An Evidence-based Approach to Phytochemicals and Other Dietary Factors

13 Isothiocyanates

Cruciferous vegetables, such as broccoli, cabbage, and kale, are rich sources of sulfur-containing compounds called glucosinolates. Isothiocyanates are biologically active hydrolysis (breakdown) products of glucosinolates. Cruciferous vegetables contain a variety of glucosinolates, each of which forms a different isothiocyanate when hydrolyzed (Fig. 13.1).¹ For example, broccoli is a good source of glucoraphanin, the glucosinolate precursor of sulforaphane (SFN), and sinigrin, the glucosinolate precursor of allyl isothiocyanate (AITC).² Watercress is a rich source of gluconasturtiin, the precursor of phenethyl isothiocyanate (PEITC), while garden cress is rich in glucotropaeolin, the precursor of benzyl isothiocyanate (BITC). At present, scientists are interested in the cancer-preventive activities of vegetables that are rich in glucosinolates (see Chapter 2), as well as individual isothiocyanates.³

Bioavailability and Metabolism

Myrosinase, a class of enzymes that catalyzes the hydrolysis of glucosinolates, is physically separated from glucosinolates in intact plant cells.4 When cruciferous vegetables are chopped or chewed, myrosinase can interact with glucosinolates and release isothiocyanates from their precursors (Fig. 13.1). Thorough chewing of raw cruciferous vegetables increases glucosinolate contact with plant myrosinase and increases the amount of isothiocyanates absorbed.⁵ Even when plant myrosinase is completely inactivated by heat, the myrosinase activity of human intestinal bacteria allows for some formation and absorption of isothiocyanates.⁶ However, the absorption and excretion of isothiocyanates is substantially lower from cooked than from raw cruciferous vegetables^5,7,8 (see the Food Sources section below). During metabolism, isothiocyanates are conjugated (bound) to glutathione, an activity that is promoted by a family of enzymes called glutathione S -transferases (GSTs), and further metabolized to mercapturic acids. These isothiocyanate metabolites can be measured in the urine and are highly correlated with dietary in-take of cruciferous vegetables.⁹ There is also some evidence that isothiocyanate metabolites contribute to the biological activity of isothiocyanates.^3,10

Fig. 13.1 Myrosinase-catalyzed hydrolysis of glucosinolates and the chemical structures of allyl isothiocyanate, benzyl isothiocyanate, phenethyl isothiocyanate, and sulforaphane.

Biological Activities

Effects on Biotransformation Enzymes Involved in Carcinogen Metabolism

Biotransformation enzymes play important roles in the metabolism and elimination of a variety of chemicals, including drugs, toxins, and carcinogens. In general, phase I biotransformation enzymes catalyze reactions that increase the reactivity of hydrophobic (fat-soluble) compounds, preparing them for reactions catalyzed by phase II biotransformation enzymes. Reactions catalyzed by phase II enzymes generally increase water solubility and promote the elimination of the compound from the body.11

Inhibition of Phase I Biotransformation Enzymes

Some procarcinogens (carcinogen precursors) require biotransformation by phase I enzymes, such as those of the cytochrome P450 (CYP) family, to become active carcinogens that are capable of binding DNA and inducing mutations. Inhibition of specific CYP enzymes involved in carcinogen activation inhibits the development of cancer in animal models.³ Isothiocyanates, including PEITC and BITC, have been found in animal studies to inhibit carcinogen activation by CYP enzymes.^12,13 Cell culture studies have also shown that SFN inhibits certain CYP enzymes.¹⁴ A small clinical trial in smokers found evidence that consumption of 170 g/day (6 oz/day) of watercress, which is rich in the glucosinolate precursor of PEITC, decreased the activation of a procarcinogen found in tobacco.¹⁵

Induction of Phase II Biotransformation Enzymes

Many isothiocyanates, particularly SFN, are potent inducers of phase II enzymes in cultured human cells.^2,14 Phase II enzymes, including GSTs, uridine diphosphate (UDP)-glucuronosyl transferases (UGTs), quinone reductase, and γ-glutamatyl cysteine ligase, play important roles in protecting cells from DNA damage by carcinogens and reactive oxygen species.¹⁶ The genes for these and other phase II enzymes contain a specific sequence of DNA called an antioxidant response element (ARE). Isothiocyanates have been shown to increase phase II enzyme activity by increasing the transcription of genes that contain an ARE.¹⁷ Limited data from clinical trials suggest that glucosinolate-rich foods can increase phase II enzyme activity in humans. When smokers consumed 170 g/day (6 oz/day) of watercress, urinary excretion of glucuronidated nicotine metabolites increased significantly, suggesting UGT activity increased.¹⁸ Brussels sprouts are rich in several glucosinolates, including precursors of AITC and SFN. Consumption of 300 g/day (11 oz/day) of Brussels sprouts for a week significantly increased plasma and intestinal GST levels in nonsmoking men.^19,20

Preservation of Normal Cell-Cycle Regulation

After a cell divides, it passes through a sequence of stages known as the cell cycle, before dividing again. Following DNA damage, the cell cycle can be transiently arrested to allow for DNA repair or, if the damage cannot be repaired, activation of pathways leading to cell death (apoptosis).21 Defective cell-cycle regulation may result in the propagation of mutations that contribute to the development of cancer. Several isothiocyanates, including AITC, BITC, PEITC, and SFN, have been found to induce cell-cycle arrest in cultured cells.²

Inhibition of Proliferation and Induction of Apoptosis

Unlike normal cells, cancer cells proliferate rapidly and lose the ability to respond to cell death signals that initiate apoptosis. Isothiocyanates have been found to inhibit proliferation and induce apoptosis in several cancer cell lines.3,22,23

Inhibition of Histone Deacetylation

In the nucleus of a cell, DNA is coiled around basic proteins called histones. In general, acetylation of histones by histone acetyl transferases pmakes DNA more accessible to transcription factors, which bind DNA and activate gene transcription. Deacetylation of histones by histone deacetylases restricts the access of transcription factors to DNA. Acetylation and deacetylation of nuclear histones is an important cellular mechanism for regulating gene transcription.24 However, the balance between histone acetyl transferase and histone deacetylase activities that exists in normal cells may be disrupted in cancer cells. Compounds that inhibit histone deacetylases can potentially suppress the development of cancer by inducing the transcription of tumor suppressor proteins that promote differentiation and apoptosis in transformed (precancerous) cells.²⁵ SFN and AITC metabolites have been found to inhibit histone deacetylase activity in cultured cancer cells.^10,26–28 Moreover, in vivo evidence for SFN inhibiting histone deacetylase came from a mouse model using prostate cancer xenografts.²⁹ In humans, histone deacetylase activity was inhibited in blood cells following ingestion of 68 g (1 cup) of SFN-rich broccoli sprouts.²⁹

Anti-inflammatory Activity

Inflammation promotes cellular proliferation and inhibits apoptosis, increasing the risk of developing cancer.30 SFN and PEITC have been found to decrease the secretion of inflammatory signaling molecules by white blood cells; these compounds have also been shown to decrease DNA binding of nuclear factor kappa B (NF-κB), a pro-inflammatory transcription factor.^31,32

Antibacterial Activity

Helicobacter pylori

Bacterial infection with Helicobacter pylori (H. pylori) is associated with a marked increase in the risk of gastric cancer.33 In the test tube and in tissue culture, purified SFN inhibited the growth and killed multiple strains of H. pylori, including antibiotic-resistant strains.³⁴ In an animal model of H. pylori infection, SFN administration for 5 days eradicated H. pylori from eight out of 11 xenografts of human gastric tissue implanted in immune-compromised mice.³⁵ However, in a small clinical trial, consumption of up to 56 g/day (2 oz/day) of glucoraphanin-rich broccoli sprouts for 1 week was associated with H. pylori eradication in only three out of nine patients with gastritis.³⁶ Further research is needed to determine whether SFN or foods rich in its precursor, glucobrassicin, will be helpful in the treatment of H. pylori infection in humans.

Disease Prevention

Cancer

Naturally occurring isothiocyanates and their metabolites have been found to inhibit the development of chemically induced cancers of the lung, liver, esophagus, stomach, small intestine, colon, and mammary gland (breast) in a variety of animal models.3,12 Although epidemiological studies provide some evidence that higher in-takes of cruciferous vegetables are associated with decreased cancer risk in humans,³⁷ it is difficult to determine whether such protective effects are related to isothiocyanates or other factors associated with consumption of cruciferous vegetables (see Chapter 2). Investigators have attempted to calculate human isothiocyanate exposure based on assessments of cruciferous vegetable intake and measurements of the maximal amounts of isothiocyanates that can be liberated from various cruciferous vegetables in the laboratory.³⁸ Case–control studies using this technique found that dietary isothiocyanate intakes were significantly lower in Chinese women³⁹ and US men⁴⁰ diagnosed with lung cancer than in cancer-free control groups.

Assessing dietary intake of cruciferous vegetables may not accurately measure an individual exposure to isothiocyanates, since other factors may alter the amount of isothiocyanates formed and absorbed (see the Bioavailability and Metabolism section above). Measuring urinary excretion of isothiocyanates and their metabolites may provide a better assessment of isothiocyanate exposure,^9,41,42 but few studies have examined the relationships between urinary isothiocyanate excretion and cancer risk. In a prospective study, Chinese men with detectable levels of urinary isothiocyanates at baseline were at significantly lower risk of developing lung cancer over the next 10 years than men with undetectable levels.⁴³ A case–control study found that urinary isothiocyanate excretion was significantly lower in Chinese women diagnosed with breast cancer than in a cancer-free control group.⁴⁴ In contrast, cruciferous vegetable intake estimated from a food frequency questionnaire was not associated with breast cancer risk in the same study.

Genetic Variation in Isothiocyanate Metabolism and Cancer Risk

GSTs are a family of phase II biotransformation enzymes that promote the metabolism and elimination of isothiocyanates and other compounds from the body. Genetic variations (polymorphisms) that affect the activity of GST enzymes have been identified in humans. Null variants of the GSTM1 gene and GSTT1 gene contain large deletions, and individuals who inherit two copies of the GSTM1-null or GSTT1-null gene cannot produce the corresponding GST enzyme.45 Lower GST activity in such individuals could result in slower elimination and thus longer exposure to isothiocyanates after cruciferous vegetable consumption.⁹ In support of this idea, several epidemiological studies found that inverse associations between isothiocyanate intake from cruciferous vegetables and the risk of lung cancer^{39,40,43,46,47} or colon cancer^48–50 were more pronounced in GSTM1-null or GSTT1-null individuals. These findings suggest a protective role for isothiocyanates that may be enhanced in individuals who eliminate them more slowly from the body.

Table 13.1 Total glucosinolate content of selected cruciferous vegetables 52

Vegetable (Raw)	Serving	Total Glucosinolates (mg)
Brussels sprouts	½ cup	104
Garden cress	½ cup	98
Mustard greens	½ cup	79
Turnip	½ cup	60
Cabbage, Savoy	½ cup	35
Kale	½ cup	34
Cabbage, red	½ cup	29
Broccoli	½ cup	27
Bok choy (pak choi)	½ cup	19
Watercress	½ cup	16

Sources

Food Sources

Cruciferous Vegetables

Cruciferous vegetables, such as bok choy, broccoli, Brussels sprouts, cabbage, cauliflower, horseradish, kale, kohlrabi, mustard, radish, rutabaga, turnip, and watercress, are rich sources of glucosinolate precursors of isothiocyanates.51 Unlike some other phytochemicals, glucosinolates are present in relatively high concentrations in commonly consumed portions of cruciferous vegetables. For example ½ cup of raw broccoli might provide more than 25 mg of total glucosinolates. Total glucosinolate contents of selected cruciferous vegetables are presented in Table 13.1.⁵² Some cruciferous vegetables are better sources of specific glucosinolates (and isothiocyanates) than others. Vegetables that are relatively good sources of some of the isothiocyanates that are currently under study for their cancer-preventive properties are listed in Table 13.2. Amounts of isothiocyanates formed from glucosinolates in foods are variable and depend partly on food processing and preparation (see the Effects of Cooking section below). Consumption of five or more weekly servings of cruciferous vegetables has been associated with significant reductions in cancer risk in some prospective cohort studies.^53–55

Broccoli Sprouts

The amount of glucoraphanin, the precursor of SFN, in broccoli seeds remains more or less constant as those seeds germinate and grow into mature plants. Thus, 3-day-old broccoli sprouts are concentrated sources of glucoraphanin, which contain 10 to 100 times more glucoraphanin by weight than mature broccoli plants.⁵⁶ Broccoli sprouts that are certified to contain at least 73 mg of glucoraphanin (also called sulforaphane glucosinolate) per 1-oz serving are available in some health food and grocery stores.

Table 13.2 Some food sources of selected isothiocyanates and their glucosinolate precursors 12

Isothiocyanate	Glucosinolate (Precursor)	Food Sources
Allyl isothiocyanate (AITC)	Sinigrin	Broccoli, Brussels sprouts, cabbage, horseradish, mustard, radish
Benzyl isothiocyanate (BITC)	Glucotropaeolin	Cabbage, garden cress, Indian cress
Phenethyl isothiocyanate (PEITC)	Gluconasturtiin	Watercress
Sulforaphane (SFN)	Glucoraphanin	Broccoli, Brussels sprouts, cabbage

Effects of Cooking

Glucosinolates are water-soluble compounds that may be leached into cooking water. Boiling cruciferous vegetables for 9–15 minutes resulted in 18%–59% decreases in the total glucosinolate content of cruciferous vegetables.52 Cooking methods that use less water, such as steaming or microwaving, may reduce glucosinolate losses.⁵⁷ However, some cooking practices, including boiling,⁵ steaming,^7,58 and microwaving at high power (750–900 W),^8,58,59 may inactivate myrosinase, the enzyme that catalyzes glucosinolate hydrolysis. Even in the absence of plant myrosinase activity, the myrosinase activity of human intestinal bacteria results in some glucosinolate hydrolysis.⁶ However, several studies in humans have found that inactivation of myrosinase in cruciferous vegetables substantially decreases the bio-availability of isothiocyanates.^5,7,8

Supplements

Dietary supplements containing extracts of broccoli sprouts, broccoli, and other cruciferous vegetables are available without a prescription. Some products are standardized to contain a minimum amount of glucosinolates and/or sulforaphane. However, the bioavailability of isothiocyanates derived from these supplements is not known.

Safety

Adverse Effects

No serious adverse effects of isothiocyanates in humans have been reported. The majority of animal studies have found that isothiocyanates inhibited the development of cancer when given prior to the chemical carcinogen (pre-initiation) However, very high intakes of PEITC or BITC (25–250 times higher than average human dietary isothiocyanate intakes) have been found to promote bladder cancer in rats when given after a chemical carcinogen (post-initiation).60 The relevance of these findings to human urinary bladder cancer is not clear, since at least one prospective cohort study found cruciferous vegetable consumption to be inversely associated with the risk of bladder cancer in men.⁵⁵

Pregnancy and Lactation

Although high dietary intakes of glucosinolates from cruciferous vegetables are not known to have adverse effects during pregnancy or lactation, there is no information on the safety of purified isothiocyanates or supplements containing high doses of glucosinolates and/or isothiocyanates during pregnancy or lactation in humans.

Drug Interactions

Isothiocyanates are not known to interact with any drugs or medications. However, the potential for isothiocyanates to inhibit various isoforms of the CYP family of enzymes raises the potential for interactions with drugs that are CYP substrates.61

Summary

• Isothiocyanates are derived from the hydrolysis (breakdown) of glucosinolates—sulfur-containing compounds found in cruciferous vegetables.

• Cruciferous vegetables contain a variety of glucosinolates, each of which forms a different isothiocyanate when hydrolyzed.

• Isothiocyanates, such as sulforaphane, may help prevent cancer by promoting the elimination of potential carcinogens from the body and by enhancing the transcription of tumor suppressor proteins.

• Epidemiological studies provide some evidence that human exposure to isothiocyanates through cruciferous vegetable consumption may decrease cancer risk, but the protective effects may be influenced by individual genetic variation in the metabolism and elimination of isothiocyanates from the body.

• Glucosinolates are present in relatively high concentrations in cruciferous vegetables, but cooking, particularly boiling and microwaving at high power, may decrease the bioavailability of isothiocyanates.