Levels Of Isothiocyanates Research Articles

Acidification and tissue disruption affect glucosinolate and S-methyl-l-cysteine sulfoxide hydrolysis and formation of amines, isothiocyanates and other organosulfur compounds in red cabbage (Brassica oleracea var. capitata f. rubra)

Cabbages are rich in sulfur-containing metabolites like glucosinolates (GLSs) and S-methyl-l-cysteine sulfoxide (SMCSO). Tissue disruption initiates hydrolysis of these compounds and bioactive volatile hydrolysis products such as isothiocyanates (ITCs), sulfides, and thiosulfinates are formed. However, nitriles, epithionitriles, or amines can also result from GLSs. Here, the influence of hydrolysis time, extent of tissue disruption (chopping vs. homogenization), and addition of lemon juice or vinegar on the outcome of enzymatic hydrolysis of GLSs and SMCSO was investigated in red cabbage. Chopping led to partial hydrolysis of GLSs, whereas homogenization completely degraded GLSs but only had a small effect on SMCSO. Homogenization increased amine formation from alkenyl and methylthioalkyl ITCs, but not from methylsulfinylalkyl ITCs. Acidification inhibited formation of products from SMCSO. Further, it reduced nitrile and epithionitrile formation and stopped amine formation, thereby increasing ITC levels. Therefore, acidification is a valuable mean to enhance ITC levels in fresh Brassica foods.

Comparing two strategies of counter-defense against plant toxins: a modeling study on plant–herbivore interactions

IntroductionVarious herbivorous insects prefer toxic plants as their hosts, although this may appear paradoxical. They have evolved specific adaptations (called counter-defenses) against the toxins. For example, the two-component chemical defense system of plants of the Brassicaceae family against herbivores consists of glucosinolates (GLSs) and the activating enzyme myrosinase. GLS hydrolysis by myrosinase leads to isothiocyanates (ITCs), which are toxic and deterrent to many insect herbivores. Two different types of counter-defenses can be distinguished: a preemptive counter-defense that prevents the GLSs from being hydrolyzed to ITCs due to metabolic redirection and direct counter-defense, where the ITCs are formed but then metabolized to non-toxic conjugates. In general, preemptive counter-defense is only a possibility if the plant stores a precursor of the toxin, which is activated upon attack. Preemptive counter-defense is believed to be more efficient due to the lower exposure to ITCs, but this has not been satisfactorily demonstrated experimentally.MethodsHere, we derive on theoretical grounds the conditions under which preemptive counter-defense reduces exposure to ITCs compared to direct counter-defense by studying the dynamics of GLS defense and counter-defense with two separate ordinary differential equation models. We model how herbivory transfers the GLSs to the insect gut with the leaf material. Thereafter, we describe the insects’ exposure to toxins by deriving the dynamics of ITCs in the gut during feeding with the two types of counter-defenses.Results and discussionBy calculating the area under the curve (AUC) of the ITC concentrations, we show, based on empirical data, that herbivores with a preemptive detoxification system are usually less exposed to ITCs. In addition, our models explain how the decline in the level of ITCs is achieved by both counter-defenses, which helps to understand the overall mechanisms and benefits of these techniques. Our results may also apply to plant defenses by inactive toxin precursors other than GLSs as well as to insects that sequester such precursors for their own defense.

Raphanus sativus functional potential: Impact of the analytical extraction technique on the bioaccessibility interpretation

AbstractRadishes (Raphanus sativus L.) are cruciferous vegetables with remarkable nutraceutical properties given their distinctive isothiocyanates (ITCs) profile. These compounds are formed after glucosinolates‐Myrosinase enzymolysis. Although it is important to characterize radishes' ITCs levels, it is also necessary to evaluate the bioactive compounds' physiological fate after radishes ingestion. To do so, the extraction techniques should adapt to such conditions of high aqueous environment. In this work, we studied the bioaccessibility of ITCs and indole‐3‐carbinol (I3C) in radishes taproots considering the analytical implications of this biological process. Results showed that ITCs and indole profiles in the radish's taproots after aqueous‐myrosinase hydrolysis followed by Dispersive Liquid‐Liquid Microextraction were distinctively different from other reports. After in vitro digestion, raphasatin showed the highest bioaccessibility despite its low quantitative yields. Notably, I3C and S‐Sulforaphene become promising phytochemicals, due to their bioaccessibility and their considerable remaining amounts after digestion.

A Pilot Intervention Designed to Increase Cruciferous Vegetable Intake to Reduce Bladder Cancer Recurrence and Progression

Study Purpose: Bladder cancer is one of the top 10 most common cancers in the US and is the most expensive cancer to treat. The majority of bladder cancers (70–80%) are diagnosed at early stages as non-muscle invasive bladder cancer (NMIBC), which can be removed surgically. However, 50–80% of NMIBC recurs within 5 years, and 15–30% progresses with poor survival. Current treatment is limited. Preclinical and epidemiologic evidence suggests that dietary isothiocyanates (ITCs) in cruciferous vegetables could be a non-invasive and cost-effective strategy to improve NMIBC prognosis. Thus, the purpose of our study was to develop and pilot test a cruciferous vegetable intervention designed to increase ITC exposure in NMIBC survivors. Methods: We used a blinded 2-arm randomized controlled trial (RCT) to test the preliminary efficacy of a 6-month cruciferous vegetable intervention for NMIBC survivors. The intervention consisted of mailed materials, a live phone-call to review the materials, and 11 Interactive Voice Responses calls. Three-day urine samples, 3-day dietary recalls, and survey data were collected at baseline and 6-months. Two-sided Wilcoxon rank-sum tests were used for between-group comparisons. Results: Forty-nine NMIBC patients diagnosed in 2018–2019 were randomized and 42 (22 treatment, 20 control) were retained for 6-month follow-up. Participants were mostly male (71.4%) and White (90.5%), with an average age of 67.4 years. Compared to the control, the treatment group reported 35.0 g (95% CI: −14.9, 85.0; P = 0.014) higher Cruciferae intake (treatment = 58.6 ± 54.5 g vs. control = 23.6 ± 96.1g) and increased urinary ITC levels by 11.1 μmol/g creatinine (treatment = 14.2 ± 24.5 vs. control = 3.1 ± 13.9, P = 0.027) at 6-month follow-up. Conclusion: Our behavioral cruciferous vegetable intervention is the first to significantly increase cruciferous vegetable intake and ITC levels in NMIBC survivors. Our evidence-based dietary intervention has the potential to offer an affordable, scalable option for NMIBC survivors to reduce their risk of recurrence and improve outcomes.

Validity of dietary isothiocyanate intake estimates from a food frequency questionnaire using 24 h urinary isothiocyanate excretion as an objective biomarker: the JPHC-NEXT protocol area.

Isothiocyanate (ITC) is formed via the hydrolysis of glucosinolates by myrosinase, found in cruciferous vegetables. Although myrosinase is inactivated by the cooking process, no studies have incorporated the effect of cooking into the estimation of dietary ITC intake or evaluated the validity. We evaluated the validity of dietary ITC intake estimated from a food frequency questionnaire (FFQ), and urinary ITC levels using 24 h urine samples or a WFR (weighed food record), and evaluated the reproducibility of dietary ITC in two FFQs administered at an interval of 1-year. The JPHC-NEXT Protocol Area included a total of 255 middle-aged participants across Japan. We calculated dietary ITC intake from WFR and two FFQs by assuming that cooked cruciferous vegetables contain zero ITC. Urinary ITC excretion was measured at two points during summer and winter. The validity and reproducibility of dietary ITC intake estimated by FFQ were assessed using Spearman's correlation coefficients. Although we observed a moderate correlation between dietary ITC intake derived from a 12-day WFR and urinary ITC excretion, notwithstanding the cooking process, the correlation between dietary ITC intake estimated by FFQ and mean urinary ITC excretion was low. However, the correlation was improved when we compared urinary ITC excretion and a 3-day WFR or FFQ collected during winter. Our FFQ showed good reproducibility. Although seasonality is a critical factor, dietary ITC intake estimated using an FFQ showed moderate validity and reproducibility and can be used in future epidemiological studies.

Effects of cooking methods on total isothiocyanate yield from cruciferous vegetables.

Cruciferous vegetables are primary sources of dietary isothiocyanates (ITCs), a group of phytochemicals showing promising cancer‐chemopreventive activities in multiple cancer models. However, no study has thoroughly examined how cooking affects the yields of ITCs from cruciferous vegetables. In this study, a high‐performance liquid chromatography (HPLC)‐based cyclocondensation assay was performed to examine the ITC yields from four major cruciferous vegetables (broccoli, cabbage, cauliflower, and kale) under six cooking conditions (stir‐frying, steaming, microwaving, boiling, stewing, and chip‐baking for kale only) and measured the level of ITCs under the raw condition for a comprehensive list of cruciferous vegetables and ITC‐containing condiments. A wide range of ITC yields was found across vegetables and condiments. Cooking significantly altered the ITC yields, showing an averagely four‐fold increase by lightly cooking (stir‐frying, steaming, and microwaving) and a 58% decrease by heavily cooking (boiling, stewing, and chip‐baking). These findings will provide the evidence‐based cooking guidance on cruciferous vegetable consumption and help better estimate dietary ITC exposure in epidemiologic studies.

Volatile Profile of Wall Rocket Baby-Leaves (Diplotaxis erucoides) Grown under Greenhouse: Main Compounds and Genotype Diversity

Wall rocket is a leafy vegetable with pungent flavor related to the presence of isothiocyanates (ITCs). Despite interest in it as a crop of high organoleptic quality, the variability of the volatile profile in the species remains unknown. Twenty-four populations grown under a greenhouse were evaluated. A considerable diversity for the total levels of volatiles was found, providing information of the aroma intensity among accessions. ITCs represented the main fraction. Allyl ITC was the main compound, and levels showed up to 6-fold difference among populations. The esters fraction was mainly represented by cis-3-hexenyl isovalerate and cis-3-hexenyl butyrate, with 20-fold differences among populations. Additionally, the content in sinigrin was evaluated as main GSL in wall rocket. Differences reached up to 13-fold. These results suggest that some populations can be used to develop highly pungent varieties, whereas some others can be selected for mild-pungent varieties, as it is the case of DER045 with low levels of ITCs and high in esters. The presence of several ITCs in the profile also suggested the presence of other novel GSLs. Overall, the work increases the knowledge in the variability of wall rocket for the volatile profile and sinigrin accumulation, a starting point for future breeding programs.

Quantification of Total Glucosinolates and Isothiocyanates for Common Brassicaceous Vegetables Consumed in the US Market Using Cyclocondensation and Thiocyanate Ion Measurement Methods

Glucosinolates are a group of important sulfur and nitrogen containing plant secondary metabolites found in Brassicaceous vegetables that are associated with many health benefits. There is a growing interest in assessing dietary glucosinolates and their degradation products’ isothiocyanates exposure to humans. Determination of glucosinolates in plants is a very challenging task. In this study, the total glucosinolates from 22 commonly consumed Brassicaceous vegetables in the US market were determined by employing 1,2-benzenedithiol cyclocondensation for the determination of isothiocyanates (ITCs) from non-indole glucosinolates and a colorimetric method for the determination of thiocyanate ion (SCN−) from indole glucosinolates. The total glucosinolates were determined from the sum of these two methods. In the 22 investigated vegetables, the highest level of total ITCs was found in horse radish (5189.0 ± 544.8 nmol/g dry weight) and the highest level of total SCN− level was found in brussel sprouts (1294.0 ± 141.6 nmol/g dry weight). The total glucosinolates ranged from 18.6 ± 3.2 to 2271.2 ± 215.3 nmol/g dry weight. The total glucosinolate and ITC data are necessary for the USDA glucosinolates database and important for establishing the relationship of glucosinolates to human nutrition.

Glucosinolates metabolism and redox state of rocket ( Eruca sativa Mill.) during germination

Isothiocyanates (ITCs) exhibit anticancer and antioxidant activity. ITCs are formed from the catalysis of glucosinolates (GLs) by myrosinase (MYR) in Brassicaceae vegetables. This study investigated the GL content, MYR activity, ITC formation, and redox state of rocket upon germination. Banye and Dongsheng were selected from four cultivars by determining growth state and ITC metabolism. In both cultivars, GL levels were the highest on the second day of germination and significantly declined on the eighth day. The 4-day-old sprouts had the highest ITC level. MYR exhibited maximal activity in Banye and Dongsheng on the fourth and the second day of germination, respectively. Regarding the antioxidant system, the sprouts had more chlorophyll, total phenol, and anthocyanin content, and they had higher DPPH scavenging effects compared with raw seeds. Moreover, Dongsheng showed greater scavenging capacity than Banye in general. Practical applications Rocket belongs to the Brassicaceae family, which has been widely considered a good source of bioactive components to prevent tumor formation and reduce the incidence of cardiovascular disease and cancer given the abundance of glucosinolate (GLs). GLs could be catalyzed by myrosinases (MYR) and converted into biologically active products including isothiocyanates (ITCs). After germination, MYR is activated and more ITCs are produced. Using rocket sprouts as a daily diet or raw material is beneficial for human health.

Hydrogen Sulfide (H₂S) Releasing Capacity of Isothiocyanates from Moringa oleifera Lam.

Moringa oleifera Lam. is rich in phytochemical compounds especially glucosinolates (GSs) and isothiocyanates (ITCs), which are active compounds for cancer chemoprevention benefits of Brassicaceae vegetables. In this study, we determined the total contents of GSs and ITCs and their specific profiles in different Moringa tissues including seeds, stems, leaves and roots. Seeds (seeds with shell and seed kernel) showed significantly higher levels of total GSs and ITCs than that of other Moringa tissues. The hydrogen sulfide (H2S) releasing capacity of total ITCs extracted from different Moringa tissues was determined by lead (II) acetate assay in 24-well plates. The H2S releasing capacity of different Moringa tissues were evaluated and compared. Moringa seeds showed the highest H2S releasing capacity, followed by roots, leaves and stems. Our results suggest that Moringa based foods may exhibit health benefits due to its GSs and ITCs contents that are the precursors for H2S, in addition to the recognized action mechanisms of ITCs.

A nested case-control study of cruciferous vegetables intake, urinary isothiocyanates level and lung cancer risk among men in urban Shanghai

Objective: To investigate the association between consumption of cruciferous vegetables (CV), level of urinary isothiocyanates (ITC) and the risk of lung cancer among man in urban Shanghai. Methods: A nested case-control study was conducted within the Shanghai Men's Health Study. Using incidence density sampling with a 2∶1 control to case selection ratio, 885 controls were selected to match 443 lung cancer cases diagnosed prior December 31, 2010. A food-frequency questionnaire was administered to estimate CV consumption. The high performance liquid chromatography method was applied to measure urinary ITC level. The CV intake and urinary ITC level were divided into quartiles according to distribution of control group. The lowest quartile was as a reference group. Conditional logistic regression model was used to analyze the relationship between CV intake, urinary ITC level and the risk of lung cancer. Results: The cruciferous vegetables intake median (P(25), P(75)) in cases and controls were 80.05 (46.89, 129.04) and 97.68 (55.25, 151.72) g/d (Z=-3.93, P<0.001). The urinary ITC level were 1.256 (0.474, 3.836) and 1.244 (0.484, 3.004) μmol/g Cr (Z=-0.39, P=0.697). After adjusting for potential confounding factors such as age, education level, smoking and alcohol consumption, for urinary ITC level, the OR(95%CI) for the highest quartile(≥3.004 μmol/g Cr) was 1.25 (0.87-1.80) compared with the lowest quartile(<0.484 μmol/g). For CV intake, the OR(95%CI) for the highest quartile(≥151.71 g/d) was 0.66 (0.43-1.02) compared with the lowest quartile(<55.25 g/d). Conclusion: No association was found between the CV intake, urinary ITC level and lung cancer risk in men.

Formation of Sulforaphane and Iberin Products from Thai Cabbage Fermented by Myrosinase-Positive Bacteria.

Myrosinase-positive bacteria from local fermented foods and beverages in Thailand with the capacity to metabolize glucosinolate and produce isothiocyanates (ITCs) were isolated and used as selected strains for Thai cabbage fermentation. Enterobacter xiangfangensis 4A-2A3.1 (EX) from fermented fish and Enterococcus casseliflavus SB2X2 (EC) from fermented cabbage were the two highest ITC producers among seventeen strains identified by 16S rRNA technique. EC and EX were used to ferment Thai cabbage (Brassica oleracea L. var. capitata) containing glucoiberin, glucoraphanin and 4-hydroxyglucobrassicin at 430.5, 615.1 and 108.5 µmol/100 g DW, respectively for 3 days at 25 °C. Different amounts of iberin nitrile, iberin, sulforaphane and indole 3-acetonitrile were produced by spontaneous, EX- and EC-induced cabbage fermentations, and significantly higher ITCs were detected (p < 0.01) with increased antioxidant activities. Iberin and sulforaphane production in EX-induced treatment peaked on day 2 at 117.4 and 294.1 µmol/100 g DW, respectively, significantly higher than iberin at 51.7 µmol/100 g DW but not significantly higher than sulforaphane at 242.6 µmol/100 g DW in EC-induced treatment at day 2. Maximum health-promoting benefits from this functional food can be obtained from consumption of a liquid portion of the fermented cabbage with higher ITC level along with a solid portion.

Hydrolysis before Stir-Frying Increases the Isothiocyanate Content of Broccoli.

Broccoli is found to be a good source of glucosinolates, which can be hydrolyzed by endogenous myrosinase to obtain chemopreventive isothiocyanates (ITCs); among them, sulforaphane (SF) is the most important agent. Studies have shown that cooking greatly affects the levels of SF and total ITCs in broccoli. However, the stability of these compounds during cooking has been infrequently examined. In this study, we proved that the half-lives of SF and total ITCs during stir-frying were 7.7 and 5.9 min, respectively, while the myrosinase activity decreased by 80% after stir-frying for 3 min; SF and total ITCs were more stable than myrosinase. Thus, the contents of SF and total ITCs decreased during stir-frying largely because myrosinase was destroyed. Subsequently, it was confirmed that compared to direct stir-frying, hydrolysis of glucosinolates in broccoli for 90 min followed by stir-frying increased the SF and total ITC concentration by 2.8 and 2.6 times, respectively. This method provides large quantities of beneficial ITCs even after cooking.

배추의 가공에 따른 glucosinolates 및 isothiocyanates 함량 변화

This study examined the total glucosinolate (GSL) and isothiocyanate (ITC) contents according to different processing conditions; fresh Kimchi cabbage (Brassica rapa L. ssp. pekinensis), salted Kimchi cabbage and kimchi (storage temperature 4℃ and 20℃) using two different cultivars (Bomatnorang and Chunkwang). Four GSL peaks representing gluconapin, glucobrassicanapin, glucobrassicin and 4-methoxyglucobrassicin were detected in Kimchi cabbage by HPLC and HPLC/MS analysis. The total GSL contents of fresh Kimchi cabbage of Bomatnorang and Chunkwang were 21.37±1.06 μg/g dry weight (DW) and 20.96±3.33 μg/g DW, respectively. After salting, the total GSL contents of salted Kimchi cabbage decreased by 39% and 52% in Bomatnorang and Chunkwang, respectively. Finally, the total GSL contents of kimchi after storage at 20℃ decreased by 83% and 56% in Bomatnorang and Chunkwang, respectively. The extracted ITC contents were analyzed by GC/MS. Three ITC peaks were detected in Kimchi cabbage representing 2-phenylethyl ITC, 3-butenyl ITC and 4-pentenyl ITC. The 2-phenylethyl ITC levels increased during the salting process but this generally fell during storage at 20℃ as kimchi. The 3-butenyl ITC levels of Kimchi cabbage according to processing decreased rapidly due to salting and then decreased slowly during storage as kimchi. The 4-pentenyl ITC of Kimchi cabbage was lost during the salting process. The results for the change in GSL and ITC contents during the kimchi making process will be used in the food industry.

Abstract 2622: A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies

Abstract Epidemiological studies have demonstrated an inverse relationship between the intake of cruciferous vegetables and risk of cancers. The protective effects may be attributed to glucosinolates and glucobrassicins in these vegetables. While glucosinolates and glucobrassicins are not chemopreventive, their metabolites, isothiocyanates (ITCs), indole-3-carbinol (I3C), and 3,3′-diindolylmethane (DIM), have been shown to inhibit tumorigenesis in rodent models. In the past studies the total ITCs intake was estimated through food frequency questionnaires and/or urinary total ITC by a HPLC-based method. Self-reported intake of cruciferous vegetables has inherent limitations in estimating the intake of ITCs because of differences in the contents of their precursors present in different kinds of cruciferous vegetables, different locations and methods of cultivation, or different methods of food preparation. Animal and cell culture studies have shown that different types of ITCs have different anticancer properties and potency, suggesting that they are not equal in protecting against the development of cancers. The lack of a reliable method to quantify specific ITCs derived from dietary intake of cruciferous vegetables hinders the investigation of the role of specific ITCs against the development of cancer in human populations. To address this deficiency, we developed a flexible, highly sensitive, fast and reliable quantitative liquid chromatography mass spectrometry method (LC-MS/MS) for detecting and quantifying common dietary ITCs plus DIM in human urine. Urinary ITCs are measured as mercapturic acid conjugates (ITCs-NAC), predominant metabolite of ITCs, using multiple reaction monitoring (MRM) technique and deuterated [2H3]-ITCs-NAC as internal standards. DIM is quantified in separate experiment using [2H2]-DIM internal standard. The method was validated by a small set of previously analyzed samples from Singapore Chinese Health Study. We found an excellent correlation between previously reported total ITCs levels and sum of ITCs detected by newly developed method. The combination of minimal sample preparation, ultra-performance liquid chromatography, and highly sensitive mass spectrometer resulted in a method that is fast (below 10 min for ITCs and 12 min for DIM) and sensitive (low femtomolar LOQ) amenable to population studies. Citation Format: Marcin Dyba, Jian-Min Yuan, Jennifer Adams-Haduch, Fung-Lung Chung. A LC-MS/MS method to quantify specific dietary isothiocyanates in human urine for epidemiological studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2622.

Myrosinase-dependent and -independent formation and control of isothiocyanate products of glucosinolate hydrolysis.

Brassicales contain a myrosinase enzyme that hydrolyzes glucosinolates to form toxic isothiocyanates (ITC), as a defense against bacteria, fungi, insects and herbivores including man. Low levels of ITC trigger a host defense system in mammals that protects them against chronic diseases. Because humans typically cook their brassica vegetables, destroying myrosinase, there is a great interest in determining how human microbiota can hydrolyze glucosinolates and release them, to provide the health benefits of ITC. ITC are highly reactive electrophiles, binding reversibly to thiols, but accumulating and causing damage when free thiols are not available. We found that addition of excess thiols released protein-thiol-bound ITC, but that the microbiome supports only poor hydrolysis unless exposed to dietary glucosinolates for a period of days. These findings explain why 3–5 servings a week of brassica vegetables may provide health effects, even if they are cooked.

Urinary Isothiocyanates Level and Liver Cancer Risk: A Nested Case-Control Study in Shanghai, China

Experimental studies have provided evidence that isothiocyanates (ITCs) from cruciferous vegetables may modulate carcinogen metabolism and facilitate carcinogen detoxification and reduce cancer risk. However, no epidemiological studies on liver cancer were reported. This study investigates the association between urinary ITCs levels and liver cancer risk among men and women in Shanghai, China. A nested case-control study of 217 incident cases of liver cancer and 427 matched controls identified from the Shanghai Women's Health Study and Shanghai Men's Health Study was conducted. Conditional logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) summarizing the association between urinary ITCs levels and liver cancer risk. Compared to those with undetectable ITCs, nonsignificantly inverse association was observed among detectable (OR = 0.80; 95% CI = 0.51–1.26), below-median (OR = 0.76; 95% CI = 0.47–1.24), and above-median concentration (OR = 0.86; 95% CI = 0.52–1.41) with liver cancer risk. Similar patterns were observed when urinary ITCs levels were categorized into tertiles or quartiles. Although our study firstly focused on the association between urinary ITCs exposure and liver cancer risk, we did not find significant results. Future multicenter prospective, different population studies are warranted to validate our findings.

Effects of a natural toxin on life history and gene expression of Eisenia andrei

Earthworms perform key functions for a healthy soil ecosystem, such as bioturbation. The soil ecosystem can be challenged by natural toxins such as isothiocyanates (ITCs), produced by many commercial crops. Therefore, the effects of 2-phenylethyl ITC were investigated on the earthworm Eisenia andrei using an ecotoxicogenomics approach. Exposure to 2-phenylethyl ITC reduced both survival and reproduction of E. andrei in a dose-dependent manner (median effective concentration [EC50] = 556 nmol/g). Cross-species comparative genomic hybridization validated the applicability of an existing 4 × 44,000 Eisenia fetida microarray to E. andrei. Gene expression profiles revealed the importance of metallothionein (MT) as an early warning signal when E. andrei was exposed to low concentrations of 2-phenylethyl ITC. Alignment of these MT genes with the MT-2 gene of Lumbricus rubellus showed that at least 2 MT gene clusters are present in the Eisenia sp. genome. At high-exposure concentrations, gene expression was mainly affected by inhibiting chitinase activity, inducing an oxidative stress response, and stimulating energy metabolism. Furthermore, analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway implied that the high concentration may have caused impaired light sensitivity, angiogenesis, olfactory perception, learning, and memory. Increased levels of ITCs may be found in the field in the near future. The results presented call for a careful investigation to quantify the risk of such compounds before allowing them to enter the soil on a large scale.

SPE for the simultaneous determination of various isothiocyanates

Several SPE sorbents were investigated for the extraction of a group of chemically diverse isothiocyanates (ITCs). They included bonded silica, carbon-based, and polymer-based sorbents with various functional groups. Results showed large differences in the ability of these sorbents to simultaneously extract ITCs from standard solutions. Recovery rates were on average the highest with divinylbenzene (DVB) based polymeric sorbents, especially with a DVB/N-vinylpyrrolidone copolymer that had recovery rates ranging between 86.7 and 95.6%. These sorbents achieved the most balanced extraction efficiency between aliphatic and aromatic, polar, and nonpolar ITCs. With graphitized carbon, C(18)-bonded silica, and amide-containing sorbent, recovery levels were higher for the two least polar aromatic ITCs (benzyl ITC and phenylethyl ITC), whereas for the polar aliphatic ITCs levels were the lowest. The least retained one, was methyl ITC that is the most polar with recoveries between 0 and 31.5%. The presence of amide groups, especially in a polyamide sorbent, appeared to be particularly unsuitable for the extraction of aliphatic ITCs. A copolymer made up of DVB and N-vinylpyrrolidone was therefore shown to be the most suited for the extraction of both aliphatic and aromatic ITCs.

Abstract B45: A preliminary study of cruciferous vegetable consumption and urinary isothiocyanate levels in African Americans and Caucasians in the eastern United States.

Abstract Cruciferous vegetable consumption is associated with decreased cancer risk for bladder, breast, cervical, colorectal, liver, lung and prostate cancer. Unique to cruciferous vegetables are glucosinolates, phytochemicals that upon contact with myrosinase are converted to isothiocyanates (ITCs). ITCs have been shown to prevent cancer by inducing apoptosis, inhibiting cell cycle progression and histone deacytelase while inducing histone hyperacetylation, and both inhibiting and inducing phase I biotransformation enzymes. Numerous cohort studies examine the association between cruciferous vegetable consumption as reported in food frequency questionnaires and cancer risk, while the Shangai and the Multi-Ethnic Cohort studies examined the association between urinary ITC levels and cancer risk. To validate the use of food frequency questionnaires as measures of cruciferous vegetable consumption in an eastern US population, we examined the correlation between self-reported cruciferous vegetable intake and urinary ITC levels in African American and Caucasian smokers from New York and Wilmington, DE. ITCs can be measured in the urine samples after cyclocondensation with 1,2-benzenedithiole to form 1,3-benzodithiole-2-thione (1,3-BDT). We developed a streamlined method for urinary ITC detection with a limit of detection of 0.1 μM, a limit of quantitation of 0.2 μM, and precision of 1.29 (%RSD) for standards and 1.99 for samples. Standard curves for 1,3-BDT detection is linear in the range of 0.2-200 μM with r2&amp;gt;0.99. The assay has less than 15% variation between trials. In samples of 119 smokers in NY, our findings indicate that African Americans consume on average of twice as much total cruciferous vegetables as Caucasians. In another sample of 91 mostly smokers, we observed detectable levels of ITC in the urine of 33% of the participants compared to 20% detection rate for the NY study. There was no statistical difference in urinary ITC levels between African Americans and Caucasians in either study. The Wilmington study did show a slight difference, with higher frequency of detection in African Americans as compared to Caucasians. Neither study show a correlation between cruciferous vegetable consumption as reported by food frequency questionnaires and urinary ITC levels. In addition, the Wilmington study did not show a correlation between urinary ITC levels and 24 hour recall of cruciferous vegetable consumption. A larger sample size may give more accurate data, but based on these preliminary findings, food frequency questionnaires may not be the best measure of exposure to cruciferous vegetables in the eastern US. Urinary ITC measures have been used to study cancer risk in mostly Asian populations. In an American population, these foods appear to be consumed too infrequently to provide reliable measures of intake. The short half-life of ITCs may also limit its validity as a biomarker in American populations. Citation Format: Anna K. Hull, Odinaka Anyanwu, Nikoia Fredericksen, Titilope Idowu, Ambata Poe, Joshua E. Muscat, John P. Richie, Derrick J. Swinton. A preliminary study of cruciferous vegetable consumption and urinary isothiocyanate levels in African Americans and Caucasians in the eastern United States. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B45.