Thermally Processing Broccoli Sprouts Impacts the Metabolism of Bioactive

Home , Broccoli sprouts, Cruciferous vegetables, Glucosinolate

Isothiocyanates in Mice

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Gregory Vincent Bricker, B.S.

Graduate Program in Food Science and Technology

The Ohio State University

2012

Master's Examination Committee:

Dr. Steven J. Schwartz, Adviser

Dr. Tatiana M. Oberyszyn

Dr. M. Monica Giusti

Copyrighted by

Gregory Vincent Bricker

2012

Abstract

Broccoli sprouts are a rich source of glucosinolates, a unique group of phytochemicals which may play a role in preventing multiple types of cancer. In order to exert physiologic activity, glucosinolates must be converted to their bioactive form, known as isothiocyanates, by the heat-labile enzyme myrosinase. This study had two objectives: First, to determine how heating broccoli sprouts affects glucosinolate content and subsequent isothiocyanate formation in vitro. Secondly, to understand how these results translate in vivo, by studying the metabolism of glucosinolates and isothiocyanates in mice fed diets containing thermally processed broccoli sprout powders (BSPs). Fresh broccoli sprouts received one of three processing treatments: (1) freeze-dried raw, (2) steamed and freeze-dried, or (3) heated at 60°C and freeze-dried. The sprouts were powdered, and then analyzed for differences in glucosinolate content and conversion to isothiocyanates. Three different mouse diets were prepared, each incorporating one of the

BSPs at 4%, and fed for 7 days (n=10 mice/group). An additional group was fed a diet containing the purified isothiocyanate sulforaphane. 24 hours prior to sacrifice, all mice were exposed dorsally to ultraviolet light. Ultra performance liquid chromatography– mass spectrometry was utilized to quantify metabolites of the two predominant isothiocyanates, sulforaphane and erucin, in the plasma, liver, skin, lungs, kidneys, and bladder. Additionally, the activity of myeloperoxidase, a biomarker for inflammation,

ii was measured in the skin. Steamed BSP had the greatest concentration of glucosinolates, followed by raw and 60°C treated BSPs. However, isothiocyanate formation from the raw and 60°C treated BSPs was approximately 5 and 23-fold greater, respectively, than the steamed BSPs, indicating that heating intensity has disparate effects on isothiocyanate formation. Mice fed the steamed BSP diet had the lowest concentrations of isothiocyanate metabolites in plasma and all tissue sites, while consumption of the 60°C- treated BSP diet generally resulted in the highest concentrations. Particularly high levels of isothiocyanate metabolites were detected in the bladder. Interestingly, we observed the isothiocyanates sulforaphane and erucin interconvert in vivo, with erucin being the vastly favored form at several tissue sites, even in mice fed the sulforaphane diet. Dietary administration of the BSPs did not reduce myeloperoxidase activity compared to the control group. In conclusion, in accordance with other studies, we show steaming severely hinders glucosinolate conversion to isothiocyanates upon consumption. Feeding broccoli sprouts treated at 60°C resulted in significantly greater concentrations of isothiocyanates at many organ sites. Lastly, once converted, chemopreventive isothiocyanates were distributed systemically to all tissue types analyzed.

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Dedicated to my family and friends

Acknowledgments

First, I’d like to thank Dr. Joseph Scheerens and Dr. Ann Chanon for being the first to teach me how to be a scientist. Also, I very much appreciate my advisor, Dr.

Steven Schwartz, for his constant support, insight, and assistance during my undergraduate and graduate years. I have additional gratitude for Dr. Tatiana Oberyszyn and Dr. Kathy Tober, both of whom were a true pleasure to collaborate with.

Thank you to all Schwartz Lab members, whose friendship and assistance have been greatly valued. In particular, I appreciate Dr. Rachel Kopec for training me in the laboratory and being a welcoming resource for whenever I’ve needed advice.

Lastly, thank you to my parents, David and Marianne Bricker, for their unceasing encouragement and support throughout my life.

Vita

April 19, 1988 ...... Born: Wooster, OH

2010...... B.S. Food Science,

The Ohio State University

2011 to Present ...... Graduate Research Associate, Department

of Food Science & Technology, The Ohio

State University

Fields of Study

Major Field: Food Science and Technology

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vi

Fields of Study ...... vi

Table of Contents ...... vii

List of Tables ...... x

List of Figures ...... xi

Chapter 1: Introduction ...... 1

Chapter 2: Literature Review ...... 2

2.1. Glucosinolate chemistry ...... 2

2.1.1. Glucosinolate biosynthesis ...... 3

2.1.2. Hydrolysis of glucosinolates ...... 4

2.1.3. Content of glucosinolates in plants ...... 5

2.2. Analysis of glucosinolates and isothiocyanates ...... 6

2.2.1. Glucosinolate extraction and analysis ...... 6

2.2.2. Isothiocyanate extraction and analysis ...... 7 vii

2.3. Absorption, distribution, metabolism, and excretion of glucosinolates and

isothiocyanates ...... 9

2.3.1. Absorption ...... 10

2.3.2. Metabolism ...... 10

2.3.3. Distribution ...... 11

2.3.4. Excretion ...... 12

2.4. Evidence for potential health benefits of cruciferous vegetables ...... 13

2.4.1 Epidemiological evidence ...... 14

2.4.2. Cell culture studies ...... 15

2.4.3. Studies with animal models ...... 17

2.4.4. Human studies ...... 21

2.5. Factors affecting isothiocyanate formation and bioactivity ...... 22

2.5.1. In vitro studies ...... 23

2.5.2. In vivo studies ...... 24

2.5.3. Role of the epithiospecifier protein ...... 25

Chapter 3: Research Plan ...... 28

Chapter 4: Materials and Methods ...... 30

4.1. Chemicals ...... 30

4.2. Processing of fresh broccoli sprouts ...... 30

viii

4.3. Glucosinolate analysis ...... 31

4.4. Sulforaphane analysis ...... 32

4.5. Creation of broccoli sprout and sulforaphane diets ...... 33

4.6. Animals, housing, and ultra violet light exposure ...... 34

4.7. Synthesis of metabolite standards ...... 34

4.8. Mouse plasma and tissue analysis ...... 35

4.9. Myeloperoxidase activity ...... 37

4.10. Statistics ...... 37

Chapter 5: Results and Discussion ...... 39

5.1. Glucosinolate analysis ...... 39

5.2. In vitro glucoraphanin conversion to sulforaphane ...... 41

5.3. Conversion to isothiocyanates in vivo ...... 42

5.4. Distribution of isothiocyanate metabolites ...... 48

5.5. Evidence and implications of sulforaphane/erucin interconversion ...... 49

5.6. Relative abundance of individual metabolites ...... 51

5.7. Myeloperoxidase activity ...... 53

Chapter 6: Conclusion...... 56

References ...... 57

List of Tables

Table 1. Glucosinolate concentration of processed broccoli sprout powders ...... 40

Table 2. In vitro formation of sulforaphane ...... 42

List of Figures

Figure 1. Glucosinolate hydrolysis ...... 3

Figure 2. Metabolism of glucosinolates to isothiocyanates and mercapturic acids ...... 11

Figure 3. Schematic of study design ...... 38

Figure 4. Mass chromatogram of the six isothiocyanate metabolites quantified...... 44

Figure 5. Concentration of isothiocyanate metabolites in plasma ...... 45

Figure 6. Concentration of isothiocyanate metabolites in liver ...... 45

Figure 7. Concentration of isothiocyanate metabolites in lungs ...... 46

Figure 8. Concentration of isothiocyanate metabolites in skin ...... 46

Figure 9. Concentration of isothiocyanate metabolites in bladder ...... 47

Figure 10. Concentration of isothiocyanate metabolites in kidneys ...... 47

Figure 11. Concentration and interconversion of sulforaphane and erucin ...... 50

Figure 12. Relative abundance of individual sulforaphane metabolites ...... 52

Figure 13. Relative abundance of individual erucin metabolites ...... 52

Figure 14. Myeloperoxidase activity ...... 55

Chapter 1: Introduction

Cancer is the second leading cause of death in the United States, contributing to approximately 23% of the total; only cardiovascular disease, at 25%, results in more fatalities (Jemal and others 2010). Undoubtedly, cancer treatments are improving, yet, the overall incidence and number of deaths by cancer is still on the rise, mainly attributed to a large, aging population (Jemal and others 2010). Taking this into account, the study of cancer prevention, by such means as maintaining a healthy diet, is becoming increasingly important, with a significant focus being placed on the potential benefits of fruits and vegetables. From antiquity and continuing to the present day, humans have exploited plants for their medicinal qualities. History shows that plants have been used in an attempt to alleviate a tremendous variety of health maladies, ranging from minor conditions to chronic diseases (Fenwick and others 1982). As opposed to the anecdotal nature by which these treatments have been passed through the generations, controlled epidemiological studies examine large populations to identify specific dietary components which may provide these health benefits. Many of these studies have linked consumption of cruciferous vegetables, such as broccoli and cabbage, to reduced risk of several types of cancer (Verhoeven and others 1996). Further research has identified unique compounds from cruciferous vegetables—glucosinolates and isothiocyanates—as the likely source for this chemoprevention.

Chapter 2: Literature Review

2.1. Glucosinolate chemistry

All cruciferous vegetables produce plant secondary metabolites called glucosinolates, the source of the vegetables’ putative chemoprevention. Structurally, glucosinolates can be described as a β-thioglucoside N-hydroxysulfate with a variable side chain. Over 120 different side chains, and, thus, glucosinolates, have been discovered, and an excellent tabulation summarizing these glucosinolates has been created (Fahey and others 2001). There is a tremendous diversity of these side chains, and they have been grouped into ten different classifications based on their structure, including aliphatic, alkene, sulfur-containing, aromatic, and indole side chains (Fahey and others 2001).

Glucosinolates themselves are not bioactive; rather, they must be hydrolyzed to bioactive products by the endogenous plant enzyme myrosinase. Myrosinase is released when the cell structure is damaged, such as from chopping, chewing, or herbivore attack.

As a result, glucosinolates are hydrolyzed to various breakdown products, including isothiocyanates, thiocyanates, and nitriles, of which, only the isothiocyanates are known to have anti-cancer activity (Figure 1) (Matusheski and Jeffery 2001). Alternatively, if myrosinase has been heat-inactivated during food preparation (e.g. from steaming or microwaving), thioglucosidases in the human gut microflora can convert glucosinolates

2 to isothiocyanates, though this conversion has repeatedly been shown to be far less efficient (Conaway and others 2000; Vermeulen and others 2008; Clarke and others

2011). Plants produce glucosinolates, which can by hydrolyzed to pungent isothiocyanates, to ward off herbivore (Giamoustaris and Mithen 1995), fungal, and bacterial attack (Brown and Morra 1997). Of note, it is only a fortuitous coincidence that glucosinolates and isothiocyanates may also convey human health altering benefits.

Figure 1. Glucosinolates are hydrolyzed to a variety of products, including isothiocyanates, nitriles, and thiocyanates (Fahey and others 2001)

2.1.1. Glucosinolate biosynthesis

While the general mechanism for glucosinolate biosynthesis has been elucidated, many details are lacking. Biosynthesis has been shown to occur in three steps: side chain elongation, formation of the glycone, and modification of the side chain. As described by

Halkier and Du, the initial building block of a glucosinolate is an amino acid, often methionine for aliphatic glucosinolates (1997). The chain is elongated through a transamination reaction and condensation with acetyl-CoA, followed by a second

3 transamination reaction, commonly creating homo-methionine or dihomo-methionine.

Next, the elongated amino acid undergoes an N-hydroyxlation, after which it is decarboxylated, creating an aldoxime. An oxygen atom is introduced, and, following a rearrangement, is converted to a thiohydroxamic acid. A glucose molecule, from uridine diphosphate glucose, is transferred and the desulfoglucosinolate is sulfated. Lastly, the side chain may be modified through a series of hydroxylations, desaturations, and oxidations, thus finalizing glucosinolate biosynthesis (Giamoustaris and Mithen 1996).

2.1.2. Hydrolysis of glucosinolates

As previously discussed, the endogenous enzyme myrosinase is responsible for hydrolyzing glucosinolates to their breakdown products. Myrosinase is stored in specialized myrosin cells which are inaccessible to glucosinolates (Thangstad and others

1990). Upon contact with glucosinolates, myrosinase cleaves the glucose moiety, resulting in an inherently unstable thiohydroxamate-O-sulfanate. Based on the pH, variable side chain, or the presence of non-enzymatic cofactors, the unstable intermediate will spontaneously rearrange to isothiocyanates, thiocyanates, nitriles, epithionitriles, or, less commonly, alcohols or cyanide (Mortazavi and others 2008). Interestingly, ascorbic acid has been shown to “activate” myrosinase, as its presence increases myrosinase activity over 100-fold (Shikita and others 1999). Near neutral pH, formation of isothiocyanates and thiocyanates is favored, while nitrile formation is favored in acid environments (Vaughn and Berhow 2005). The presence of the epithiospecifier protein, which is present in some cruciferous vegetables, such as broccoli, drives formation to nitriles. However, this requires the presence of iron (Williams and others 2010).

2.1.3. Content of glucosinolates in plants

Cruciferous vegetables are not taxonomically confined to a single genus, or even family. While most cruciferous vegetables reside in the family Brassicaceae, the closely related Capparaceae family also contains several glucosinolate containing species (Fahey and others 2001). A taxonomic breakdown of glucosinolate-containing plants is provided in a recent review article (Clarke 2010).

Interestingly, the most commonly consumed cruciferous vegetables, including cabbage, Brussels sprouts, cauliflower, and broccoli, are members of the same species,

Brassica oleracea, differing only in cultivar. Typically, each cultivar has its own unique glucosinolate profile. In broccoli, the glucosinolate glucoraphanin predominates, but in

Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolate is sinigrin (Kushad and others 1999). Arugula is known for its high concentration of glucoerucin (Barillari and others 2005; Cataldi and others 2007). From a chemopreventive standpoint, most research has focused on glucoraphanin-rich vegetable sources, as glucoraphanin is hydrolyzed to the potent isothiocyanate sulforaphane.

In mature broccoli, the florets contain the highest concentrations of glucoraphanin on a dry weight basis, followed by leaves and stalks (Campas-Baypoli and others 2009).

Interestingly, Fahey and others discovered that immature broccoli, otherwise known as broccoli sprouts, contains levels of glucosinolates that are 10-100 times that of mature broccoli (Fahey and others 1997). For this reason, broccoli sprouts are often used to make glucosinolate or isothiocyanate rich extracts for use in animal and human feeding studies.

In addition, the discovery that broccoli sprouts contain extremely high levels of glucosinolates underscores the importance of analytical analysis techniques, as discussed below.

2.2. Analysis of glucosinolates and isothiocyanates

Identification and quantification of compounds from plants are critical steps to studying their potential as chemopreventive agents. Identification of individual chemicals from an extract can be used to reveal the active compound, such as isothiocyanates in a broccoli sprout extract (Zhang and others 1992). Furthermore, quantitative analysis can be used as a tool to determine which vegetables are the optimal sources of the chemicals of interest.

2.2.1. Glucosinolate extraction and analysis

In order to accurately quantify glucosinolates, it is essential to preserve them in their glucosinolate form. To accomplish this, the endogenous enzyme myrosinase must first be inactivated to prevent conversion to hydrolysis products. Often, this is done by steaming a sample prior to extraction or extracting directly into boiling water (Fahey and others 1997; Clarke and others 2011). The majority of studies have shown glucosinolates to be quite stable (Song and Thornalley 2007; Fahey and others 2012), however prolonged exposure to heat may cause degradation (Hanschen and others 2012).

Extraction techniques for glucosinolates are typically straightforward, and extraction into polar solvents, such as water or aqueous methanol solutions, is typical. Fresh plant tissue can be extracted as is, or, alternatively, plant tissue can be lyophilized and ground to a powder, which increases the concentration, homogeneity, and stability of a sample

(Fahey and others 2012). Previous reports have shown success extracting glucosinolates into water, centrifuging, and decanting (Clarke and others 2011).

Unfortunately, glucosinolates can be difficult to identify, separate, and quantify intact, due to the lack of pure standards, relatively small differences in polarities, and low extinction coefficients (Mortazavi and others 2008). A high performance liquid chromatography (HPLC) method in which glucosinolates are enzymatically desulfated has gained popularity. However, sample preparation for this method is time-consuming, as it requires on-column desulfation for 16 hours before anion-exchange chromatography can be performed (Matthäus and Luftmann 2000). Thus, the need for rapid, sensitive glucosinolate analysis methods remained. Several qualitative mass spectrometry methods have been developed, including matrix-assisted laser desorption/ionization-time of flight

(Botting and others 2002), and negative ion electrospray mass spectrometry (Bennett and others 2004). However, the need for quantitative analysis still existed, so our lab developed a selective and rapid method to separate intact glucosinolates with HPLC, followed by detection and quantification with electrospray ionization-tandem mass spectrometry (Tian and others 2005).

2.2.2. Isothiocyanate extraction and analysis

Isothiocyanate extraction and determination has also proven to be difficult. First, glucosinolates must be converted to isothiocyanates. This can be accomplished by homogenizing fresh plant material, in which endogenous myrosinase is still active (Zhu and others 2010). Alternatively, if myrosinase has been denatured, exogenous sources of myrosinase, such as commercially available myrosinase or daikon sprouts, can be added

(Getahun and Chung 1999; Matusheski and others 2001; Shapiro and others 2001).

However, one must realize the implications of these two techniques, as they may produce differing proportions of hydrolysis products. If, for example, broccoli has been steamed prior to analysis, myrosinase and the epithiospecifier protein will be denatured. Upon addition of exogenous myrosinase, nearly 100% of glucosinolates will likely be converted to isothiocyanates. However, if fresh plant material is hydrolyzed, endogenous myrosinase and epithiospecifier protein will still be present and, as a result, large portions of the nitrile product will be formed at the expense of isothiocyanates (Matusheski and others 2006).

The two most common techniques for isothiocyanate analysis are gas chromatography and high pressure liquid chromatography. For both techniques, converted isothiocyanates are typically extracted into nonpolar solvents, such as dichloromethane. For gas chromatography analysis, a method using flame ionization detection is commonly used (Chiang 1998). This method has been successful in quantifying both isothiocyanates and nitriles (Matusheski and others 2001).

Unfortunately, the high heat inherent with gas chromatography is reported to degrade isothiocyanates (Śmiechowska and others 2010).

Several non-thermal liquid chromatography techniques have been developed to separate isothiocyanates, followed by photo diode array detection. However, because of their low molar absorptivities, isothiocyanates must first be subjected to cyclocondensation or conjugation reactions. One of the most commonly used techniques exploits the electrophilic nature of the isothiocyanate’s N=C=S backbone. Addition of the

8 chemical 1,2-benzenedithiol to isothiocyanates causes the creation of a cyclocondensation product with significantly higher molar absorptivity (Zhang and others

1992). All isothiocyanates in this mixture will form the same cyclocondensation product.

Thus, this method provides a convenient bulk measurement of total isothiocyanates.

However, this convenience can also be a detriment, as the ability to differentiate and quantify individual isothiocyanates is lost (Zhang 2012). Alternatively, conjugation of isothiocyanates to the reducing agent 2-mercaptoethanol increases molar absorptivity, while retaining the ability to separate and quantify individual isothiocyanates in a mixture

(Vermeulen and others 2006).

Lastly, sensitive analysis techniques are necessary for quantification of isothiocyanates and their metabolites in biological samples. Mass spectrometry techniques, employing electrospray ionization, have been shown to provide quantitative measurements of the isothiocyanates sulforaphane, iberin, and their metabolites in plasma

(Al Janobi and others 2006). A similar method has been used to quantify these metabolites in tissue (Clarke and others 2011). Our lab has developed a method measuring both sulforaphane and erucin metabolites, which is critical in the study of isothiocyanate metabolism, as will be discussed below (Clarke and others 2011).

2.3. Absorption, distribution, metabolism, and excretion of glucosinolates and isothiocyanates

Several animal and human studies have provided details on the absorption, distribution, metabolism, and excretion of glucosinolates and isothiocyanates. In general, the metabolic pathway has been shown to be similar between species. Briefly, after

9 glucosinolates are converted to isothiocyanates, either by endogenous myrosinase or thioglucosidases in the gut, they are rapidly absorbed and converted to water soluble conjugates, transformed through the mercapturic acid pathway, and excreted in the urine.

2.3.1. Absorption

The absorption site of glucosinolates and/or isothiocyanates is dependent upon which form in consumed, as well as whether they are consumed with myrosinase. If glucosinolates have been converted to isothiocyanates, they are likely to pass through the stomach and be absorbed in the small intestine (Petri and others 2003). Alternatively, if glucosinolates have not been converted by this point, they will pass to the large intestine and, to an extent, be hydrolyzed to isothiocyanates by gut microflora and absorbed in the colon (Shapiro and others 1998). Traditionally, glucosinolates have not been thought to be absorbed intact, though at least one animal study shows that it is indeed possible, at least with relatively large doses (Bheemreddy and Jeffery 2007).

2.3.2. Metabolism

Early animal studies gave a glimpse of isothiocyanate metabolism in rats, dogs,

(Brüsewitz and others 1977) guinea pigs, and rabbits (Görler and others 1982). These studies clearly established that, following absorption, isothiocyanate metabolism is initiated by conjugation to glutathione, followed by sequential conversion to cysteinylglycine, cysteine, and N-acetylcysteine conjugates (Figure 2). An identical pathway has been observed in humans, as well (Gasper and others 2005). Notably, it has been reported that some isothiocyanate conjugates have biological activity themselves

(Bhattacharya and others 2012). However, this may be a result of the isothiocyanate

10 dissociating from its conjugate. Thus, the conjugate may serve as a “vehicle” to transport the isothiocyanate through circulation (Conaway and others 1996).

Figure 2. Metabolism of glucosinolates to isothiocyanates and mercapturic acids (Al Janobi and others 2006)

2.3.3. Distribution

The distribution of isothiocyanates is critical to understanding the potential efficacy as chemopreventive agents, as it is less likely a tissue or organ will derive a benefit if it is inaccessible to the compounds. A recent study investigated the systemic distribution of sulforaphane metabolites following oral gavage of the compound. In this study, sulforaphane metabolites were detected and quantified in plasma, liver, kidney, small intestine, colon, lung, brain and prostate, demonstrating the vast systemic 11 distribution of the compound (Clarke and others 2011). However, because sulforaphane is known to be reduced to the isothiocyanate erucin (Kassahun and others 1997), in vivo, this study failed to accurately measure total isothiocyanate distribution, as only sulforaphane and its metabolites were measured. Other studies have reported isothiocyanates to be distributed to the bladder and bladder tumor tissue in particularly high concentrations (Munday and others 2008; Abbaoui and others 2012), likely because the bladder comes into direct contact with urine, the primary excretory medium of isothiocyanates. It can be difficult to study the distribution of glucosinolates and isothiocyanates in humans, as the extent of samples available for analysis are typically restricted to plasma and urine. However, one study has reported finding isothiocyanate metabolites in mammary tissue (Cornblatt and others 2007).

2.3.4. Excretion

Excretion of isothiocyanates and their metabolites primarily occurs through the urine. However, small amounts may be excreted through fecal and respiratory routes

(Bollard and others 1997). In human urine, the N-acetylcysteine-isothiocyanate conjugate contributes the greatest proportion of isothiocyanate metabolites. It is not detected in human plasma, which is not altogether surprising, since it is the final mercapturic acid of the pathway (Conaway and others 2000). Several studies have demonstrated that when cruciferous vegetables are consumed with active myrosinase, the absorption, metabolism, and excretion of isothiocyanate metabolites occurs very rapidly, and is associated with a sharp increase, and subsequent decrease, in plasma isothiocyanate levels (Shapiro and others 1998). Conversely, if intact glucosinolates are consumed without myrosinase, they

12 are not absorbed until hydrolysis in the colon. As a result, isothiocyanate metabolites take much longer to show up in the plasma, and they are present at lower concentrations for longer periods of time (Shapiro and others 2006; Egner and others 2011). These disparate rates of excretion may have implications in the potential chemopreventive benefits afforded by cruciferous vegetable consumption. Various targets and mechanisms of isothiocyanate-mediated chemoprevention require relatively high physiologic concentrations, such as induction of apoptosis, which may be achieved from after a high isothiocyanate dose. However, other targets, such as phase II enzyme induction, may respond well to lower, more steady-state levels, such as those provided by glucosinolate doses (Egner and others 2011). Further study into the potential health affects afforded from cruciferous vegetable consumption will be discussed below.

2.4. Evidence for potential health benefits of cruciferous vegetables

Dating back to 4000 B.C., cruciferous vegetable have been consumed for their spicy, pungent flavor characteristics, as well as for medicinal qualities (Fenwick and others 1982). However, it was not until 1992 that researchers from Johns Hopkins discovered that broccoli extracts could significantly induce phase II detoxification enzymes, and further discerned that isothiocyanates were the major source of this induction (Zhang and others 1992). Since these findings, hundreds of studies have been performed, seeking to identify potential health benefits of cruciferous vegetables, as well as the mechanisms by which they may provide these benefits. Epidemiological, cell culture, animal, and human models have been employed and, overall, results have been promising. Thus, the purpose of this section is to review selected studies to establish the

13 grounds for cruciferous vegetable research, as well as to explore the progress that has been made to date.

2.4.1 Epidemiological evidence

Epidemiological studies can be credited with first establishing the link between increased cruciferous vegetable consumption and reduced risk of cancer. Unfortunately, a common issue with epidemiological studies is attributing an observed benefit to a single factor or variable. Those who frequently consume cruciferous vegetables often consume many vegetables in general (Verhoeven and others 1996). Thus, it is often difficult to determine if an observation, such as reduced risk of cancer, is due solely to the cruciferous vegetable consumption. For this reason, individuals whose total vegetable intake consists primarily of cruciferous vegetables are of particular usefulness for epidemiological studies (Verhoeven and others 1996).

An early epidemiological study reported a decreased risk of colon cancer with higher vegetable consumption, and this risk was reduced even more in those who consumed cabbage, Brussels sprouts, and broccoli, all of which are crucifers (Graham and others 1978). Similarly, consumption of hakusai, a Chinese cabbage, was inversely linked to colon cancer risk in a Japanese population (Haenszel and others 1980). A meta- analysis aggregated results from 87 case-control studies, and determined 67% of which found an inverse correlation between cruciferous vegetable consumption and risk of various cancers, particularly cancer of the lungs, stomach, and colon (Verhoeven and others 1996). However, the authors caution that many factors influencing the results of the individual studies, including selection bias, recall bias, and publication bias, may

14 affect their overall conclusion. Taking these factors into account, properly designed and managed epidemiological studies remain important for future study.

2.4.2. Cell culture studies

Cell culture has been an extremely useful tool for identifying chemopreventive compounds from cruciferous vegetables, as well as discovering potential mechanisms by which they exert their benefits. One particular mechanism that has received significant attention is the ability of cruciferous vegetables to induce phase II enzymes. In general, the purpose of the phase II enzyme system is to modify xenobiotics, such as carcinogens, by conjugating them to hydrophilic molecules, such as glutathione (Prochaska and others

1992). In turn, this allows the xenobiotic to be more rapidly excreted. Therefore, induction of these enzymes accelerates xenobiotic disposal, which is advantageous if the xenobiotic compound is carcinogenic. An initial study determined that extracts from various cruciferous vegetables significantly induced phase II enzymes in murine liver cancer cells (Prochaska and others 1992). Soon after, the same researchers determined that isothiocyanates, particularly sulforaphane, were the source of the enzyme induction

(Zhang and others 1992). Since the activity of sulforaphane was discovered, it has been considered by many as the most potent isothiocyanate, with respect to chemopreventive capabilities.

One concern with isothiocyanates was that the compounds would have similar inducing ability with phase I enzymes, such as cytochrome p450. This is of great importance when seeking chemopreventive compounds, because phase I enzymes have the potential to “activate” carcinogens (Fahey and others 2001). If a compound induces

15 both phase I and phase II enzymes, it is known as a bifunctional inducer. If it only induces phase II enzymes, it is known as a monofunctional, or selective, inducer

(Prochaska and others 1992). Fortunately, sulforaphane and other isothiocyanates have been shown to be monofunctional inducers. In fact, research has demonstrated that some isothiocyanates actually have the ability to inhibit certain phase I enzymes. In cultured human liver cells, sulforaphane competitively interacted with deleterious phase I enzymes, while simultaneously up-regulating beneficial phase II enzyme systems

(Mahéo and others 1997). Three other isothiocyanates, erucin, benzyl, and phenethyl isothiocyanate, have also been shown to inhibit phase I enzymes in liver cell lines

(Mersch-Sundermann and others 2004; Lamy and others 2008).

In addition to modulating phase I and II enzyme systems, isothiocyanates convey other potentially beneficial outcomes in both animal and human cancer cell cultures. In human colon cancer cells, sulforaphane induces both cell cycle arrest and apoptosis

(Gamet-Payrastre and others 2000). Sulforaphane is also able to induce apoptosis in human prostate cancer cells, through an intricate array of mechanisms involving the protein Apaf-1 (Choi and others 2007). Further research shows the oxidation state of the sulfur in an isothiocyanates’ side chain has a significant effect on apoptotic potential, as isothiocyanates with an oxidized sulfur were more potent (Kim and others 2010). While isothiocyanates were originally thought to only play a role in cancer prevention, these studies suggest isothiocyanates may have anti-cancer activity even after the initiation phase of carcinogenesis.

2.4.3. Studies with animal models

Arguably, the most promising studies demonstrating the potential of cruciferous vegetables as chemopreventive agents have employed animal models. Traditionally, most animal studies have used murine models to study the health benefits of cruciferous vegetables. Several mechanisms initially observed in cell culture have been confirmed using animal studies, such as induction of phase II enzymes, which has shown to be a consistent and repeatable effect of isothiocyanate consumption in rat colon, liver, kidney,

(Hwang and Jeffery 2004) duodenum, forestomach, bladder, (Munday and Munday 2004) and mouse skin (Dinkova-Kostova and others 2007). Also from a mechanistic approach, sulforaphane has been shown to act as a histone deacetylase inhibitor, and thus may have a role in cancer chemoprevention and/or therapy (Myzak and others 2006).

Many animal studies have also reported positive effects of cruciferous vegetables with respect to cancer outcomes, including tumor incidence, multiplicity, and total volume. In one commonly used study design, an animal is challenged with a known carcinogen, and then placed into a control or treatment group. Often, several treatment groups may be employed, to determine if other variables, such as the dosage or particular compound administered, have an effect on cancer outcomes. Results are viewed as positive if animals from the treated group have significantly lower cancer incidence than those in the control group.

Chemoprevention with isothiocyanates has been observed at several organ sites.

Sulforaphane has been shown to decrease the incidence of 9, 10-dimethyl-1, 2- benzanthracene-induced mammary cancer in rats by over 60%, compared to the control

17 group (Zhang and others 1994). Munday and others (2008) investigated the effects of consuming a broccoli sprout extract on nitrosamine-induced bladder cancer in rats and discovered an inverse relationship between tumor incidence and dose. While 95% of rats in the control group developed bladder tumors, tumors were observed in 74% and 38% of rats fed 40 and 160 μmol isothiocyanates/kg body weight, respectively. In a study on prostate cancer, a 10% broccoli powder diet decreased tumor weight by 42% compared to the control (Canene-Adams and others 2007). Generally, sulforaphane has been thought to be the most potent isothiocyanate, with respect to chemopreventive potential.

However, a recently published report suggests that erucin has similar, and possibly even better, potential to reduce tumor size in a murine bladder cancer xenograft model

(Abbaoui and others 2012). Thus, the need to research multiple isothiocyanates, not just sulforaphane, remains great.

Several animal studies have provided compelling evidence that isothiocyanates may provide protection against skin cancer. In the United States, there are more diagnosed cases of non-melanoma skin cancer than all other cancer types combined

(Oberyszyn 2011). While deaths from non-melanoma skin cancer are rare, an individual that develops skin cancer may be twice as likely to develop other forms of cancer later in life (Chen and others 2008). Thus, there is a need for additional approaches to mitigate the risk of developing skin cancer.

Several recent reports suggest that topical administration of isothiocyanate-rich broccoli sprout extracts have potential as chemopreventive agents for skin cancer. In a commonly used model, hairless, immunocompetent mice are exposed to ultraviolet (UV)

18 light twice weekly for 20 weeks. This UV treatment results in initiated, high-risk mice that are extremely susceptible to developing skin tumors (Dinkova-Kostova and others

2006). After the 20 weeks of UV exposure, topical applications of broccoli sprout extract, containing 1.0 µmol sulforaphane, reduced tumor incidence by 50%, compared to the control group, during the subsequent 11 weeks (Dinkova-Kostova and others 2006). Of note, this intervention occurred after exposure to the UV light, so the effects of the broccoli sprout extract were on tumor progression, not initiation. This model of a post- initiation intervention is extremely relevant to humans, as many individuals do not adequately protect themselves from UV-induced damage during the early years of their life (Dinkova-Kostova and others 2006). Thus, this report provides evidence that steps may be taken later in life to prevent the development of skin cancer. From a mechanistic basis, follow-up studies report an identical topical application increases levels of three phase II enzymes, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S- transferase (GST), and heme oxygenase 1 (Dinkova-Kostova and others 2007). Further study has demonstrated that topical applications, prior to UV exposure, reduce erythema and myeloperoxidase levels in mouse skin, indicating reduced inflammation (Talalay and others 2007). This is notable, as chronic inflammation may play a role in skin cancer development. Interesting gender differences in the inflammatory response have been observed. In male and female mice receiving equal doses of UV light, female mice exhibit a greater inflammatory response, but have fewer and less advanced tumors than male mice, which exhibit a decreased inflammatory response (Thomas-Ahner and others

2007). Thus, the role of inflammation in skin cancer development remains unclear.

While topical applications of broccoli sprout extracts have shown great promise, ideally, the same beneficial effects could be achieved through dietary means, which would be the preferred administration route for several reasons. First, many individuals already fail to apply sunscreen regularly, so it is reasonable that a similar obstacle would occur for the use of a topical, isothiocyanate-rich product. Similarly, isothiocyanate containing foods are already commonly found in the human diet, so a lifestyle change, aside from possibly consuming more isothiocyanates, would not be necessary. Lastly, a topical application would likely serve to benefit only the skin, while a dietary approach could potentially result in systemic benefits (Dinkova-Kostova and others 2010). For these reasons, investigators recently studied the ability of broccoli sprout extracts to prevent UV-induced skin tumorigenesis through dietary means. In this study, mice were exposed to UV light twice weekly for 17 weeks. After which, they were placed on a diet containing broccoli sprout extracts. Compared to the control group, tumor incidence was reduced by 25%. Furthermore, tumor multiplicity and total volume were reduced 47% and 70%, respectively, indicating that if mice did form tumors, they were typically smaller in size (Dinkova-Kostova and others 2010). Taken as a whole, these results are exceptionally promising and certainly warrant future research. However, care must be taken when extrapolating results from any animal study to humans. Dissimilarities between human and animal species, differing treatment regimes, and dosages greater than typical human consumption can potentially produce results that favor the efficacy of a chemopreventive substance (Steinmetz and Potter 1996).

2.4.4. Human studies

Aside from epidemiological studies, human trials seeking to identify and establish the chemopreventive properties of cruciferous vegetables have been relatively rare, likely due to the costs and regulations associated with experiments on humans. Nonetheless, a small number of studies have been performed. As briefly discussed above, topical application of a broccoli sprout extract prior to UV exposure reduced inflammation in mice. A similar model was used on humans. Briefly, for the three days prior to UV exposure, broccoli sprout extract was topically applied once per day to the back of subjects. On an adjacent section of skin, only the vehicle, acetone, was applied. Subjects were exposed to UV radiation and 24 hours later, the redness, or erythema, of the exposed skin was measured. On average, erythema was reduced 37.7% compared to the untreated control sections of skin, with a range of 8.4% to 78% erythema reduction.

Notably, this effect was not due to a UV-blocking effect of the extract, though a mechanism was not conclusively identified (Talalay and others 2007). It may be related to induction of the phase II response, which was elevated in human skin from a similar topical application (Dinkova-Kostova and others 2007).

A clinical trial was performed in a region of China known for air pollution and consumption of alfatoxin-containing foods. As a result, the population from this region is at high risk for hepatocellular carcinoma. In the study, participants consumed either a placebo or a glucosinolate-rich broccoli sprout beverage. Urine was collected, and biomarkers of alfatoxin and phenanthrene were quantitatively measured, along with isothiocyanate metabolites. Significant inverse associations were observed between levels

21 of these biomarkers and isothiocyanate metabolites excreted (Kensler and others 2005).

The authors suggested that the decreased biomarker levels were a result of altered phase

II metabolism, as the biomarkers were redirected to less toxic compounds due to the consumption of the broccoli sprout beverage. However, this theory requires more definitive evidence.

The health benefits of cruciferous vegetables have been known for many millennia, yet, only in the past three decades has significant effort been aimed at understanding how these benefits are achieved. We now know that cruciferous vegetables are particularly unique, as they are excellent sources of glucosinolates and isothiocyanates. We have learned a great deal about how these compounds behave in the body, as well as many mechanisms by which they provide anti-cancer benefits. However, much work is still needed to better understand why these compounds are effective for particular cancer types and why results of epidemiological studies have been, in some cases, inconsistent.

2.5. Factors affecting isothiocyanate formation and bioactivity

Glucosinolates are not bioactive, thus, conversion to isothiocyanates is necessary, which can be accomplished through two routes. First, the endogenous enzyme myrosinase, which is physically separated from glucosinolates in the plant cell, is released upon damage to the cell, such as during chopping or chewing, resulting in glucosinolate hydrolysis (Shapiro and others 2001). Alternatively, if glucosinolates reach the gut intact, thioglucosidases in the enteric microbiota can also convert glucosinolates to isothiocyanates, though this conversion has been reported to be inefficient (Shapiro

22 and others 1998). Past research has shown that the cooking methods used to prepare cruciferous vegetables for consumption may have drastic effects on the concentrations of glucosinolate and isothiocyanates delivered to the body, as discussed below.

2.5.1. In vitro studies

Many common home-processing techniques are not conducive to glucosinolate retention and/or isothiocyanate formation. In a study in which cruciferous vegetables were boiled, steamed, microwaved, or stir-fried, boiling resulted in the greatest loss of glucosinolates, while the other methods did not produce significant glucosinolate loss

(Song and Thornalley 2007). After analysis of the water used for boiling, it was determined that approximately 90% of the starting glucosinolate content had leached into the cooking water. Obviously, unless the cooking water is consumed or otherwise incorporated in a meal, these glucosinolates are irrecoverable. Even if a preparation technique retains glucosinolates, the heat itself may have negative effects on their ultimate availability as isothiocyanates. Heat intensive processing techniques have been shown to inactivate myrosinase, thus preventing glucosinolate conversion to isothiocyanates, as demonstrated in vitro, as well as with in vivo animal and human studies. Jones and others (2010) measured in vitro formation of the isothiocyanate sulforaphane in broccoli that had been steamed, microwaved, or boiled and compared their findings to raw broccoli. Steaming for 2 minutes resulted in ~50% decreased sulforaphane formation, while steaming for 5 minutes caused a 90% reduction.

Strikingly, both microwaving and boiling for 2 minutes resulted in nearly 99% reduction in sulforaphane production compared to the raw broccoli. Regarding the thermal stability

23 of myrosinase, one report indicates the enzyme is thermally inactivated in broccoli at approximately 70°C, while in immature broccoli sprouts, myrosinase remains active at temperatures as high as 100°C (Matusheski and others 2004). Interestingly, this discrepancy suggests that myrosinase from broccoli sprouts is more heat resistant than that from mature broccoli, perhaps due to a different myrosinase isoform being present in the sprouts.

2.5.2. In vivo studies

Zhu and others (2010) report that dietary administration of the glucosinolate glucoraphanin to rats, without any myrosinase source, results in urinary recovery of

23.8% of the dose as isothiocyanate metabolites. In contrast, rats fed a powdered broccoli diet, with active endogenous myrosinase, excreted 62.5% of the administered glucosinolate dose (Zhu et al. 2010). Numerous human studies have proven that consuming cruciferous vegetables devoid of myrosinase results in decreased conversion and excretion of isothiocyanates and their metabolites. Feeding 200 g of fresh or steamed broccoli resulted in urinary excretion of 32.3% and 10.2%, respectively, of the ingested dose of glucosinolates (Conaway and others 2000). Interestingly, thorough chewing of broccoli sprouts has been shown to result in significantly greater excretion (42.4%) than sprouts swallowed whole (28.8%), suggesting that mastication may release more myrosinase from plant tissue and permit greater glucosinolate hydrolysis (Shapiro and others 2001). In subjects consuming fresh or cooked watercress, urinary isothiocyanate excretion was 45.2% and 4.02%, of the total glucosinolate dose, respectively (Getahun and Chung 1999). Similar results are observed for plant extracts or supplements. Urinary

24 recovery of broccoli sprout extracts containing either 25 μmol glucoraphanin or 25 μmol sulforaphane was 17.8% and 70.6%, respectively. Variability was much greater in those subjects consuming glucoraphanin than sulforaphane, demonstrating the inconsistent behavior of enteric thioglucosidases between individuals (Shapiro and others 2006).

There are many broccoli supplement powders available on the market, but they do not contain a myrosinase source. Not surprisingly, isothiocyanate excretion from these powders supplements is substantially lower than an equivalent dose of glucosinolates from fresh broccoli sprouts (Clarke and others 2011). Clearly, consumption of cooked cruciferous vegetables or glucosinolates, without the presence of myrosinase, is associated with lower isothiocyanate excretion levels compared to consumption of raw vegetables or isothiocyanates.

2.5.3. Role of the epithiospecifier protein

As previously discussed, glucosinolates can be converted to a variety of hydrolysis products. At the expense of isothiocyanates, glucosinolates may be hydrolyzed to nitriles instead, which do not have bioactivity (Matusheski and Jeffery 2001). In fact, after in vitro hydrolysis of broccoli, the ratio of nitrile to isothiocyanate has been reported to be nearly 9:1 (Matusheski and others 2004). Thus, it is important to understand what factors cause the overwhelming production of nitriles, as well as if they can be modified to shift production to isothiocyanates. Several reports establish that the presence of the epithiospecifier protein (ESP), present in some cruciferous vegetables, results in formation of nitrile products (Matusheski and others 2006; Williams and others 2008).

Interestingly, Matusheski and others determined mild heating can selectively destroy

ESP, while retaining the activity of myrosinase. As a result, in vitro analyses indicate that, after heating broccoli to approximately 60°C, nitrile formation is minimized, with a concomitant increase in the formation of isothiocyanates (Matusheski and others 2004).

However, it is unreasonable to expect the common consumer to carefully heat their broccoli to 60°C. Therefore, it is of interest to understand how commonly used cooking techniques affect the activity of ESP and myrosinase, and to determine whether any of these techniques can be used to optimize isothiocyanate yield. In addition, various varieties of broccoli may have inherently different levels of ESP and myrosinase, which could alter the relative proportions of hydrolysis products formed (Wang and others

2012). A recent study examined the effects of broccoli cultivar and cooking techniques on sulforaphane and sulforaphane nitrile production. In unheated Pinnacle broccoli, 91% of the hydrolysis products were nitriles. Alternatively, in the Brigadier variety, this value was only 52%, indicating the vast differences in the ratio of hydrolysis products formed between broccoli varieties. The authors also compared the effects of microwaving, boiling, or steaming, and determined that steaming for a short time (1-3 minutes) is likely the best method to maximize isothiocyanate conversion (Wang and others 2012). In the future, developing cultivars of broccoli with low ESP activity should be a priority.

Delivering the maximum dose of bioactive isothiocyanates is of interest when considering the chemopreventive potential of cruciferous vegetables. Unfortunately, many common preparation methods used by today’s consumers ultimately have a negative effect on glucosinolate retention and/or conversion to isothiocyanates.

Furthermore, research has shown that glucosinolates in some cruciferous vegetables, such

26 as broccoli, are converted to non-isothiocyanate hydrolysis products that have no biological activity. Clearly, understanding how to maximize the dose of isothiocyanates delivered to the body is of interest for both researchers and health-conscious consumers.

Chapter 3: Research Plan

Previously, in vitro studies have shown that thermally processing broccoli sprouts at

60°C can greatly increase the yield of isothiocyanates upon hydrolysis, at the expense of nitrile formation (Matusheski and others 2004). This is due to selective inactivation of the epithiospecifier protein (ESP), which drives conversion of glucosinolates to nitriles.

Despite these interesting in vitro results, no study has been performed to determine if this increased yield is observed, in vivo, if ESP-inactivated broccoli sprouts are consumed. In addition, information is lacking on the metabolism and distribution of isothiocyanate metabolites. While the general metabolic pathway has been elucidated, little is known about accumulation of individual metabolites at specific tissue sites. A recent study of isothiocyanate tissue distribution only considered the isothiocyanate sulforaphane (Clarke and others 2011). However, because sulforaphane is reduced to the isothiocyanate erucin, in vivo (Kassahun and others 1997)¸ this study failed to report the true isothiocyanate concentration in various tissues. Other studies have used the cyclocondensation assay, which provides a bulk measurement but fails to allow differentiation between isothiocyanate species. Lastly, a recent study reported that dietary administration of a broccoli sprout extract reduced the incidence, multiplicity, and volume of UV-induced skin tumors (Dinkova-Kostova and others 2010). However, while the authors

28 hypothesized various mechanisms for this effect, none were investigated. Therefore, we have sought to address these gaps in the knowledge, through the following research aims:

1. Determine how steaming or heating broccoli sprouts at 60°C affects glucosinolate

conversion to isothiocyanates, in vivo, compared to unheated sprouts, by feeding

mice diets containing thermally-processed BSPs. Understand how these results

relate to those from previous in vitro work. We hypothesize that results from past

in vitro studies will mirror our in vivo animal study. We expect mice consuming

steamed BSP to have the lowest concentrations of isothiocyanates in plasma and

tissues, followed by mice consuming the unheated and 60°C heated BSPs.

2. Investigate how the isothiocyanate metabolites of sulforaphane and erucin are

distributed to various mouse tissues, examine relative proportions of the

individual metabolites in these tissues, and determine if isothiocyanate

consumption influences inflammatory biomarkers in the skin of mice exposed to

ultraviolet light. We expect isothiocyanate metabolites to be distributed

systemically and predict the relative abundance of individual metabolites to be

affected by their tissue location. Lastly, because isothiocyanates are thought to

exhibit anti-inflammatory activity, we hypothesize that feeding BSPs will

decrease the activity of inflammatory biomarkers in the skin of mice exposed to

ultraviolet light.

Chapter 4: Materials and Methods

4.1. Chemicals

Optima grade solvents (Fisher Scientific; Pittsburgh PA) were used for all analyses involving mass spectrometry. HPLC (high performance liquid chromatography) grade solvents were used for all other analyses (Fisher Scientific, Pittsburgh PA). 2- mercaptoethanol, triflouroacetic acid, and formic acid were purchased from Sigma-

Aldrich. Triethylamine was from EMD Chemical (Gibbstown, NJ). D,L-Sulforaphane used for external standard calibration was from Toronto Research Chemicals (Toronto,

Ontario, Canada) and D,L Sulforaphane used for the mouse diet was from Santa Cruz

Biotechnology (Santa Cruz, California). Glucoraphanin and glucoerucin standards were purchased from the Royal Veterinary and Agricultural University (Copenhagen,

Denmark). Isothiocyanate metabolite standards were synthesized in lab, as described below, with the initial isothiocyanate standards purchased from LKT laboratories (St.

Paul, MN)

4.2. Processing of fresh broccoli sprouts

Fresh, 6-day old broccoli sprouts were acquired from a local grower and stored refrigerated until processing the same day. The sprouts were subjected to one of three processing treatments: freeze-dried raw, steamed and freeze-dried, or heated at 60°C and freeze-dried. To steam, fresh broccoli sprouts were placed in a sieve, covered, and set

30 over a boiling water bath for 5 min. To facilitate equal heating, the sprouts were tossed occasionally. The 60°C heat treatment was performed by placing fresh sprouts in zipper storage bags, removing the air, and submerging in a 60°C water bath for 10 min. All sprouts were then flash frozen by plunging in liquid nitrogen and freeze-dried for 48 h.

After lyophilization, all broccoli sprouts were ground to a powder with a mortar and pestle.

4.3. Glucosinolate analysis

The glucosinolates glucoraphanin and glucoerucin were quantified in the three freeze-dried broccoli sprout powders (BSPs). Approximately 40 mg of the freeze-dried powder was added to a glass vial, followed by addition of 3 mL boiling water. The loosely-capped vial was immediately placed in a boiling water bath for 5 min, after which it was vortexed and sonicated for 5 min. The sample was centrifuged for 5 min at 600 x g and the aqueous extract was decanted and set aside. Twice more, the pellet was resuspended in 3 mL of room temperature water and vortexed/sonicated for 10 min, followed by centrifugation, decanting, and combining of the extracts, yielding approximately 9 mL aqueous extract. This extract was brought up to 10 mL with water, and then centrifuged at 21,000 x g prior to analysis.

Glucoraphanin and glucoerucin were separated on a Zorbax SB-CN reversed phase column (4.6 x 250 mm; 5 um; Agilent Technologies, Santa Clara, CA) with and

Agilent 1100 Series HPLC. A binary gradient, consisting of 0.1% formic acid in water

(A) and 0.1% formic acid in acetonitrile (B), was used at a flow rate of 1.5 mL/min.

Initial conditions consisted of 0%B for three minutes, followed by linear increases to

10% B by four minutes, 50% B by eight minutes, and 95% B by 9 minutes, after which the column was equilibrated at 0%B for three minutes. An AB Sciex QTrap 5500 mass spectrometer (Concord, Canada), operated in electrospray negative mode, was used for quantitation. Selected reaction monitoring was based on the common liberation of HSO4¯ anion from the glucosinolate. Thus, the transitions for glucoraphanin (436 > 97) and glucoerucin (420>97) were used, with dwell times of 140 ms. Other relevant mass spectrometry parameters include a desolvation gas temperature of 550°C, declustering potential of 70 V, entrance potential was 10 V, collision cell exit potential was 11 V, collision energy of 30eV, ion spray potential of 4.5 kV, gas 1 at 60 psi, gas 2 at 55 psi, and the curtain gas pressure at 30 psi. An external standard curve was used to determine concentrations of the glucosinolates.

4.4. Sulforaphane analysis

Sulforaphane formation in the three BSPs was measured using a method described by Vermeulen et al., with minor modifications. Approximately 25 mg of the freeze-dried powder were combined with 5 mL HPLC-grade water and incubated at 45°C for 2 hours to allow glucosinolate hydrolysis. Next, 10 mL dichloromethane was added and the mixture was mechanically shaken for 20 minutes and centrifuged at 600 x g for

10 min, followed by removal of the dichloromethane layer. Once more, the sample was extracted in dichloromethane, and the dichloromethane layers were combined, creating

~20 mL extract, which was eventually brought up to 25 mL with dichloromethane. A 3 mL aliquot was mixed with 1 mL of conjugating reagent (20 mM triethylamine and 200

32 mM 2-mercaptoethanol in dichloromethane). This mixture was incubated at 30° C for 60 min and then dried under gaseous nitrogen.

Samples were reconstituted in 1:1 water:acetonitrile, then passed through 0.45 μM nylon filters into HPLC vials. Separation and quantification of the 2-mercaptoethanol conjugate of sulforaphane was achieved using a Waters 2695 separations module coupled to a Waters 996 photo diode array detector. 10 μL were injected onto a Waters Symmetry

C18 column (3.5 μm, 4.6 x 75 mm). A linear gradient, consisting of solution A (0.1% formic acid) and solution B (0.1% formic acid in acetonitrile) was employed. Initial conditions of 0% B increased to 50% B over 15 minutes. The 2-mercaptoethanol conjugate of sulforaphane was quantified by creating a standard curve. The 2- mercaptoethanol erucin conjugate was detected, but could not be consistently quantified, so it was excluded from analysis.

4.5. Creation of broccoli sprout and sulforaphane diets

Five diets were produced for this study: a control diet, diets containing either the raw, steamed, or 60°C-treated BSPs, and a diet containing purified sulforaphane. AIN-

93G powder mouse diet was used as the control diet, and a modified AIN-93G diet, equalized for total calories and macronutrient content, was used as the base for the broccoli sprout and sulforaphane diets. The freeze-dried, powdered broccoli sprouts were incorporated into their respective diets at 4% (w/w). For the sulforaphane diet, the diet manufacturer dispersed sulforaphane in the soybean oil prior to blending in the other ingredients, creating a diet with a final concentration of 3 mmol sulforaphane/kg diet.

Isothiocyanate analysis of the sulforaphane diet confirmed that only sulforaphane was present (data not shown). All diets were stored refrigerated until use.

4.6. Animals, housing, and ultra violet light exposure

60 female SKH-1 hairless mice, eight weeks of age, were purchased from Charles

Rivers Laboratories (Wilmington, MA). Mice were divided into five groups, with 20 mice consuming the control diet, and 10 mice each consuming the raw, steamed, 60°C- treated, and sulforaphane diets. Mice were housed five per cage on a 12 hour light/dark cycle in a room maintained at 21-23°C. Prior to beginning of the study, all procedures were approved by the Ohio State Institutional Animal Care and Use Committee. Mice were acclimated to a powder AIN-93G control diet for six days, followed by being fed experimental diets for one week. Fresh food was supplied daily and water was provided ad libitum. 24 hours prior to sacrificing, all mice were exposed dorsally to one minimal erythemic dose, previously determined to be 2240 J/m2. Body weights were measured at several points during the experiment. There were no significant differences in weight gain between the mice consuming the treatment diets. However, all groups consuming treatment diets gained significantly less weight than the control group (data not shown).

4.7. Synthesis of metabolite standards

Isothiocyanate metabolite standards were synthesized based on the method of

Vermeulen and others (2003). Briefly, this method exploits the spontaneous reaction between an isothiocyanate and glutathione, cysteinylglycine, cysteine, or N- acetylcysteine. In short, these mixtures were prepared, which formed the isothiocyanate metabolic conjugates. Next, the mixtures were purified using semipreparative reversed

34 phase chromatography on a 10 x 250 mm, 5 µm C18 Bondpak column (Waters) with a water and acetonitrile mobile phase. Acetonitrile was removed through rotary evaporation, and the remaining aqueous portion was freeze dried, resulting in a powder.

This powder was determined to be greater than 95% pure. Next, it was weighed, and extinction coefficients were determined in triplicate. In methanol at 270 nm, these molar extinction coefficients (M-1 cm-1) were determined to be 7,334 sulforaphane-glutathione,

4,796 sulforaphane-cyteinylglycine, 7,551 sulforaphane-cysteine, 6,832 sulforaphane-N- acetylcysteine, 1,653 erucin-glutathione, 2,302 erucin-cysteinylglycine, 1,609 erucin- cysteine, and 3,391 erucin-N-acetylcysteine.

4.8. Mouse plasma and tissue analysis

Mice were killed by carbon dioxide inhalation. Blood, skin, liver, lung, kidney, and bladders were collected. Blood was collected into EDTA-coated tubes, placed on ice, fractionated within 2 hours of collection, and stored at -80°C until analysis. A 10 mm skin punch was taken for myeloperoxidase analysis, along with an additional section of dorsal skin for isothiocyanate analysis. All tissue samples were frozen in liquid nitrogen immediately after collection and stored at -80°C until analysis.

Plasma samples were prepared for analysis by thawing on ice, then acidifying to

10% with ice cold trifluoroacetic acid. The sample was vortexed, centrifuged for 5 min at

21,000 x g, and the supernatant was removed. With the exception of bladders, all solid tissue samples were prepared by freezing in liquid nitrogen and grinding to a powder with a tissue grinder. 100 mg of tissue powder was transferred to a microcentrifuge tube and

400 μL of 1% formic acid was added. The sample was vortexed, and then probe sonicated

35 for 3 seconds. 50 μL ice cold trifluoroacetic acid was added to precipitate proteins and the sample was vortexed again, followed by centrifugation at 21,000 x g for 5 min. The supernatant was removed and placed on ice. Next, the pellet was resuspended in 450 μL of a 10% trifluoroacetic, 1% formic acid solution and probe sonicated for 3 seconds. The sample was centrifuged and the supernatants were pooled. For kidney only, the pellet was extracted a third time, as the third fraction contained >10% of the total isothiocyanate metabolite content. For bladder analysis, the above procedure was used, except the bladders were weighed and extracted whole, as probe sonication was sufficient to break up the bladder tissue.

Sulforaphane and its metabolic conjugates, as well as the erucin conjugates were separated with a Waters Acuity Ultra Performance Liquid Chromatography (UPLC) system (Milford, MA) interfaced, splitless, with a triple quadruple mass spectrometer

(Quattro Ultima, Micromass, UK). 5 μL of the supernatant was injected directly onto an

Acquity BEH UPLC column (100 x 2.1 mm, 1.7 μM). The mobile phase consisted of

0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Initial conditions consisted of 0% B, which increased linearly to 10% B at 1 min, 33.3% B at 2.5 min, 72%

B at 4 min, and equilibrated back to 0% B by 6 min (curve=1). Selected reaction monitoring of the collision induced dissociation transitions for nine analytes were as follows: sulforaphane (178 > 114), sulforaphane-glutathione (485 > 136), sulforaphane- cysteine (299 > 136), sulforaphane-N-acetylcysteine (341 > 114), erucin-glutathione (469

> 179), erucin-cysteinylglycine (340 > 103), erucin-cysteine (283 > 103), and erucin-N- acetylcysteine (325 > 164). Dwell times ranged from 80-150 ms. The desolvation

36 temperature was 450°C, cone voltage was 35 V, RF1 was 12.5 V, and collision energy was 10-17 eV. The CID argon pressure was 3 x 3-10 mbar. Mixed standard curves were prepared for quantification on the conjugates.

4.9. Myeloperoxidase activity

Myeloperoxidase, an enzymatic marker of neutrophil infiltration and inflammation, was quantified in the skin using a method previously described by Wilgus and others (2003). Briefly, a 10 mm skin punch was homogenized in 1.25 mL of a 0.5% hexadecyltrimethylammonium bromide 50 mM potassium phosphate solution (pH 6.0), with care taken to keep the sample cool during homogenization. Three times, the homogenate was subjected to a cycle of sonicating, freezing, and thawing. Next, the homogenate was centrifuged and the supernatant was removed. Myeloperoxidase activity was quantified spectrophotometrically over 5 minutes at 450 nm.

4.10. Statistics

In vitro glucosinolate and sulforaphane data is the result of thee replicates, and is presented as the average ± standard deviation. All biological data is presented as the average, with standard error of the mean as ± error bars. IBM SPSS Statistics 19

(Chicago, IL) was used to determine the statistical significance of results. One-way

ANOVA was employed, followed by Tukey’s post-hoc test, and results were considered significant at the p<0.05 level.

Figure 3. Schematic of study design

Chapter 5: Results and Discussion

In this study, we were interested in understanding how various processing techniques affect the ability for glucosinolates to convert to isothiocyanates, both in vitro and in vivo. In addition, we sought to determine how the isothiocyanates sulforaphane and erucin are metabolized and distributed in vivo. To accomplish this, we performed several experiments to quantify glucosinolate and isothiocyanate contents of heat processed/freeze-dried BSPs, as well as isothiocyanate metabolite levels in plasma and tissue samples from mice who consumed these BSPs or a diet containing purified sulforaphane. Lastly, we were interested to determine if dietary administration of BSPs or the purified isothiocyanate sulforaphane would affect levels of an inflammatory biomarker in the skin of mice, following exposure to ultraviolet light.

5.1. Glucosinolate analysis

The glucosinolates glucoraphanin and glucoerucin were quantified in the freeze- dried BSPs (Table 1). In many reports, glucoraphanin is the predominant glucosinolate in broccoli and broccoli sprouts (Kensler and others 2005; Kushad and others 1999).

Interestingly, we found the concentration of glucoerucin to be slightly higher than glucoraphanin in all broccoli sprout samples, which may be an effect of the variety of broccoli used in this study. The various processing techniques employed resulted in significantly different glucosinolate levels. Steamed broccoli sprouts had the greatest

glucosinolate concentration, while the “fresh” and 60°C processing techniques resulted in

reduced levels.

Table 1. Glucosinolate concentration of processed broccoli sprout powders; Different letters (glucoraphanin) or symbols (glucoerucin) represent significant differences between processing treatments

The disparity in glucosinolate content is likely caused by glucosinolate hydrolysis

during processing. We noted that the 60°C heat treatment caused a decrease in the

physical structure of the broccoli sprouts, likely releasing some glucosinolates which

mixed with myrosinase and formed hydrolysis products. In the raw and 60°C-treated

sprouts, additional glucosinolate hydrolysis may have occurred during the freeze-drying

process, as any thawing in the freeze-dry flask would have also allowed glucosinolates to

mix with myrosinase. It is unlikely that any glucosinolate hydrolysis occurred in the

steamed broccoli sprouts, as the steaming process inactivated myrosinase.

Going forward, care should be taken to prevent this glucosinolate degradation

during processing. Additional thermal methods for inactivating the epithiospecifier

protein (ESP) which do not cause partial destruction of the physical structure of the

40 sample must be considered. One potential idea would be to first freeze-dry the sample, followed by application of a dry heat. However, glucosinolate degradation in the freeze- dried fresh sample indicates that hydrolysis may have occurred during the freeze-drying process, as well. It is possible that evaporative cooling, which should theoretically keep the sample frozen when under vacuum, was insufficient in actually doing so. This may have been a result of overloading the freeze-dryer flask.

5.2. In vitro glucoraphanin conversion to sulforaphane

Glucosinolates from the BSPs were hydrolyzed in vitro, and the 2- mercaptoethanol conjugate of sulforaphane was quantified (Table 2). The main benefit of the 2-mercaptoethanol conjugation method is that it permits the separation and quantification of individual isothiocyanates, as opposed to the often used cyclocondensation assay, which supplies the bulk sum of all isothiocyanates in a sample.

Unfortunately, only sulforaphane could be accurately quantified in our analysis. The 2- mercaptoethanol conjugate of erucin was detected; however, our analyses did not produce consistently quantitative results. For this reason, it was excluded from analysis. The percent conversion from glucoraphanin to sulforaphane is also reported in Table 2. As discussed above, glucosinolate loss was observed during processing and freeze-drying of the fresh and 60°C-treated BSPs, likely due to degradation to hydrolysis products. Thus, to accurately reflect sulforaphane yields, the percent conversion was calculated by expressing the amount of sulforaphane formed as a percent of the total glucoraphanin content in the steamed broccoli sprouts. This value, 22.5 µmol/g DW, is considered the initial glucoraphanin concentration in all BSPs prior to any loss during processing. As

41 anticipated, steaming resulted in the lowest conversion to sulforaphane, approximately

4%. In the freeze-dried fresh and 60°C-treated BSPs, approximately 23% and 98% conversion was observed, respectively. We believe these vastly different conversion rates reflect the presence or absence of myrosinase and/or ESP. It is apparent that steaming inactivated nearly all myrosinase, as reflected by the extremely low sulforaphane yield.

Conversely, both myrosinase and ESP were active in the fresh BSP. As a result, a moderate amount of glucoraphanin was converted to sulforaphane. Lastly, as has previously been shown, the 60°C heat treatment resulted in substantially higher conversion from glucoraphanin to sulforaphane (Matusheski and others 2004). Though we did not measure ESP activity, we are confident this increase was due to the selective inactivation of ESP, which drives formation to nitriles at the expense of isothiocyanates.

Table 2. In vitro formation of sulforaphane and percent conversion from glucoraphanin; Different letters represent significant differences between processing treatments.

5.3. Conversion to isothiocyanates in vivo

As discussed in Chapter 2, many studies have reported low bioavailability of isothiocyanates after feeding broccoli whose endogenous myrosinase has been

42 inactivated (Clark and others 2011, Egner and others 2011). To our knowledge, no study has reported the effect of feeding ESP-inactivated broccoli or broccoli sprouts. While it is clear that, in vitro, inactivating ESP enhances isothiocyanate formation, one cannot assume the same for in vivo consumption. Conditions affecting in vitro hydrolysis, including pH, temperature, presence of iron, and incubation time are likely different than those for in vivo digestion (Matusheski and others 2004; Vaughn and Berhow 2005;

Williams and others 2010). Thus, we sought to determine differences in the metabolism of mice consuming ESP-inactivated BSP, compared to results from mice consuming steamed BSP, with no active myrosinase, and “fresh” non-heated BSP, containing active myrosinase and ESP. Specifically, we were interested in determining how the various heat treatments would affect the relative conversion of glucosinolates to isothiocyanates in vivo.

Mice were fed the control, broccoli sprout, and sulforaphane diets for one week, after which they were sacrificed. Plasma, liver, skin, bladder, lung, and kidney samples were analyzed, and the sulforaphane and erucin metabolites were quantified in each sample. Data presented are the sum of the glutathione, cysteine, and N-acetylcysteine conjugates for either sulforaphane or erucin. Our method also measured free sulforaphane and the isothiocyanate-cysteinylglycine conjugates, but levels of these compounds were, at most, negligible, and thus excluded from calculations. A sample chromatogram, demonstrating the separation and detection of these metabolites, is shown in Figure 4.

Figure 4. Mass chromatogram of the six isothiocyanate metabolites quantified.

Isothiocyanate metabolites were detected in all plasma and tissue samples of mice which consumed the broccoli sprout diets, indicating that isothiocyanates are readily distributed throughout the body. No isothiocyanate metabolites were detected in mice consuming the control diet. For most tissues, significant differences between metabolite concentrations were observed as a result of the diet consumed, as shown in Figures 5-10.

In all figures, differing letters (sulforaphane) or symbols (erucin) represent statistically significant differences between diets. Variability within mice consuming the same diet was relatively high, particularly in the lung, plasma, and bladder. In the lung, this may just be a result of the extremely low levels distributed to that tissue. Concentrations in the plasma and bladder are more volatile and may be more sensitive to the time passed since the mice last ate, while concentrations in other organs may be more steady state and less affected by the duration since last consumption.

Figure 5. Concentration of isothiocyanate metabolites in plasma; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets

Figure 6. Concentration of isothiocyanate metabolites in liver; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets

Figure 7. Concentration of isothiocyanate metabolites in lungs; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets

Figure 8. Concentration of isothiocyanate metabolites in skin; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets

Figure 9. Concentration of isothiocyanate metabolites in bladder; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets

Figure 10. Concentration of isothiocyanate metabolites in kidneys; Different letters (sulforaphane) or symbols (erucin) represent significant differences between diets. Note: For total sulforaphane, the p-value between steamed and fresh was 0.051

As expected, mice consuming the steamed BSP had the lowest levels of isothiocyanate metabolites in all sample types analyzed. In most cases, these levels were statistically significantly lower than those from mice consuming the “fresh” and 60°C- treated BSPs. Thus, these data affirm the importance of consuming glucosinolates with active myrosinase. Comparing metabolite levels between mice consuming the “fresh” or

60°C-treated BSPs, we observed statistically significant differences at some, but not all tissue sites. As previously discussed, the decrease in glucosinolate concentration in the raw and 60°C-treated BSPs was likely due to the formation of glucosinolate hydrolysis products, such as isothiocyanates. Thus, one limitation of the present study is the inability to conclusively distinguish whether increased isothiocyanate metabolite concentrations in mice which consumed the 60°C-treated BSP diet is due to better conversion to isothiocyanates in vivo, or because some isothiocyanates had already been pre-formed in the BSP during processing, and were thus more bioavailable. For future study, this issue could be avoided by ensuring that glucosinolates remain intact up until consumption.

5.4. Distribution of isothiocyanate metabolites

A pharmacokinetic study of isothiocyanate metabolism and distribution following a single oral dose was recently performed, which gave a glimpse of the isothiocyanate profile in plasma and various tissues at a specific time point (Clarke and others 2011). In contrast, this study provides a more long-term observation on the distribution of isothiocyanate metabolites after consuming an isothiocyanate-rich diet for a relatively longer period of time.

In the present study, a wide range of metabolite concentrations were observed in different tissues varying 50 to 100-fold in concentration. Our data indicate that isothiocyanates are inclined to accumulate at specific sites, particularly the liver, bladder, and kidney. Lower concentrations were detected in plasma, skin, and lung tissue. To our knowledge, this is the first report of detecting isothiocyanate metabolites in the skin.

Considering the liver, bladder, and kidney are all key sites directly related to isothiocyanate metabolism or excretion, it is not surprising that concentrations were higher in these tissues. Previously, Munday and others showed that dietary administration of a broccoli sprout extract significantly inhibited chemically-induced bladder cancer in rats. They note that the bladder epithelium, the major site of bladder cancer development, comes into direct contact with isothiocyanate metabolites found in urine (Munday and others 2008).

5.5. Evidence and implications of sulforaphane/erucin interconversion

The presence of both sulforaphane and erucin metabolites in the plasma and tissue of mice which consumed the sulforaphane diet confirm that sulforaphane is reduced to erucin, in vivo (Figure 11). Previous studies have reported this phenomenon in both animals and humans (Kassahun and others 1997; Bheemreddy and Jeffery 2007; Clarke and others 2011; Abbaoui and others 2012). However, these studies have been limited to plasma and urine analysis. Conversely, by analyzing several tissue sites, we have made the discovery that the sulforaphane to erucin ratio is dependent on the isothiocyanate’s tissue location. In plasma and skin, approximately equal quantities of the two isothiocyanates are present. However, in liver, bladder, and kidney, erucin is the

49 predominant form. Notably, the concentration of erucin was over 11-fold higher than sulforaphane in the liver, despite no erucin being ingested. Only in the lung (the site of respiration) was sulforaphane, the oxidized form, present in statistically significantly greater concentrations. In plasma and at most tissue sites, similar sulforaphane to erucin ratios were observed between mice consuming the BSP diets and mice fed the sulforaphane diet, even though the BSP diets contained both glucoraphanin and glucoerucin, while the sulforaphane diet contained just the purified isothiocyanate. These findings suggest that equilibrium between the two isothiocyanate species is reached, independent of whether individual or multiple isothiocyanates/glucosinolates are consumed.

Figure 11. Concentration and interconversion of sulforaphane and erucin in mice fed a diet containing sulforaphane; an asterisk indicates a significant difference between total sulforaphane and total erucin at each tissue site

These findings have several implications for future work with cruciferous vegetables and isothiocyanates. First, while sulforaphane is widely thought to be the most potent isothiocyanate, the large extent converted to erucin brings into question the importance of which form is consumed. For future study, it would be interesting to feed a diet containing erucin, to determine if the ratio of sulforaphane to erucin at various organs is independent of the isothiocyanate ingested. At this time, it is unknown what drives this interconversion to occur. Additionally it remains to be seen whether conversion from sulforaphane to erucin is relevant to the biological activity of isothiocyanates and cruciferous vegetables.

5.6. Relative abundance of individual metabolites

Figures 12 and 13 compare the relative amounts of individual sulforaphane and erucin metabolites, respectively, in the plasma and tissues analyzed. The relative abundance of individual metabolites was very comparable between all treatment diets, thus, for simplicity, only data from mice consuming the sulforaphane diet is shown.

Figure 12. Relative abundance of individual sulforaphane metabolites in mouse plasma and tissue

Figure 13. Relative abundance of individual erucin metabolites in mouse plasma and tissue 52

In the plasma, skin, liver, and lung, the relative proportions of the glutathione, cysteine, and N-acetylcysteine sulforaphane metabolites are comparable to their matching erucin counterparts. In the plasma, for both sulforaphane and erucin, approximately equal amounts of the three metabolites were present. In the liver, the glutathione conjugate predominates. Interestingly, major differences appear between the sulforaphane and erucin metabolite profiles in kidney and bladder. In the kidney, the relative abundance of sulforaphane-glutathione, cysteine, and N-acetylcysteine conjugates was approximately

5%, 35%, and 60%, respectively. Interestingly, the glutathione, cysteine, and N- acetylcysteine conjugates for erucin accounted for 25%, 60%, and 14% of the total. In the bladder, approximately equal quantities of the three sulforaphane conjugates were observed. However, for erucin, the N-acetylcysteine metabolite was almost non-existent, and the glutathione conjugate accounted for over 60% of the total. This is surprising, considering the N-acetylcysteine conjugate is the predominant form shown to be excreted in the urine (Al Janobi and others 2006; Egner and others 2008).

5.7. Myeloperoxidase activity

Previous work with mouse models has shown that both topical and dietary administration of broccoli sprout extracts may play a role in preventing UV-induced skin carcinogenesis, though no definitive mechanism has been elucidated (Dinkova-Kostova and others 2006; Dinkova-Kostova and others 2010). It is well known that chronic exposure to ultraviolet light increases the likelihood of skin cancer development, and there are concerns that chronic inflammation may increase this risk (Matsumura and

Ananthaswamy 2004; Muller and others 2008). Thus, myeloperoxidase, a biomarker for skin inflammation, was quantified in the skin of mice consuming the control and treatment diets. Previously, Talalay and others (2007) demonstrated that a topical application of a broccoli sprout extract prior to UV exposure reduced myeloperoxidase activity in the skin of mice. We sought to determine if a dietary administration would have a similar impact. Somewhat surprisingly, mice with higher total concentrations of isothiocyanate metabolites in the skin (i.e. those consuming the 60C and SF diets) had, on average, greater myeloperoxidase levels (Figure 14). From our results, it is difficult to determine whether this effect is random or caused by isothiocyanates, because of the inherent biological variability in myeloperoxidase activity. Of note, the concentration of sulforaphane topically applied in the Talalay study (0.5 M) was approximately six orders of magnitude greater than the concentration achieved through the dietary administration.

For future work, mice could be fed diets containing wide ranges of sulforaphane, as doses that vary more will increase the probability of observing an effect if isothiocyanates truly do increase UV-induced myeloperoxidase activity.

Figure 14. Myeloperoxidase activity in the skin of mice consuming control and treatment diets; different letters represent significant differences between diets

Chapter 6: Conclusion

In accordance with other studies, our findings reveal that steaming broccoli sprouts severely hinders glucosinolate conversion to isothiocyanates in vivo. We also found that targeted inactivation of ESP resulted in significantly greater levels of isothiocyanate metabolites at several tissue sites, though it is unclear if this effect was due to greater isothiocyanate conversion, in vivo, or because some isothiocyanates were already formed in the diet, as a result of processing, and were thus more bioavailable.

Isothiocyanate metabolites were distributed to all tissues analyzed, indicating the potential for systemic benefits of cruciferous vegetable consumption. Dietary administration of broccoli sprouts or sulforaphane did not reduce activity of myeloperoxidase, as had been hypothesized, perhaps due to the relatively low levels delivered to the skin. Interestingly, sulforaphane and erucin interconvert in vivo, with erucin being the favored form in certain tissues, particularly the liver. Future research is needed to establish the significance of these findings and to determine whether this interconversion is relevant to the biological activity of isothiocyanates.

References

Abbaoui B, Riedl KM, Ralston RA, Thomas-Ahner JM, Schwartz SJ, Clinton SK, Mortazavi A. 2012. Inhibition of bladder cancer by broccoli isothiocyanates sulforaphane and erucin: Characterization, metabolism, and interconversion. Molecular Nutrition & Food Research 56(11) 1675-87.

Al Janobi AA, Mithen RF, Gasper AV, Shaw NP, Middleton RJ, Ortori CA, Barrett DA. 2006. Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry. Journal of Chromatography B. 844(2):223-34.

Barillari J, Canistro D, Paolini M, Ferroni F, Pedulli GF, Iori R, Valgimigli L. 2005. Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds and sprouts. Journal of Agricultural and Food Chemistry 53(7):2475-82.

Bennett RN, Mellon FA, Kroon PA. 2004. Screening crucifer seeds as sources of specific intact glucosinolates using ion-pair high-performance liquid chromatography negative ion electrospray mass spectrometry. Journal of Agricultural and Food Chemistry 52(3):428-3.

Bhattacharya A, Li Y, Geng F, Munday R, Zhang Y. 2012. The principal urinary metabolite of allyl isothiocyanate, N-acetyl-S-(N-allylthiocarbamoyl)cysteine, inhibits the growth and muscle invasion of bladder cancer. Carcinogenesis 33(2):394-8.

Bheemreddy RM, Jeffery EH. 2007. The metabolic fate of purified glucoraphanin in F344 rats. Journal of Agricultural and Food Chemistry 55(8):2861-6.

Bollard M, Stribbling S, Mitchell S, Caldwell J. 1997. The disposition of allyl isothiocyanate in the rat and mouse. Food and Chemical Toxicology 35(10-11):933-4.

Botting CH, Davidson NE, Griffiths DW, Bennett RN, Botting NP. 2002. Analysis of intact glucosinolates by MALDI-TOF mass spectrometry. Journal of Agricultural and Food Chemistry 50(5):983-8.

Brown PD, Morra MJ. 1997. Control of soil-borne plant pests using glucosinolate- containing plants. Advances in Agronomy 61:167-231. 57

Brüsewitz G, Cameron BD, Chasseaud LF, Görler K, Hawkins DR, Koch H, Mennicke WH. 1977. The metabolism of benzyl isothiocyanate and its cysteine conjugate. Biochemical Journal 162(1):99-107.

Campas-Baypoli ON, Sánchez-Machado DI, Bueno-Solano C, Ramírez-Wong B, López- Cervantes J. 2009. HPLC method validation for measurement of sulforaphane level in broccoli by-products. Biomedical Chromatography 24(4):387-92.

Canene-Adams K, Lindshield BL, Wang S, Jeffery EH, Clinton SK, Erdman JW. 2007. Combinations of tomato and broccoli enhance antitumor activity in dunning r3327-h prostate adenocarcinomas. Cancer Research 67(2):836-43.

Cataldi TRI, Rubino A, Lelario F, Bufo SA. 2007. Naturally occurring glucosinolates in plant extracts of rocket salad (Eruca sativa L.) identified by liquid chromatography coupled with negative ion electrospray ionization and quadrupole ion-trap mass spectrometry. Rapid Communications in Mass Spectrometry 21(14):2374-88.

Chen J, Ruczinski I, Jorgensen TJ, Yenokyan G, Yao Y, Alani R, Liegeois NJ, Hoffman SC, Hoffman-Bolton J, Strickland PT, Helzlsouer KJ, Alberg AJ. 2008. Nonmelanoma skin cancer and risk for subsequent malignancy. Journal of the National Cancer Institute 100(17):1215-22.

Chiang WCK, Pusateri DJ, Leitz REA. 1998. Gas chromatography/mass spectrometry method for the determination of sulforaphane and sulforaphane nitrile in broccoli. Journal of Agricultural and Food Chemistry 46(3):1018-21.

Choi S, Lew KL, Xiao H, Herman-Antosiewicz A, Xiao D, Brown CK, Singh SV. 2007. D,L-Sulforaphane-induced cell death in human prostate cancer cells is regulated by inhibitor of apoptosis family proteins and Apaf-1. Carcinogenesis 28(1):151-62.

Clarke DB. 2010. Glucosinolates, structures and analysis in food. Analytical Methods 2(4):310-25.

Clarke JD, Riedl K, Bella D, Schwartz SJ, Stevens JF, Ho E. 2011. Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement. Journal of Agricultural and Food Chemistry 59(20):10955-63.

Clarke JD, Hsu A, Riedl K, Bella D, Schwartz SJ, Stevens JF, Ho E. 2011. Bioavailability and inter-conversion of sulforaphane and erucin in human subjects consuming broccoli sprouts or broccoli supplement in a cross-over study design. Pharmacological Research 64(5):456-63.

Clarke JD, Hsu A, Williams DE, Dashwood RH, Stevens JF, Yamamoto M, Ho E. 2011. Metabolism and tissue distribution of sulforaphane in Nrf2 knockout and wild-type mice. Pharmaceutical Research 28(12):3171-9.

Conaway CC, Jiao D, Chung FL. 1996. Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study. Carcinogenesis 17(11):2423-7.

Conaway CC, Getahun SM, Liebes LL, Pusateri DJ, Topham DK, Botero-Omary M, Chung FL. 2000. Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli. Nutrition and Cancer 38(2):168-78.

Cornblatt BS, Ye L, Dinkova-Kostova AT, Erb M, Fahey JW, Singh NK, Chen MA, Stierer T, Garrett-Mayer E, Argani P, Davidson NE, Talalay P, Kensler TW, Visvanathan K. 2007. Preclinical and clinical evaluation of sulforaphane for chemoprevention in the breast. Carcinogenesis 28(7):1485-90.

Dinkova-Kostova AT, Fahey JW, Benedict AL, Jenkins SN, Ye L, Wehage SL, Talalay P. 2010. Dietary glucoraphanin-rich broccoli sprout extracts protect against UV radiation- induced skin carcinogenesis in SKH-1 hairless mice. Photochemical & Photobiological Sciences 9(4):597-600.

Dinkova-Kostova AT, Fahey JW, Wade KL, Jenkins SN, Shapiro Ta, Fuchs EJ, Kerns ML, Talalay P. 2007. Induction of the phase 2 response in mouse and human skin by sulforaphane-containing broccoli sprout extracts. Cancer Epidemiology, Biomarkers & Prevention 16(4):847-51.

Dinkova-Kostova AT, Jenkins SN, Fahey JW, Ye L, Wehage SL, Liby KT, Stephenson KK, Wade KL, Talalay P. 2006. Protection against UV-light-induced skin carcinogenesis in SKH-1 high-risk mice by sulforaphane-containing broccoli sprout extracts. Cancer Letters 240(2):243-52.

Egner PA, Chen JG, Wang JB, Wu Y, Sun Y, Lu JH, Zhu J, Zhang YH, Chen YS, Friesen MD, Jacobson LP, Muñoz A, Ng D, Qian GS, Zhu YR, Chen TY, Botting NP, Zhang Q, Fahey JW, Talalay P, Groopman JD, Kensler TW. 2011. Bioavailability of sulforaphane from two broccoli sprout beverages: results of a short-term, cross-over clinical trial in Qidong, China. Cancer Prevention Research 4(3):384-95.

Egner PA, Kensler TW, Chen J, Gange SJ, Groopman JD, Friesen MD. 2008. Quantification of sulforaphane mercapturic acid pathway conjugates in human urine by high-performance liquid chromatography and isotope-dilution tandem mass spectrometry. Chemical Research in Toxicology 21(10):1991-6.

Fahey JW, Talalay P, Kensler TW. 2012. Notes from the field: green chemoprevention as frugal medicine. Cancer Prevention Research 5(2):179-88.

Fahey JW, Zalcmann AT, Talalay P. 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56(1):5-51.

Fahey JW, Zhang Y, Talalay P. 1997. Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proceedings of the National Academy of Sciences 94(19):10367-72.

Fenwick GR, Heaney R, Mullin WJ, VanEtten C. 1982. Glucosinolates and their breakdown products in food and food plants. Critical Reviews in Food Science and Nutrition 18(2):123-201.

Gamet-Payrastre L, Li P, Lumeau S, Cassar G, Dupont M, Chevolleau S, Gasc N, Tulliez J, Terce F. 2000. Sulforaphane, a naturally occurring isothiocyanate, induces cell cycle arrest and apoptosis in HT29 human colon cancer cells. Cancer Research 60(5):1426-33.

Gasper AV, Al-Janobi A, Smith JA, Bacon JR, Fortun P, Atherton C, Taylor MA, Hawkey CJ, Barrett DA, Mithen RF. 2005. Glutathione S-transferase M1 polymorphism and metabolism of sulforaphane from standard and high-glucosinolate broccoli. The American Journal of Clinical Nutrition 82(6):1283-91.

Getahun SM, Chung FL. 1999. Conversion of glucosinolates to isothiocyanates in humans after ingestion of cooked watercress. Cancer Epidemiology, Biomarkers & Prevention 8(5):447-51.

Giamoustaris A, Mithen R. 1996. Genetics of aliphatic glucosinolates. IV. Side-chain modification in Brassica oleracea. Theoretical Applied Genetics 93(5):1006-10.

Giamoustaris A, Mithen R. 1995. The effect of modifying the glucosinolate content of leaves of oilseed rape (Brassica napus ssp. oleifera) on its interaction with specialist and generalist pests. Annals of Applied Biology 126(2):347-63.

Görler K, Krumbiegel G, Mennicke WH, Siehl HU. 1982. The metabolism of benzyl isothiocyanate and its cysteine conjugate in guinea-pigs and rabbits. Xenobiotica 12(9):535-42.

Graham S, Dayal H, Swanson M, Mittelman A, Wilkinson G. 1978. Diet in the epidemiology of cancer of the colon and rectum. Journal of the National Cancer Institute 61(3):709-14.

Haenszel W, Locke FB, Segi M. 1980. A case-control study of large bowel cancer in Japan. Journal of the National Cancer Institute 64(1):17-22. 60

Halkier BA, Du L. 1997. The biosynthesis of glucosinolates. Trends in Plant Science 2(11):425-31.

Hanschen FS, Platz S, Mewis I, Schreiner M, Rohn S, Kroh LW. 2012. Thermally induced degradation of sulfur-containing aliphatic glucosinolates in broccoli sprouts (Brassica oleracea var. italica) and model systems. Journal of Agricultural and Food Chemistry 60(39):9890-9.

Hwang E, Jeffery EH. 2004. Effects of different processing methods on induction of quinone reductase by dietary broccoli in rats. Journal of Medicinal Food 7(1):95-9.

Jemal A, Siegel R, Xu J, Ward E. 2010. Cancer statistics, 2010. CA: A Cancer Journal for Clinicians 60(5):277-300.

Jones RB, Frisina CL, Winkler S, Imsic M, Tomkins RB. 2010. Cooking method significantly effects glucosinolate content and sulforaphane production in broccoli florets. Food Chemistry 123(2):237-42.

Kassahun K, Davis M, Hu P, Martin B, Baillie T. 1997. Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: identification of phase I metabolites and glutathione conjugates. Chemical Research in Toxicology 10(11):1228- 33.

Kensler TW, Chen J, Egner PA, Fahey JW, Jacobson LP, Stephenson KK, Ye L, Coady JL, Wang J, Wu Y, Sun Y, Zhang Q, Zhang B, Zhu Y, Qian G, Carmella SG, Hecht SS, Benning L, Gange SJ, Groopman JD, Talalay P. 2005. Effects of glucosinolate-rich broccoli sprouts on urinary levels of aflatoxin-DNA adducts and phenanthrene tetraols in a randomized clinical trial in He Zuo township, Qidong, People's Republic of China. Cancer Epidemiology, Biomarkers, & Prevention 14(1):2605-13.

Kim MJ, Kim SH, Lim S. 2010. Comparison of the apoptosis-inducing capability of sulforaphane analogues in human colon cancer cells. Anticancer Research 30(9):3611-9.

Kushad M, Brown A, Kurilich A, Juvik J, Klein B, Wallig M, Jeffery E. 1999. Variation of glucosinolates in vegetable crops of Brassica oleracea. Journal of Agricultural and Food Chemistry 47(4):1541-8.

Lamy E, Schröder J, Paulus S, Brenk P, Stahl T, Mersch-Sundermann V. 2008. Antigenotoxic properties of Eruca sativa (rocket plant), erucin and erysolin in human hepatoma (HepG2) cells towards benzo(a)pyrene and their mode of action. Food and Chemical Toxicology 46(7):2415-21.

Mahéo K, Morel F, Langouët,Sophie, Ferrec EL. 1997. Inhibition of cytochromes p-450 and induction of glutathione s -transferases by sulforaphane in primary human and rat hepatocytes. Cancer Research 57(1):3649-52.

Matsumura Y, Ananthaswamy HN. 2004. Toxic effects of ultraviolet radiation on the skin. Toxicology and Applied Pharmacology 195(3):298-308.

Matthäus B, Luftmann H. 2000. Glucosinolates in members of the family brassicaceae: separation and identification by LC/ESI-MS-MS. Journal of Agricultural and Food Chemistry 48(6):2234-9.

Matusheski NV, Jeffery EH. 2001. Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile. Journal of Agricultural and Food Chemistry 49(12):5743-9.

Matusheski NV, Juvik JA, Jeffery EH. 2004. Heating decreases epithiospecifier protein activity and increases sulforaphane formation in broccoli. Phytochemistry 65(9):1273-81.

Matusheski NV, Swarup R, Juvik Ja, Mithen RF, Bennett M, Jeffery EH. 2006. Epithiospecifier protein from broccoli (Brassica oleracea L. ssp. italica) inhibits formation of the anticancer agent sulforaphane. Journal of Agricultural and Food Chemistry 54(6):2069-76.

Matusheski NV, Wallig Ma, Juvik Ja, Klein BP, Kushad MM, Jeffery EH. 2001. Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea. Journal of Agricultural and Food Chemistry 49(4):1867-72.

Mersch-Sundermann V, Knasmüller S, Wu X, Darroudi F, Kassie F. 2004. Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents. Toxicology 198(1-3):329-40.

Mortazavi A, Ralston R, Grainger E, Schwartz S, Clinton SK. 2008. Cruciferous vegetables and bladder cancer. In: Ronald R. Watson, Victor R. Preedy, editors. Botanical Medicine in Clinical Practice. CAB International. p 278-92.

Muller AJ, Sharma MD, Chandler PR, Duhadaway JB, Everhart ME, Johnson BA,3rd, Kahler DJ, Pihkala J, Soler AP, Munn DH, Prendergast GC, Mellor AL. 2008. Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase. Proceedings of the National Academy of Sciences 105(44):17073-8.

Munday R, Munday CM. 2004. Induction of phase II detoxification enzymes in rats by plant-derived isothiocyanates: comparison of allyl isothiocyanate with sulforaphane and related compounds. Journal of Agricultural and Food Chemistry 52(7):1867-71. 62

Munday R, Mhawech-Fauceglia P, Munday CM, Paonessa JD, Tang L, Munday JS, Lister C, Wilson P, Fahey JW, Davis W, Zhang Y. 2008. Inhibition of urinary bladder carcinogenesis by broccoli sprouts. Cancer Research 68(5):1593-600.

Myzak MC, Dashwood WM, Orner Ga, Ho E, Dashwood RH. 2006. Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apc-minus mice. FASEB Journal 20(3):506-8.

Oberyszyn T. 2011. Effects of black raspberries on UV-induced cutaneous inflammation and tumor development. In: Navindra P. Seeram, Gary D. Stoner, editors. Berries and Cancer Prevention. New York, NY: Springer. p 131-42.

Petri N, Tannergren C, Holst B, Mellon FA, Bao Y, Plumb GW, Bacon J, O'Leary KA, Kroon PA, Knutson L, Forsell P, Eriksson T, Lennernas H, Williamson G. 2003. Absorption/metabolism of sulforaphane and quercetin, and regulation of phase II enzymes, in human jejunum in vivo. Drug Metabolism and Disposition 31(6):805-13.

Prochaska HJ, Santamaria AB, Talalay P. 1992. Rapid detection of inducers of enzymes that protect against carcinogens. Proceedings of the National Academy of Sciences 89(6):2394-8.

Shapiro TA, Fahey JW, Wade KL, Stephenson KK, Talalay P. 2001. Chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. Cancer Epidemiology, Biomarkers & Prevention 10(5):501-8.

Shapiro TA, Fahey JW, Wade KL, Stephenson KK, Talalay P. 1998. Human metabolism and excretion of cancer chemoprotective glucosinolates and isothiocyanates of cruciferous vegetables. Cancer Epidemiology, Biomarkers & Prevention 7(12):1091-100.

Shapiro TA, Fahey JW, Dinkova-Kostova AT, Holtzclaw WD, Stephenson KK, Wade KL, Ye L, Talalay P. 2006. Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study. Nutrition and Cancer 55(1):53-62.

Shikita M, Fahey JW, Golden TR, Holtzclaw WD, Talalay P. 1999. An unusual case of 'uncompetitive activation' by ascorbic acid: purification and kinetic properties of a myrosinase from Raphanus sativus seedlings. Biochemical Journal 341(3):725-32.

Śmiechowska A, Bartoszek A, Namieśnik J. 2010. Determination of glucosinolates and their decomposition products—indoles and isothiocyanates in cruciferous vegetables. Critical Reviews in Analytical Chemistry 40(3):202-16.

Song L, Thornalley PJ. 2007. Effect of storage, processing and cooking on glucosinolate content of Brassica vegetables. Food and Chemical Toxicology 45(2):216-24. 63

Steinmetz KA, Potter JD. 1996. Vegetables, fruit, and cancer prevention: a review. Journal of the American Dietetic Association 96(10):1027-39.

Talalay P, Fahey JW, Healy ZR, Wehage SL, Benedict AL, Min C, Dinkova-Kostova AT. 2007. Sulforaphane mobilizes cellular defenses that protect skin against damage by UV radiation. Proceedings of the National Academy of Sciences 104(44):17500-5.

Thangstad OP, Iversen TH, Slupphaug G, Bones A. 1990. Immunocytochemical localization of myrosinase in Brassica napus L. Planta 180(2):245-8.

Thomas-Ahner JM, Wulff BC, Tober KL, Kusewitt DF, Riggenbach Ja, Oberyszyn TM. 2007. Gender differences in UVB-induced skin carcinogenesis, inflammation, and DNA damage. Cancer Research 67(7):3468-74.

Tian Q, Rosselot RA, Schwartz SJ. 2005. Quantitative determination of intact glucosinolates in broccoli, broccoli sprouts, Brussels sprouts, and cauliflower by high- performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Analytical Biochemistry 343(1):93-9.

Vaughn SF, Berhow MA. 2005. Glucosinolate hydrolysis products from various plant sources: pH effects, isolation, and purification. Industrial Crops and Products 21(2):193- 202.

Verhoeven D, Goldbohm RA, van Poppel G. 1996. Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiology, Biomarkers & Prevention 5(9):733-48.

Vermeulen M. 2003. Synthesis of isothiocyanate-derived mercapturic acids. European Journal Of Medicinal Chemistry 38(7-8):729-37.

Vermeulen M, Klo pping-Ketelaars I, van den Berg R, Vaes WHJ. 2008. Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli. Journal of Agricultural and Food Chemistry 56(22):10505-9.

Vermeulen M, van den Berg R, Freidig AP, van Bladeren PJ, Vaes WHJ. 2006. Association between consumption of cruciferous vegetables and condiments and excretion in urine of isothiocyanate mercapturic acids. Journal of Agricultural and Food Chemistry 54(15):5350-8.

Wang GC, Farnham M, Jeffery EH. 2012. Impact of thermal processing on sulforaphane yield from broccoli ( Brassica oleracea L. ssp. italica). Journal of Agricultural and Food Chemistry 60(27):6743-8.

Wilgus TA, Koki AT, Zweifel BS, Kusewitt DF, Rubal PA, Oberyszyn TM. 2003. Inhibition of cutaneous ultraviolet light B mediated inflammation and tumor formation with topical celecoxib treatment. Molecular Carcinogenesis 38(2):49-58.

Williams DJ, Critchley C, Pun S, Chaliha M, O'Hare TJ. 2010. Key role of Fe(2+) in epithiospecifier protein activity. Journal of Agricultural and Food Chemistry 58(15):8512-21.

Williams DJ, Critchley C, Pun S, Nottingham S, O'Hare TJ. 2008. Epithiospecifier protein activity in broccoli: the link between terminal alkenyl glucosinolates and sulphoraphane nitrile. Phytochemistry 69(16):2765-73.

Zhang Y, Talalay P, Cho CG, Posner GH. 1992. A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proceedings of the National Academy of Sciences 89(6):2399-403.

Zhang Y, Cho CG, Posner GH, Talalay P. 1992. Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Analytical Biochemistry 205(1):100-7.

Zhang Y, Kensler TW, Cho CG, Posner GH, Talalay P. 1994. Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proceedings of the National Academy of Sciences 91(8):3147-50.

Zhang Y. 2012. The 1,2-benzenedithiole-based cyclocondensation assay: a valuable tool for the measurement of chemopreventive isothiocyanates. Critical Reviews in Food Science and Nutrition 52(6):525-32.

Zhu N, Soendergaard M, Jeffery EH, Lai R. 2010. The impact of loss of myrosinase on the bioactivity of broccoli products in F344 rats. Journal of Agricultural and Food Chemistry 58(3):1558-63.