2.1.2. Goitrogenic Effects

The reduction of the livestock feeding quality of seed meal following oil extraction is largely due to the presence of thiocyanate ion (SCN-), isothiocyanates and oxazolidine-2 thiones, which all have been shown to be goitrogenic [64].

At neutral pH, myrosinase-initiated hydrolysis of glucosinolates mainly yield isothiocyanates which are relatively stable in aqueous solutions. In comparison and especially at higher pH, isothiocyanates from glucobrassicin and neoglucobrassicin, which belong to indolylglucosinolates, as well as sinalbin may degrade to free thiocyanate ion and further metabolites [65]. McGregor et al. reported in oil-free seed meal from four cultivars of *B. juncea* thiocyanate ion concentration of 5.4 to 6.9 μmol/g whereas the amount of volatile isothiocyanates correspond to 116.9 to 145.9 μmol/g [47]. Mustard seeds of *S. alba* contain the glucosinolate sinalbin, which under physiological condition (pH 5–7) is

decomposed mainly to 4-hydroxybenzylalcohol and thiocyanate ion. Under acidic conditions other degradation compounds were identified, such as 4-hydroxybenzyl cyanide or 4-hydroxybenzylnitrile (2-(4-hydroxyphenyl)acetonitrile) [66]. Paunovic et al. investigated sinalbin degradation products in ground yellow mustard seeds and paste. They identified 2-(4-hydroxyphenyl)acetonitrile as the major degradation product in ground seeds, whereas the most abundant sinalbin degradation product in mustard paste was 4-(hydroxymethyl)phenol. Other degradation compounds identified were 4-methyl phenol, 4-ethyl phenol, 4-(2-hydroxyethyl)phenol and 2-(4-hydroxyphenyl) ethanoic acid [67].

The thiocyanate ion formed by degradation of glucosinolates is known to be a competitive inhibitor of the sodium/iodide (NIS) symporter located on the basolateral membrane of the thyroid follicular cell. Consequently, the iodide uptake by the thyroid gland is impaired which can lead to reduced synthesis of thyroid hormones [68]. The effect is only evident in iodine-deficient diets and increased iodine intake can prevent the adverse effects.

This is not the case for goitrin. Isothiocyanates that are derived from progoitrin (2 hydroxy-3-butenyl glucosinolate) cyclize to produce 5-vinyloxazolidine-2-thione, known as goitrin. Goitrin is a potent inhibitor of the thyroid peroxidase, which plays a central role in the thyroid hormone biosynthesis by regulating organification, iodination and coupling reactions to form thyroid hormones (T3 and T4) in the thyroid gland. This leads to suppressed thyroxine secretion and reduced serum tetraiodothyronine (T4) concentration and can result in a compensatory increase in thyroid glandular mass (known as goiterhyperplasia and hypertrophy). This has been shown in several animal species. Adverse effects in animals were observed with different concentrations of progoitrin/goitrin in the diet, ranging from 71 to 755 μmol/100 g depending on species investigated. In general, ruminants seem to be less sensitive to dietary glucosinolates, unlike pigs, which are severely affected by dietary glucosinolates compared to rabbit, poultry, and fish [63,69].

Besides isothiocyanates, nitriles have been reported to exert goitrogenic effects [70] however only sparse descriptions exist about the mode of action of nitriles on the thyroid gland. More reliable data report adverse effects on liver and kidney functions of this substance class [62]. The formation of nitriles from glucosinolates is favoured under acid conditions or in the presence of Fe(II) ions, whereas epithiospecific protein (ESP) promotes the formation of epithionitriles.

It was shown that nitriles were the dominant glucosinolate products from hydrolysis of aliphatic glucosinolates in cabbage presumably due to the effect of endogenous Fe(II) ions [71]. However, limited data exist regarding the amount or formation of nitriles consuming mustard seeds. Cole et al. showed negligible concentrations of nitriles in seeds of *B. juncea* or *B. nigra* [72] and Choi et al. detected only very small amounts of organic nitriles in serum of rats treated with sinigrin, the predominant glucosinolate in mustard seeds [73].

In principle, exposure of isothiocyanates and goitrin derived from glucosinolates, predominantly sinigrin and gluconapin occurring in mustard seeds, might also be capable of inducing goitrogenic effects in humans. However, no reliable studies exist regarding exposure of goitrogens due to the consumption of mustard and mustard products and higher risk of goiter or related pathologies.

Studies in human volunteers indicate that exposure of goitrogens (e.g., progoitrin) through the intake of realistic amounts of Brassica vegetables does not alter thyroid function. However, if consumed in considerable quantities it may contribute to development of goiter [74,75].

The absence of epidemiological evidence regarding goitrogenic effects after consumption of Brassica vegetables containing high amount of these toxic compounds, such as goitrin from Brussels sprouts, is partly due to the fact that cooking inactivates myrosinase responsible for activation of progoitrin to goitrin, thus reduces the biological availability of goitrogenic breakdown products to sub-clinical levels.

It needs to be emphasized, that mustard seeds or mustard condiment are generally not heat-treated and damage of the seeds might form high amounts of goitrogens whose exposition should still be considered and monitored, especially in iodine-deficient population groups or in subjects where thyroid hormone production is already impaired. Additionally, even if myrosinase activity is diminished, the ability of intestinal microorganisms to readily convert inactive precursors, such as progoitrin, into goitrogenic compounds, should also be considered.

#### 2.1.3. Genotoxic and Carcinogenic Effects

Certain glucosinolate breakdown products have been under observation as possible human carcinogens. Several of these compounds are electrophilically reactive, for example, due to the highly electrophilic central carbon in the isothiocyanate group (-N=C=S), and possibly undergo addition as well as substitution reactions with nucleophiles.

In the past, several isothiocyanates from Brassica vegetables including broccoli tested as freshly prepared juice were investigated using in vitro models and partially demonstrated dose-dependent genotoxic effects in bacterial and mammalian cells [76–80].

Musk et al. observed significant clastogenic activity (chromosome aberration and sister chromatid exchanges) in in vitro cell lines for phenyl isothiocyanate (PITC), phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC), which is one of the major breakdown products of the glucosinolate sinalbin contained in white (*S. alba*) mustard seeds. No mutagenic effects were observed for allyl-isothiocyanate (AITC), known as the predominant hydrolysis product of sinigrin and the major flavour constituent of brown (*B. juncea*) and black mustard (*B. nigra*) seeds [81,82].

With regard to AITC, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) summarized in its scientific opinion on the safety of AITC used as a food preservative genotoxicity tests in in vitro and in vivo studies (summary of the studies in [83]). Positive as well as negative results have been observed in genotoxicity tests with AITC in bacterial and mammalian cells in vitro when no metabolic activation occurred. Mutagenic effects mostly occurred when cytotoxic concentrations of AITC were used. In the presence of metabolic activation, the results were generally negative, even in modified Ames tests with extended preincubation time suggesting that cytochrome P450 isoenzymes seem to be able to reduce markedly the occurrence of mutagenic derivatives. According to the EFSA Panel, reactive oxygen species might be involved in the genotoxic effects of AITC in vitro. On the other hand, no convincing evidence on genotoxicity was shown in in vivo studies performed on different genetic endpoints. Therefore, the Panel did not raise concern regarding genotoxic effects of AITC [83].

Moreover, AITC administered via gavage was not carcinogenic for B6C3F1 mice of either sex, however an increased incidence of transitional cell papillomas of the urinary bladder of F344/N male rats was reported [84]. Possible explanations for the carcinogenic effects of AITC at repeated high doses in male rats consider the accumulation of the corresponding mercapturic acid conjugate of AITC. The AITC conjugate *N*-acetyl-S-(*N*-allylthiocarbamoyl)-L-cysteine is the major urinary metabolite in humans and rats whereby in rats the AITC clearance is slower compared to humans. High concentrations of the N-*acetyl*-cysteine conjugate in the bladder might therefore be a direct irritant on the bladder epithelium or dissociate into free AITC likewise act as irritating substance resulting in regenerative hyperplasia and subsequent benign papillomas. Considering that no genotoxicity in vivo was observed, a threshold mechanism (high-dose response) of AITC underlying these effects in the urinary bladder was assumed [83].

Several aliphatic, aromatic and indole glucosinolates were also tested by Baasanjav-Geber et al. for their mutagenic potential using Ames test and two different *S. typhimurium* strains. Mutagenicity was observed only when myrosinase was added to the test compounds demonstrating the predominant role of the glucosinolate breakdown products with respect to the mutagenic potency. The indole glucosinolate neoglucobrassicin, mainly occurring in broccoli and kale, had the highest increases in the number of revertants in both strains. Sinalbin, which is the main glucosinolate in white mustard (*S. alba*) seeds and hydrolysed to 4-benzyl isothiocyanate (BITC), likewise had a high number of revertants. However, the mutagenic potency of sinalbin was much lower and similar to other glucosinolates tested [85].

In summary, high concentrations of several glucosinolates, especially indolic glucosinolates and their degradation products, isothiocyanates have been shown to be mutagenic in vitro and the results might give some cause for concern. However, clear genotoxic or carcinogenic effects in humans have not been reported [86,87]. In this context, it has to be emphasized that under in vivo test conditions isothiocyanates may be efficiently detoxified [26,76,78].

#### 2.1.4. Evaluation of Toxicological Effects

The characteristic hot and pungent flavour of mustard is based predominantly on isothiocyanates produced from the parent compounds sinigrin and sinalbin, though various types of other glucosinolate-derived products, such as goitrin, may also occur but in much smaller quantities. The described toxic and antinutritional effects of these biologically active breakdown products seem predominantly occur in animal species rather than in humans and with the administration of high doses.

In 2008, EFSA considered the Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluations of miscellaneous nitrogen-containing substances including allylisothiocyanate and benzyl-isothiocyanate. The panel agreed, based on a Maximised Surveyderived Daily Intake (MSDI) approach, with the JECFA conclusion that "No safety concern at estimated levels of intake as flavouring substances" is made, which also applied to allyl-isothiocyanate and benzyl-isothiocyanate, and that "the available data on genotoxicity and carcinogenicity do not preclude the evaluation of the flavouring substances through the Procedure" [88,89].

Except for AITC [83], no comprehensive toxicological evaluation and derivation of health-based guidance values of glucosinolates or their degradation products predominantly occurring in mustard seeds have been identified. In its opinion dealing with the safety of AITC for the proposed use as food additive, EFSA established an acceptable daily intake (ADI) of 20 μg/kg body weight. AITC concentrations in mustard seeds and products vary widely depending on mustard species and process conditions [10,57,83,90]. Assuming an average mustard consumption of 1 g/day containing 1000 mg AITC per kg mustard product, the intake of AITC would be 1 mg per day and would not exceed the ADI of 1.4 mg for an adult weighing 70 kg. However, if the body weight is lower or the AITC concentration is higher, up to 15,000 mg/kg mustard, as it is documented by EFSA [83], would result in exceedance of the ADI, e.g., for children or adolescents.

Numerous factors, such as the activity of myrosinase and specifier proteins as well as processing conditions (temperature, pH, acidic environment) may influence the conversion rate of glucosinolates to isothiocyanates and the formation of more or less, partially not yet known, toxic derivatives.

Early experimental data have indicated that isothiocyanates might be mutagenic and genotoxic in various assays, however evidence of carcinogenicity in experimental animals is inconclusive and human studies are limited. On the opposite, research has recently focused on the anti-carcinogenic properties of constituents of Brassica vegetables, especially of isothiocyanates. Anti-carcinogenic effects have been attributed to the inhibition of cytochrome P450 enzymes, and a concomitant induction of phase II detoxifying enzymes, thereby preventing the activation of pro-carcinogens and improving their conjugation and elimination [91–93].

Therefore, it is not surprising that glucosinolates and their hydrolysis products are already on the market as ingredients in dietary supplements and concentrated herbal preparations. However, most evidence concerning anti-carcinogenic effects and the mechanism of action has come from in vitro or animal studies.

Despite the huge number of epidemiological studies reporting on decreased cancer risk in humans along with a higher intake of Brassica vegetables, an increasing (therapeutic) use of high-dose, partially isolated glucosinolates (isothiocyanates) should be questioned

when clear dose–effect relationships are not clinically proven and the tissue bioavailability is largely unknown. It is certainly possible that some glucosinolates have properties that are both carcinogenic and chemopreventive [94].

Furthermore, it has to be noticed that volatile mustard oils including AITC and goitrin are listed as undesirable substances in animal feed [61]. Pointing out the harmfulness of these compounds and the need of monitoring including exposure assessments when used by humans in concentrated forms is necessary because human studies focusing on adverse effects are still scarce. However, the intake of biologically active glucosinolate breakdown products from mustard seeds is comparatively low and could be considered to be tolerated when for example the ADI established for allyl isothiocyanates from whatever source is not exceeded.
