The metabolism of SFN is summarized in . The initial reaction involves enzymatic hydrolysis of glucoraphanin, the glucosinolate precursor of SFN, found in the plant. This reaction is catalyzed by myrosinase, a β-thioglucosidase, which cleaves the glycone from the glucosinolate forming glucose, hydrogen sulfate and one of many different aglycones (e.g. thiocyanate, ITC, or a nitrile) depending on the glucosinolate, reaction pH, and availability of ions [50
]. At neutral pH, the major glucosinolate hydrolysis products are stable isothiocyanates. After absorption, SFN is predominantly metabolized via the mercapturic acid pathway. In these reactions, the electrophilic central carbon of the -N=C=S group in SFN reacts with the sulfhydryl group of GSH to form a dithiocarbamate GSH conjugate. The enzymes that catalyze GSH conjugation to SFN are the family of GST enzymes, and polymorphisms in these enzymes have a significant impact on overall ITC metabolism (discussed below). Interestingly, SFN is also able to induce its own metabolism via induction of GSTs. The final steps in SFN metabolism is formation of SFN-Cys and ultimately SFN-NAC [51
The absorption and bioavailability of SFN is affected by several factors. The first factor involves the hydrolysis of SFN from glucoraphanin via myrosinase activity. This initial step is critical because only the ITC form is thought to be biologically active and exhibits the desired anticancer properties. Importantly, mammalian cells do not have endogenous myrosinase activity. Instead myrosinases are found in the plant or the gut microbial flora. In the cruciferous plant, the myrosinase enzyme is physically separated from the glucosinolate by the plant cell wall, but upon physical disruption through chopping, cutting, and/or chewing, the enzyme is released and the ITC is released. However, myrosinase is heat labile and thus cooking procedures can inactivate the enzyme and significantly reduce the bioavailability of SFN up to 3-fold [52
]. Another source of myrosinase activity is the intestinal microbial flora. Evidence from experiments done with isolated human fecal bacteria [53
] and others using F344 rats dosed ip with glucoraphanin, indicates that glucoraphanin can be converted to SFN by colonic microbial flora and that enterohepatic circulation is requisite for efficient metabolism [54
]. However, the bioavailability of SFN is six times less when metabolism of the glucosinolate to the ITC had not occurred prior to ingestion [55
], revealing a strong reliance on plant myrosinase activity, as opposed to the intestinal gut flora. Nonetheless, an important factor determining inter-individual SFN bioavailability is variability in the gut microbial flora when the non-hydrolyzed glucosinolate is consumed.
The last factor that affects SFN bioavailability is related to polymorphisms in phase 2 SFN metabolizing genes, such as GSTs, which play a significant role in determining the detoxifying ability of an organism. In general, GST enzymes catalyze the conjugation of GSH to electrophiles such as SFN. There are six different classes of GST isoenzymes; α, µ, π, θ, σ, and κ, and each functional unit is composed of two subunits. Each is designated by the abbreviated Roman capital of each Greek letter followed by a number indicating subunit composition (ex. GSTµ1=GSTM1). In general, the substrate specificity for the different isoenzymes overlaps but, specifically, each class has varying degrees of reactivity for different substrates. GST null genotypes are quite prevalent in the population, with up to 50% of people being GSTM1 null and 47% GSTT1 null.
The effect that GST genotype has on cancer development and chemoprevention is complex. In the context of high cruciferous vegetable intake, evidence is mounting in favor of a GST null genotype providing a protective effect against lung, colon, and breast cancers [56
]. Since GST activity plays a critical role in SFN metabolism and subsequent excretion, lower GST activity in individuals with GST polymorphisms could result in slower elimination and longer exposure to isothiocyanates after cruciferous vegetable consumption. For example, one study investigating a population in Singapore reported higher ITC excretion among GSTT1 positive individuals in comparison to GSTT1 null [58
], implying shorter exposure times to the potential beneficial metabolites of SFN for GSTT1 positive individuals. This finding corroborates with a later study in Singapore, indicating that GST null genotype coupled with high cruciferous vegetable intake reduced the risk of colorectal cancer [59
]. However, other studies have shown that the GSTM1 null genotype produced a slight increase in the area under time-concentration curve (AUC) for metabolite concentrations in plasma, a significantly higher rate of SFN metabolite excretion, and a higher percentage of SFN excreted 24 h after ingestion, indicating shorter retention times of SFN and its metabolites [60
]. In addition, in other studies, a GSTM1 null genotype had a non-significant increase in prostate cancer risk, while men in the high vegetable consuming group that have GSTM1 present exhibited the greatest reduction in cancer risk [1
]. These inconsistencies may be explained by differences in the predominant ITC consumed (3-butenyl-ITC and4-pentenyl-ITC versus SFN), the class of GST null genotype present (GSTT1 versus GSTM1), or the cancer of interest (colorectal versus prostate). Despite these conflicting results, the impact of polymorphism on nutrient bioavailability is an important area of research that will aid in our understanding of response variability to SFN and other phytochemicals in human populations.