

VIII. THE MAMMALIAN DETOXIFICATION SYSTEM Mammalian cells are continuously exposed to endogenous and exogenous

toxins, either as by-products of metabolism or as environmental agents. These compounds are usually highly electrophilic and disrupt normal cellular function by reacting with nucleophilic centers located in and on proteins and DNA. In the extreme case, DNA adducts can be formed that result in the formation of a neoplastic cell and subsequently a cancerous cell can develop. To prevent these deleterious effects the mammalian system has developed specific pathways to stabilize and subsequently excrete xenobiotics. These pathways rely on the expression and activity of several groups of proteins known as Phase I and Phase II detoxification enzymes, such as cytochrome P450s [EC 1. 14.14.1], glutathione-S-transferases [EC 2.5.1.18], quinone reductase [EC 1.6.99.2], and UGT-glucoronosyltransferases [EC 2. 4.1.17]. The coordinate regulation of the latter group is generally controlled through the same transcriptional mechanism ensuring that several different Phase II detoxification enzymes may be induced by a single xenobiotic insult (112,113).

The association between Phase II detoxification enzymes and cancer risk has been the focus of much study. Deficiencies as a result of genetic polymorphisms can often lead to increased susceptibility to toxins and chemically induced carcinogenesis. These factors are emphasized in the reported increased susceptibility of smokers null for GST M1, GSTT1, and GSTP1and additional associations with increased incidence of colon cancer, skin cancer, and ovarian cancers for individuals who are GSTM1-null (115– 119). One possible means to reduce cancer risk, representing the basic principle of chemoprevention, is to modulate the activities of cellular protec- tive enzymes using dietary supplements or dietary intervention. An increased consumption of cruciferous vegetables containing ITCs that can potentially stimulate the induction of Phase II detoxification agents may offer aid in improving human health.

382 Rose et al.

Inducers of Phase II detoxification enzymes are categorized in to two main groups based on the hypothesis first highlighted by Prochaska and Talalay (114). The first group, deemed bifunctional inducers, is comprised of chemical agents, such as polycyclic aromatics and h-napthoflavone, that induce gene expression either through the antioxidant responsive element (ARE) or xenobiotic-responsive element (XRE) present within the promoter region of many of these genes. In contrast, monofunctional inducers such as ITCs induce Phase II enzymes gene expression via the ARE. Of the several groups of enzymes studied the involvement and mechanisms for CYP450s, GSTs, and NQO1have been widely addressed and it is these enzymes that will

be the focus of discussion.