NUTR*4510 Toxicology, Nutrition & Food PDF

NUTR*4510 Toxicology, Nutrition & Food Unit 7: Natural Toxins in Plant Products  Phytoalexins (plant pesticides) Plant defense mechanisms Physical Thorns Spines C...

NUTR*4510 Toxicology, Nutrition & Food Unit 7: Natural Toxins in Plant Products  Phytoalexins (plant pesticides) Plant defense mechanisms Physical Thorns Spines Chemical Release of toxins (e.g. phytoalexins) to defend against the attacking organism or adverse environmental conditions Deter herbivores (animals, insects) Prevent plant infection by pathogens (fungus, bacteria) Is there increased risk of toxicity from organic crops/foods compared to those grown with synthetic/man-made pesticides? Active area of research Research Question: could organic foods (grown with no added pesticides/herbicides) develop higher levels of phytoalexins…particularly with successive generations (seeds from one year used for the following years crop)? PROBLEM: the concept of phytoalexins is NOT communicated to the public in the same way concerns regarding synthetic pesticides are…..(more on this later)  Phytochemicals & Glycosides - Phyto = plant-derived - Most phytochemicals contain a sugar molecule = “glycoside” - If the sugar is glucose = “glucoside” Glucose or Glycoside other sugar(s) Aglycone (contains any sugar(s)) (without sugar) Myrosinase / Glucoside * Thioglucosidase Aglucone (contains glucose) (without glucose) * - Reaction Catalyzed by endogenous plant enzymes - Heat (e.g., during cooking) may interrupt enzyme activity - (Human) digestive enzymes - secreted from Plant pancreas or small intestine enzyme disruptors - (Human) bacterial enzymes - Bacteria in colon (i.e., the gut microbiota) - Spontaneous chemical reaction - Heat (e.g. during cooking) may enhance these chemical reactions - Depending on the X, the glycoside/glucoside may be more toxic or the aglycone/aglucone may be more toxic - ** some bacteria have myrosinase activity, e.g. Citrobacter species  Glucosinolates - Phytoalexin produced by plants of the Cruciferae/Brassicaceae family, Brassica genus “Brassica Vegetables” , also called Cruciferous Vegetables - Brussel sprouts have highest glucosinolate content - Cabbage has significant (though not the most) glucosinolate content and is a staple food (i.e., consumed regularly and in large quantities) in many countries - > 80 different compounds - Give plants a bitter taste Cooking/heat generally ↓glucosinolate content  Glucosinolate Breakdown Products Glucosinolate Intermediate (a glucoside) Aglucone Myrosinase / Thioglucosidase + D-glucose H2 O + HSO4- glucose Spontaneous Rearrangement Thiocyanates Isothiocyanates (ITC) Cyanogens (Nitriles) - Goitrogenic: - Regulates phase I and competitive phase II metabolic - Very toxic, but a enzymes inhibition of limited amount iodine uptake by formed - Anti-carcinogenic the thyroid gland, leading to effects at low to enlargement of moderate doses, the thyroid gland, - BUT can be Remember the dose i.e., goiter makes the poison!- pro-carcinogenic at high doses - The plant myrosinase enzyme can be activated by cutting or chewing the vegetables, but heating can destroy its activity. - Microbial myrosinase from gut microbiota can also release ITCs in gastrointestinal tract after ingestion of cruciferous vegetables.  Thiocyanates & goiter Development - Thiocyanates, formed from the breakdown of glucosinolates or metabolism of cyanide, inhibit the uptake of iodide by the active transporter on thyroid gland cells - Mimics dietary iodide deficiency and leads to goiter - Conditional toxicity: may be overcome by higher dietary iodide intake (to out compete the thiocyantates). - The EXCEPTION is goitrin (effects of goitrin cannot be overcome by ↑ iodide dietary intake) - ~ 2 billion people worldwide have iodide deficiency - 10% in the North America; 60% in Europe - Lead to a global iodized salt program - Congenital hypothyroidism, caused by the absence or deficiency of normal thyroid secretion affects ~50 million people worldwide - Main preventable cause of cognitive impairment - Presents with cognitive impairment and reduced growth - Iodide deficiency causes ~96% of goiters; thiocyanates cause ~4% of goiters worldwide  Thiocyanates and Goiter Development Diet (source of Iodine, converted to Iodide (I-) in the GIT) 1 Iodide (I-) Glucosinolates Cyanogens Goitrin Thiocyanates Cyanide (HCN) Active transporter 2 3 I- T3/T4 Signals various tissues to regulate metabolic rate Thyroid gland ↑ TSH 4 Low T3/T4 levels 5 goiter, congenital hypothyroidism… thyroid cancer hypothalamus Pituitary TSH = thyroid stimulating hormone  Thiocyanates and goiter Development Iodine is converted to iodide ion (I-) in the GIT - Iodine’s sole function in the body is for synthesis of thyroid hormones: triiodothyronine (T3) and thyroxine (T4), which affect several tissues to regulate metabolic rate 1. Iodide ion (I-) is well absorbed into the blood from dietary sources (e.g., seafood, grains, dairy, etc.) 2. Iodide is actively transported into the thyroid gland 3. Iodide is used to synthesize T3 and T4 via a series of enzymatic reactions 4. Production and secretion of T3/T4 is regulated by thyroid stimulating hormone (TSH) secreted by the pituitary gland - The hypothalamus senses T3/T4 status; when low, it signals the pituitary gland to secrete TSH 5. During dietary iodide deficiency, T3/T4 production decreases, leading to increased TSH secretion from the pituitary gland - Chronic TSH stimulation of the thyroid gland promotes hyperplasia (cell division) in an attempt to capture more iodide and produce more T3/T4; clinically, this swelling of then neck is termed “goiter” - Chronic hyperplasia leads to thyroid cancer - Severe goiter (i.e., iodide and thyroid hormone deficiency) during pregnancy has teratogenic effect during fetal development, causing congenital hypothyroidism resulting in cognitive impairment and reduced growth Thiocyanates & Animal Intakes In humans, ~4% of goiters are attributable to glucosinolates intake, however, there are also concerns for animal intakes High glucosinolate content in feed provided to livestock animals can be dangerous and cause adverse health effects: Reduced feed intake and animal growth Gastrointestinal irritation (which can perpetuate reduced intake and growth) Goiter (effect of thiocyanate formation) Anemia Hepatic (liver) and renal (kidney) lesions (effect of Nitrile formation) Adverse effects are greater in non-ruminant animals (e.g. pigs and chickens) compared to ruminants (e.g. cows). High glucosinolate ingestion in chickens increases mortality and lowers both egg production and egg weight. Glucosinolates can be fatal if consumed by pigs Young animals are more sensitive to the effects of glucosinolates versus older animals.  Other Goiter Inducing Phytochemicals: Rapeseed & Canola - Major world-wide oilseed crop - Grows better in cool/cold temperatures than soy - Contains the glucosinolate: progoitrin, which is converted to goitrin - Goitrin is strongest goiter-inducing metabolite of any glucosinolate due to strong inhibition of iodine uptake by the thyroid gland - Goitrin is goitrogenic - Effects of goitrin cannot be overcome by iodine supplementation Sulfate release can bind trace minerals and contribute to mineral deficiencies glucose Myrosinase / Thioglucosidase H2O Goitrin Progoitrin (+ sulfate (SO4-2) + ( a glucoside) glucose) - “Canola” is a new strain of rapeseed, bred to be LOW in progoitrin - Developed at the University of Manitoba, Canada Canola was originally bred from rapeseed and the plants are similar in appearance. Canola has much lower levels of erucic acid and glucosinolates (such as progoitrin) – the characteristics that make rapeseed undesirable. Erucic acid – monounsaturated omega 9 fatty acid found in many Brassicaceae family plants – highest levels in rapeseed and mustard (over 40% of total fatty acids in these plants is erucic acid) Over time, higher consumption can lead to a heart condition called myocardial lipidosis. This is temporary and reversible. Lipid accumulation (erucic acid) in the heart tissue. Within cells the lipid droplets are physically in contact with the mitochondria → leads to myocardial injury and degeneration Tolerable daily intake of erucic acid is 7 mg/kg of body weight (bw) per day…Children from high consuming populations are more likely to have intakes that exceed this tolerable cut off Most adults consume, on average, between 0.3 to 4.4 mg/kg bw per day or erucic acid Rapeseed meal is used in animal feed → toxicity risk to chickens…develop adverse effects faster compared to larger animal stocks (e.g. pigs and cows)  Glucosinolate Breakdown Products Glucosinolate Intermediate (a glucoside) Aglucone Myrosinase / Thioglucosidase + D-glucose H2 O + HSO4- glucose Spontaneous Rearrangement Thiocyanates Isothiocyanates (ITC) Cyanogens (Nitriles) - Goitrogenic: - Regulates phase I and competitive phase II metabolic - Very toxic, but a enzymes inhibition of limited amount iodine uptake by formed - Anti-carcinogenic the thyroid gland, leading to effects at low to enlargement of moderate doses, the thyroid gland, - BUT can be i.e., goiter - pro-carcinogenic at high doses Remember the dose makes the poison! Cyanogenic Glycosides/Glucosides - a.k.a. Cyanogens - Contain a nitrile group (-CN) - Release hydrogen cyanide (HCN; a.k.a. HCN) - toxic to humans - Lethal Dose, 50% (LD50, amount of toxin required to kill 50% of the population): ~1 mg/kg body weight = ~50-70 mg total - Volatile – rapidly absorbed through GIT, lung, skin 25 g lima beans → 50-75 mg HCN - > 25 different glycosides 10 g bitter almonds → ~50 mg HCN Food Major cyanogen HCN yield (mg/kg food) Cassava roots Linamarin 15-1000 Sorghum leaves Dhurrin 750-790 Flax seed meal Linamarin, Linustatin, 360-390 Neolinustatin Lima beans Linamarin 2000-3000 Bamboo shoots Taxiphylin 100-8000 Apple seeds Amygdalin 690-790 Apricot kernel Amygdalin 785-813 Bitter almonds Amygdalin 4700 -Amongst the various cyanogens: -Amygdalin most common cause of acute HCN toxicity in people living in developed countries -Linamarin most common cause of chronic HCN toxicity worldwide  Cyanogen Metabolism - Requires specific glucosidase AND lyase enzymes - Extracellular - Exposed to intracellular glucoside or glycoside by bruising or mashing - Leads to release of hydrogen cyanide (HCN) Many bacteria in the human intestinal microbiota express the enzyme beta-glucosidase  Cyanide Metabolism HCN can be metabolized via 2 routes: A. Conversion to thiocyanate B. Binding to hydroxy-cobalamin (vitamin B12) A. Conversion to thiocyanate 1. Thiosulfate is formed from SAA - SAA are also substrates for GSH and PAPS production 2. Rhodanase conjugates HCN with thiosulfate, forming thiocyanate 3. Thiocyanate can be excreted via urine; however, if excretion processes are saturated, thiocyanate can competitively inhibit iodide (I-) active transport into the thyroid gland - Chronic thiocyanate production can lead to goiter or thyroid cancer (adults) or congenital hypothyroidism in the developing fetus GSH Cysteine Mercaptopyruvate Thiosulfate 1 (S2O32-) HCN (SO42-) SAA pool Rhodanase (e.g. Methionine) Size of SAA pool Thiocyanate 2 PAPS (HS-CN) can be limiting! Urine 3 Competitive inhibitor of I- active transport Into thyroid gland goiter Thiocyanates and Goiter Development Diet Iodide (I-) Glucosinolates Cyanogens Thiocyanates Cyanide (HCN) Active transporter I- T3/T4 Signals various tissues to regulate metabolic rate Thyroid gland ↑ TSH Low T3/T4 levels goiter, congenital hypothyroidism… thyroid cancer hypothalamus Pituitary  Cyanide Metabolism HCN can be metabolized via 2 routes: A. Conversion to thiocyanate B. Binding to hydroxycobalamin B. Binding to Hydroxycobalamin (vitamin B12) Hydroxycobalamin + HCN → cyanocobalamin → urine - Cyanocobalamin is the supplemental form of vitamin B12 due to its effective absorption - Converted to hydroxy-=cobalamin or methylcobalamin in the body, releasing HCN. ** methylcobalamin is the bioactive form of vitamin B12 - Released HCN is conjugated with thiosulfate, forming thiocyanate - HCN is a conditional toxicity, dependent on: - SAA status for a source of cysteine and sulfate in the production of thiosulfate - Vitamin B12 status to facilitate HCN excretion via urine and prevent thiocyanate production - Iodine status to outcompete thiocyanate for active transport into the thyroid gland, preventing goiter development  Acute Cyanide Toxicity Acute = Immediate and life-threatening effects; can result from a single exposure, higher the dose the more severe the adverse health effects - HCN is a volatile chemical – rapidly absorbed through GIT, lung, skin - Enters cell and binds the heme iron in cytochrome oxidase a3 of complex IV in the Electron Transport Chain… Cytochrome oxidase a3 cannot be reduced, which blocks ATP formation and leads to a build-up of H+ = ↓ pH H+ Builds up; pH decreases - Adverse Health Effects: ontinuum of increasing severity: - Dyspnea (shortness of breath) - Tachycardia - Metabolic acidosis - Coma … Death in 20 minutes – 3 hours Treatment Hydroxocobalamin + cyanide = cyancobalamin (non-toxic) Amyl nitrite or sodium nitrite → both causes formation of methemoglobinemia (methemoglobin binds cyanide) Sodium thiosulfate – enhances the conversion of cyanide to thiocyanate (renally excreted) Problem – methemoglobin cannot bind oxygen (it binds cyanide instead) …risk of cyanide toxicity is the greater threat (versus reduced oxygen carry capacity in the body) so the risk of the intervention is worth the risk.  Taxiphylin Prevention: Processing bamboo shoots through peeling, slicing, washing, and boiling can reduce or remove the cyanogenic glycosides and HCN  Amygdalin ** use of a combination of treatment interventions Cyanide antidote kit = administration of sodium nitrite in addition to a high dose of hydroxocobalamin - Cyanide antidote kits often contain sodium nitrite, but they are risky: Nitrite + Hemoglobin (Hb) → Methemoglobin (metHb) - In MetHb, Iron in the heme group is in the Fe3+ (ferric) state, not the Fe2+ (ferrous) of normal Hb - MetHb cannot bind oxygen; therefore, it cannot carry oxygen to tissues Research Perspective: Swine are commonly used in research as an animal model that is more similar physiologically to humans vs. rodents  Chronic = exposure Chronic Cyanide Toxicity to smaller amounts over time - Due to cyanide exposure in smaller doses over time…the cyanide is still binding to cytochrome a3 of the electron transport chain. 1. Tropical Ataxia Neuropathy (Konzo) (primary effect) - Endemic in many areas of Africa - Demyelination of peripheral nerves → neurotoxicity - Associated with low vitamin B12 status 2. Tropical Amblyopia (primary effect) - Common in West Africa - Optic nerve damage causing a form a blindness - Associated with low vitamin B12 status - Similar to tobacco amblyopia (due to HCN in smoke) 3. Goiter…can lead to thyroid cancer - Secondary effect to thiocyanate production (to clear the HCN from the body) - Common in cassava-dependent populations - Associated with low iodine status Cassava Benefits: - Easily grown – low fertilizer requirement - Extremely drought resistant - Consumed around the world in tropical and subtropical areas as a carbohydrate dietary staple - Depended on by ~500 million people worldwide Problems: - High in the cyanogen: linamarin - Low protein (only 1-2% energy from protein), therefore, limited SAA content - Vitamin B12 is found primarily in animal meat, which is not often abundant in areas that are dependent on cassava as a dietary staple ….therefore, tend to COMBINE low B12 status with high linamarin intakes - Cassava is often grown in areas of low soil iodine and typically these regions have no dietary iodine fortification programs  Cyanogen Processing Methods are Important! Considerations: - Cyanogens: Not toxic as parent glycoside Heat stable Resistant to digestion in stomach and small intestine May be cleaved by colonic bacteria! - Βeta-Glucosidase and hydroxynitrile lyase enzymes: Sensitive to heat →denatured with cooking - HCN: Volatile – easily absorbed in the GIT, but also easily lost in cooking/drying food preparation processes Dangerous processing technique: crush raw plant material in water - Mixes cyanogenic glycoside with the endogenous plant glycosidase and lyase enzymes, releasing HCN Safe processing technique #1 (often used for cassava): 1. Remove skin, which contains higher levels of cyanogens 2. Crush raw root in water and incubate: endogenous plant glucosidases and lyases release HCN 3. Dry and grind to a flour: volatizes HCN. Volatilize means evaporate or disperse in a vapour 4. Add water and ferment again in case plant enzymes are still active 5. Cook: volatize residual HCN - Process is labour intensive and requires water, which may not be available, particularly during drought conditions Safe processing technique #2 (often used for lima beans): 1. Boil food in water: heat inactivates plant glucosidase 2. Cross fingers! Colonic bacteria may release HCN BEWARE OF MISINFORMATION Examples of unsafe amygdalin use to combat cancer…. - Vitamin C increases cyanogen metabolism and HCN release, and reduces body stores of cysteine…think of the implications? - Nutrient interactions are rarely considered when combining alternative medicines or supplements by the general public. - More research is required to test the possible adverse interactions between nutrients and phytochemicals (or phytoalexins!!!)  Glucosinolate Breakdown Products Glucosinolate Intermediate (a glucoside) Aglucone Myrosinase / Thioglucosidase + D-glucose H2 O + HSO4- glucose Spontaneous Rearrangement Thiocyanates Isothiocyanates (ITC) Cyanogens (Nitriles) - Goitrogenic: - Regulates phase I and competitive phase II metabolic - Very toxic, but a enzymes inhibition of limited amount iodine uptake by formed - Anti-carcinogenic the thyroid gland, leading to effects at low to enlargement of moderate doses, the thyroid gland, - BUT can be i.e., goiter - pro-carcinogenic at high doses Remember the dose makes the poison!  Isothiocyanates (ITC) Anti-carcinogenic mechanisms: 1. Bind AhR and block large induction of CYP1A1 and CYP1A2 by PAH, HCA, PCBs and dioxins (i.e. AhR antagonist via competitive inhibition) - Phytochemicals are often in higher concentrations than other X - Phytochemicals only mildly induce expression of CYP1A1 and CYP1A2 (i.e. weak AhR agonist) * ‘The dose makes the poison’ Phytochemicals (e.g. ITCs) PAH, HCA, PCBs, dioxins, etc. Mild CYP induction CYP1A1 AhR Large CYP induction CYP1A2 2. Bind and inhibit CYP activity (i.e. CYP antagonist via competitive inhibition) CYP Phytochemicals (e.g. ITCs) Fe // PAH, HCA, etc. ITCs (e.g. sulphorophane) bind to CYP1A1 or CYP1A2 BUT the enzyme is not catalytically active 3. Induce phase II metabolic enzymes and the enzymes that produce the conjugating agents - E.g. via AhR-driven changes in gene expression (which gets translated into protein) General Pathway for Isothiocyanate (ITC) Metabolism Majority are NOT metabolized by P450 enzymes (although binding to AhR and CYP1A1 and 1A2 is possible)…instead many BLOCK the enzymes Direct metabolism by glutathione conjugation “other NAT” γ-glutamyl dipeptidase Mercapturic Acid transpeptidase Conjugate Isothiocyanate (parent compound) We will focus on 3 examples of isothiocyanates: Glucosinolate Isothiocyanate Myrosinase / Thioglucosidase - Glucobarassican → Indole-3-carbinol (I3C) - Gluconasturtiin → Phenethyl isothiocyanate (PEITC) - Glucoraphanin → Sulforaphane  Isothiocyanates (ITC) - Isothiocyanates (ITC) are typically stable in the blood with the exception of 3-indolylmethyl-isothiocyante, which spontaneously degrades to form indole-3-carbinol (I3C) - I3C dimerizes to form diindolylmethane (DIM) for bioactivity Intermediate is NOT stable Thioglucosidase Spontaneous degradation or rearrangement (Dimerization) via condensation reaction 2 I3C molecules → 1 DIM (I3C) - I3C and other ITCs [e.g. 4- methylsulphinylbutyl isothiocyanate (sulforaphane) and phenethyl isothiocyanate (PEITC)]; have been shown to decrease cancer incidence in animal models @ lower doses - Are ITCs the bioactive responsible for association between vegetable consumption and decreased cancer incidence in human studies? Toxicities associated with chronic high dose purified ITC supplements is an active area of research…too much may NOT be good, but low doses (in foods) are beneficial  Indole-3-Carbinol and Condensation Products…not so simple after all - Reactions occur faster in acidic conditions i.e., the stomach!!! Higdon et al., 2007 Research Questions (to still be answered….) What are the biological activities of these other I3C condensation products? What possible interactions exist with other xenobiotics? What is a “safe” level of each product in the body? Do you think I3C supplements are a good idea…or do you need more information?  Additional Influences of I3C and DIM on Xenobiotic Metabolism cytoplasm **review ALL the transcriptional programs of Nrf2 and the Ahr from unit 2! nucleus cytoplasm ** I3C/DIM has “Chaperone” for the AhR = a MILD complex of proteins induction of AhR downstream transcriptional program versus the STRONG induction with PAH or HCA nucleus https://lpi.oregonstate.edu/mic/dietary- factors/phytochemicals/indole-3-carbinol  Anti-Carcinogenic Effects of I3C and DIM *** at LOW doses/intakes Strongest evidence in the literature supporting anti- carcinogenic effects in these types of cancer https://researchoutreach.org/articles/exciting-advancements- ovarian-cancer-treatment/  HIGH Doses of Indole -3-Carbinol (I3C) Estimated intake of glucobrassicin in the western diet is approximately 12.5 mg (fresh sources) and 7 mg (cooked sources) per person/day Average daily consumption of I3C is approximately 0.1 mg/kg body weight. Brassica consumption in the traditional Japanese diet is approximately 1.6 mg/kg body weight of I3C for a 70 kg person The daily dose of 200 to 400 mg of I3C has been used in clinical trials. These daily exposures are equivalent to 2.9 to 5.7 mg/kg for a 70 kg person. Higher doses in long term animal studies (receive daily gavage dose equal to 12.5 to 50 mg/kg body weight in a human) caused: liver cancer benign tumors in the small intestine, mesenteric lymph node, thyroid gland , uterus, stomach and nose Hepatoblastoma (arrows) = liver tumor USA Dept Health & Human Services, Toxicology Studies of I3C, 2017 REVIEW: General Pathway for Isothiocyanate (ITC) Metabolism Are not metabolized by P450 enzymes Direct metabolism by glutathione conjugation “other NAT” γ-glutamyl dipeptidase transferase Isothiocyanate (parent compound) Note: gamma-glutamyl transferase is also called transpeptidase (see our phase II unit notes) We will focus on 3 examples of isothiocyanates: Myrosinase / Glucosinolate Thioglucosidase Isothiocyanate - Glucobarassican → Indole-3-carbinol (I3C) - Gluconasturtiin → Phenethyl isothiocyanate (PEITC) - Glucoraphanin → Sulforaphane Phenethyl isothiocyanate (PEITC) An ITC found in cruciferous vegetables such as broccoli and watercress (richest sources) but also brussel sprouts, cabbage, turnips 1 ounce of watercress = 2-6 mg PEITC High bioavailability Potent inducer of phase II enzymes (GST, SULT) + antioxidants (NQO1) In animal GIT cancer models PEITC decreases formation of aberrant crypt foci (ACF; colon tumor precursors) chemically induced tumors in stomach and colon (co- administration of PEITC + various X…including HCA) PEITC anti-cancer mechanisms (summary…many signaling mechanisms involved) Increases apoptosis of cancer cells Decreases cancer cell proliferation and tumor growth Inhibits angiogenesis (limits the blood supply to a developing tumor) Inhibitor of the CYP2 family → alters the metabolism of some X Inactivates CYP2E1 Can COMPETE for binding to CYP2E1 with other xenobiotics NEGATIVE OUTCOME – PEITC binds to GSH and reduces the amount of GSH available within the tumor microenvironment to quench ROS reactions…more ROS produced by tumor cells can cause more DNA damage (see next slide) Observational data show that polymorphisms in GST are associated with altered cancer risk GST-M1 null or GST-T1 null → metabolize or eliminate ITCs at a slower rate (protective against cancer) Prevalence = ~10-21% in Caucasian populations vs. ~64% in Asian populations  Metabolism of PEITC Dissociate from glutathione and influence cell signaling (anti-cancer effects, many mechanisms here) Encoded by GST-M1, GST-T1 & others) Enzymes cleave Glu and Gly **still biologically Excretion active form (similar process activity levels to the parent compound) …still able to exert anti- Mercapturic acid conjugate cancer effects prior to excretion NOTE: while PEITC is bound to GSH….GSH is NOT AVAILABLE to bind to reactive intermediates or quench ROS (e.g. produced during Phase I reactions and within tumors), leaving these ROS free to continue to cause tissue, cellular and/or DNA damage…the higher the intake of PEITC, the greater the demand on cellular GSH availability Sulforaphane Physical damage to the plant (i.e. chewing) mixes the glucosinolate with the enzyme Glucoraphanin Sulforaphane (a glucosinolate) (an ITC) Induces many of the same anti-cancer effects as PEITC Inhibits CYP1A2 activity Activator/inducer of Nrf2 (via interaction with sulfhydryl groups → releases Nrf2 from Keap1) to ↑ mRNA expression of oxidant defense enzymes (plus others we’ve discussed!) Increase GSH production (if SAA are available) via increased expression of glutamate-cysteine ligase (rate limiting enzyme in GSH synthesis) Increases GST expression/activity (Phase II enzyme) Sulforaphane also induces the expression of other phase II enzymes (SULT, UDPGT, NAT) Stimulates expression of other anti-oxidant enzymes → quench ROS via electron reductions: glutathione reductase (GSR), glutathione peroxidase (GPX), thioredoxin (TXN), NAD(P)H:quinone oxidoreductase 1 (NQO1) Comparative BIOAVAILABILITY of Common Phytochemicals (absorbed and reaches the blood stream) Variety of Quercetin-rich foods Andrographis Plant (South Asia) Tumeric Milk thistle Houghton 2019, Ox Med Cell Long Glucoraphanin Sulforaphane Glutathione - Anti-cancer effects conjugation - Anti-inflammatory effects - Anti-oxidant effects (including reducing oxidized LDL- cholesterol) Excretion Many studies demonstrating anti-inflammatory effects of sulforaphane in animals and humans… let’s look at one study example…. IL-6 CRP - Overweight/obese subjects - 30 g fresh broccoli sprouts per day = 51 mg of Glucoraphanin → Sulforaphane (confirmed in the blood and urine) - Measured plasma levels of two well documented inflammatory mediators (CRP, IL-6) over time (± dietary intervention)  Sulforaphane – Low Doses Beneficial, High Doses lead to Toxicity Toxicity studies showed that at high doses of sulforaphane can result in: Sedation (at 150–300 mg/kg) Hypothermia (at 150–300 mg/kg) Impairment of motor coordination (at 200–300 mg/kg) Decreased skeletal muscle strength (at 250–300 mg/kg) Death (at 200–300 mg/kg) Some supplements contain BOTH purified Sulforaphane AND Glucoraphanin Some supplements also contain myrosinase, which will increase the conversion of glucoraphanin to sulforaphane Given the bioavailability of sulforaphane…COULD BE EXPOSED TO UNSAFE INTAKE LEVELS when consuming a supplement? GSTM1 & GSTT1 positive → wildtype, normal GST enzyme expression and function GSTM1 null & GSTT1 null - SNPs for reduced expression and/or low enzymatic activity - Slower phase II (glutathione conjugation reactions) - Beneficial SNP for the effects of ITCs…perhaps not a beneficial SNP for the phase II reaction clearance of reactive intermediates produced from other xenobiotics Do you see anything wrong with the interpretation of the results in this abstract?  Glycoalkaloids - Potatoes are the world’s 3rd largest crop with >374 million tons produced every year - Largest vegetable crop in Canada with > 4 million tons produced every year - Per capita intake: 86 kg / year in Europe 63 kg / year in North America 14 kg / year in Asia - Family: Solanaceae…also called the “nightshade family” - Other Solanaceae family members (besides potatoes) = tomato, egg plant, tobacco - Many Solanaceae family members contain the phytoalexins called glycoalkaloids, these include - Solanine → bitter tasting, found in all nightshade plants, most toxic; pesticidal properties to protect the plant - Tomatine → fungicidal properties, is toxic and high in wild tomatoes and under-ripe (green) tomatoes - Chaconine → found in all nightshade plants (second most toxic) - Quantitatively, potato GAs are the most abundant natural toxin in the food supply -Potatoes → solanine and chaconine (90%, but lower amounts -Egg Plant → α-solasonine and α-solamargine (infrequently studied) -Tomatoes → tomatine and deyhdrotomatine (in green tissue), esculeosides (in the red tissue)  A) plant biosynthesis of tomatidine and solanidine from cholesterol, catalyzed by GAME enzymes (glycoalkaloid metabolizing enzymes) B) Conversion of tomatidine to α-tomatine C) Conversion of solanidine to α-solanine, and α-chaconine Reactions utilize SGT enzymes (solanidine glycosyltransferases) -Factors affecting potato GA content: -Most abundant in the skin (typical of phytoalexins) – i.e., peeling potatoes reduces their GA content -Higher with breeding for disease resistance -Increased by any pest/environmental/mechanical damage (e.g. insects, frost, during harvest, etc.) * Potatoes continue to produce GA during storage -Not affected by cooking, except deep frying (due to high heat and oil)…this doesn’t mean eat only french fries!!! -Not well absorbed in the GIT -Effectively excreted Where are the glycoalkaloids located in the potato? Potatoes are tubers = the underground part of the plant stem https://www.slideshare.net/Adrienna/glycoalkaloids-in-potatoes-hk-tcf-2016  Glycoalkaloids (GA) Nitrogen containing Sugar (i.e., glucose) containing e.g. Solanine - GAs are also glycosides - The parent GA/glycoside is toxic – i.e., GAs do not need to be cleaved to be bioactive - Solanine is most abundant GA in potatoes: ~2-15 mg/100g potato - Other glycoalkaloids found in potatoes = chaconine  Glycoalkaloids & Toxic Effects Adverse health effects from higher intakes of glycoalkaloids are usually related to consumption of potatoes that show signs of physical change or damage (e.g. sprouting, greening, bruising). Symptoms = bitter or burning sensation in the mouth flu-like symptoms such as nausea, vomiting, stomach and abdominal cramps, and diarrhea. More severe cases may be accompanied by a variety of neurological effects (i.e., drowsiness, apathy, restlessness, shaking, confusion, weakness, and disturbed vision). There are a few reports of deaths being attributed to glycoalkaloid exposure from the consumption of potatoes, potato leaves, and potato berries. Health Canada has established a health-based maximum level of 20 mg total glycoalkaloids per 100 g (fresh weight) of potato tuber. This maximum level is applicable to all potatoes that are commercially sold in Canada. Potatoes continue to produce glycoalkaloids during storage, to avoid keep in a cook, dark, dry place and cut away sprouts and green areas on the potato surface  Glycoalkaloids: Mechanisms of Action 1. Inhibition of acetylcholinesterase - Acetylcholinesterase rapidly degrades excess acetylcholine within cholinergic synapses - Solanine inhibits acetylcholinesterase, causing hyperactive cholinergic synapses Solanine and other GAs 2. Detergent effects on cell membranes - The alkaloid portion of GAs is lipid-soluble – i.e. has affinity for cholesterol - GAs bind cholesterol and disrupt cell membrane organization, which disrupts cell signaling pathways/signal transduction 3. Hemolysis of Red Blood Cells Overall, toxicity is due to a combination of these mechanisms -Toxicity is rare, but occurs and is sometimes fatal -Acute health effects: Vomiting, dyspnea, tachycardia, coma…rarely death -Chronic health effects: Teratogen, e.g. neural tube defects (although the data is controversial)  Tomatine – high in unripe (i.e., green) and wild tomato varieties Toxic effects of α-tomatine include vomiting, diarrhea, lethargy, confusion, weakness and depression In the unripe (green) tomato → phytosterols (structure similar to cholesterol) is converted to tomatine by the family of plant enzymes called GAME enzymes (glycoalkaloid metabolizing enzymes). Tomatoes of varying degrees of ripeness → α-tomatine content and toxicity risk decreases as the fruit ripens As the tomato ripens, α- tomatine is converted to either saponins or aescins. **Only tomatine shown here, there are other glycoalkaloids in tomatoes a saponin; we discuss this class or “anti-nutrient” later in the unit! Tomatine Content Higher concentrations of tomatine in the flower, leaf and stem of the plant (should be avoided). LIMIT unripe (green) Gives a bitter tomato intake flavour/taste LD50 stands for "lethal dose 50" and is a measurement of how much of a substance is required to kill half of a group of test animals Average mouse weight = 28 g …or 0.5 mg/g BW Works out to 14 g of tomatine Unripe tomato has 465 mg/kg fresh weight = 0.465 mg/g of tomato OR 30 g of green tomato to reach the oral LD50 Antinutrients - Toxic factors that act by interfering with the absorption or function of nutrients - Classification (by type of nutrient involved): i. Proteins or amino acids - E.g. Soybean protease inhibitors (discussed in this unit) ii. Minerals - E.g. Phytate – binds and interferes with metal ion utilization (discussed in this unit) - E.g. Thiocyanates – interfere with iodide utilization (already discussed) iii. Vitamins - E.g. Thiaminases – impair bioactivity of thiamin (vitamin B1) (discussed in this unit) - E.g. Avidin – binds and inactivates biotin (vitamin B7) - By mechanism: i. Prevent absorption – e.g., Phytate, avidin ii. Destroy nutrient – e.g., Thiaminases iii. Interfere with tissue utilization – e.g., Thiocyanates Soybeans - World soybean supply: ~150 million metric tons - 90 million metric tons in the USA with estimated value of 10.6 billion dollars - Best converter of sun energy to dietary protein - High in dietary protein and oil: each soybean seed contains 20-23% oil and 39-45% protein - Oil is converted to margarine, shortening, mayonnaise, salad oils, and salad dressing - Meal is used as high-protein animal feed for the production of eggs, poultry, and pork - AA content compliments that of grains - Easily grown and maintained - Does not require fertile soil - Effective pest resistance due to protease inhibitors, lectins, saponins, and phytate  Soybean Protease Inhibitors 2 families: 1. Kunitz inhibitors: Anti-trypsin 2. Bowman-Birk inhibitors: Anti-trypsin and anti- chymotrypsin Roles in plants: 1. Inhibit proteolysis of seed protein until germination 2. Phytoalexin – i.e., pesticide Effects on animals: - Growth depression due to poor dietary protein absorption - Pancreatic enlargement in an attempt to increase protease secretion into the small intestine for increased dietary protein absorption - Heat (e.g., cooking) denatures soybean protease inhibitors; therefore, humans rarely experience detrimental health effects, but still a concern for animals - Soybean protease inhibitors are acid stable – i.e., resistant to denaturing by stomach acid ~70% of the soybeans grown in the USA are used for animal feed, with poultry being the number one livestock sector consuming soybeans, followed by hogs, dairy, beef and aquaculture. Soybean protease inhibitors: Mechanism of action 1. Trypsin (a protease) converts zymogens into their active enzyme form, which cleave peptide bonds to release free amino acids and small peptides for absorption → block trypsin, block protein digestion 2. Chewing raw soybeans releases their protease inhibitors 3. Protease inhibitors are acid stable – i.e. resistant to denaturing by stomach acid 4. Protease inhibitors inhibit trypsin, which inhibits the digestion of peptides to amino acids and smaller peptides for absorption Raw soybean 5. Undigested peptides stimulate an enteric nervous system receptor to MOUTH signal to the pancreas to produce and Mechanical breakdown 2 secrete more zymogens into the small intestine, in an attempt to release more Protease inhibitors free amino acids and small peptides for absorption STOMACH - In response, the pancreas undergoes hyperplasia in an attempt to increase its HCl secretion 3 capacity to produce and secrete zymogens Zymogens (inactive) 4 Peptides 1 Absorption Signals for more zymogen 5 production and secretion Enteric nervous system receptor - Undigested peptides are digested by colonic bacteria, releasing ammonia (NH3) which is absorbed and incorporated into the urea cycle Phytic Acid/Phytate Roles in plants: 1. Phytic acid is principal storage form of phosphorus in many plant tissues, especially bran and seeds – i.e. rich in whole grains and legumes 2. Phytoalexin - At physiological pH, the phytic acid phosphates are partially ionized, resulting in the phytate anion - Undigestible – non-ruminant animals (i.e., monogastrics, humans; pigs) lack the required phytase enzyme Effects on humans: - Phytate has strong binding affinity and capacity (with 6 binding sites) for dietary minerals: calcium, magnesium, iron, and zinc, ultimately inhibiting their absorption - Major contributor to world-wide mineral deficiency Phytate Processing - Milling, soaking, and/or fermentation, e.g.: 1. Mill wheat to a flour - Mechanically disrupts phytate 2. Soak in water with added yeast and sugar = dough 3. Incubate at warm temperature - Yeast decreases pH which activates wheat phytase enzyme, ultimately digesting phytate and releasing wheat minerals for absorption (and preventing phytate from binding to other dietary minerals) - Still, unleavened breads (i.e. made without yeast) are staple foods in several parts of the world… - No activation of plant phytase enzymes - Still have the effects of phytate and the binding of other minerals to phytate (thereby decreasing their bioavailability)  Phytate & Mineral Absorption/Risk of Mineral Deficiencies Susceptible minerals include zinc, calcium, magnesium, iron…they are commonly bound to phytate. Humans do not express the phytase enzyme 1. Zinc (Zn) - higher levels of zinc in animal proteins vs. plant sources - Oysters (4 oz = 40+ mg Zn…≥UL (40 mg/day) **can also reach UL with supplemental Zn Zn TOXICITY: -intestinal pain, nausea, vomiting, diarrhea, headache - Depressed immune function - ↓HDL cholesterol levels - @ 5-6 times UL = zinc binds other minerals and decreases their absorption (e.g. copper, iron ) - ~20-30% of dietary zinc is absorbed…poor/low body Zn status results in increased Zn absorption (typically) - Higher requirements during growth, sexual development and pregnancy Phytate and Minerals Zinc (Zn) ~20-30% of dietary zinc is absorbed, which can be further reduced by dietary phytate Iron supplements (non-heme iron) and calcium can inhibit zinc absorption Vegetarians will consume less dietary zinc (@greater risk for deficiency vs. animal protein consumers) Particularly true for high phytate consumers Animal protein, methionine and histidine can INCREASE zinc absorption Vegetarians consuming unleavened breads (no yeast to inactivate phytate) + limited dietary zinc + high phytate = greater risk for deficiency Zinc can bind other minerals and decreases their absorption (e.g. copper, iron) **nutrient interactions when co-consumed (as they always are) can be very complicated Zinc Deficiency: Disrupts cell division Delayed sexual maturation & causes impotence Eye and skin lesions, hair loss Impaired immune function → increased incidence of infection  Phytate and Minerals 2. Magnesium Phytate induced magnesium deficiency causes: Muscle cramps, seizure, nausea weakness, cognitive impairment Hypomagnesemia (low blood Mg) Osteoporosis (increased risk of bone fractures)_ 3. Calcium Absorption reduced by binding factors found in plant sources of Ca+2 (seeds, nuts, spinach , swiss chard) – phytate – oxalates – other minerals bind Ca+2 and ↓ its bioavailability (Zinc , magnesium, iron, phosphorus) – Low vitamin D status will adversely impact dietary calcium absorption (vitamin D stimulates expression of intestinal calcium transporters) Calcium deficiency → loss of bone structure leading to osteopenia and osteoporosis (increased risk of fractures) **Common Mg+2 and Ca+2 deficiencies also in chronic alcohol consumers Phytate and Minerals 4. Iron (Fe) Heme iron found in animal-based foods (hemoglobin, myoglobin) Non-heme iron found in animal products (without hemoglobin or myoglobin) or plant-based foods (90- 95% of dietary iron) Absorption efficiency rate of non-heme iron = 2-4%; ~1%...very low in vegans Iron absorption is decreased by: Phytates Oxalates (oxalic acid) found in spinach, some whole grains, chard, rhubarb Fibre Vegetable proteins (e.g. soy protein) Other Minerals: zinc, calcium Iron Deficiency Erythropoiesis (↓ work capacity, low transferrin, reduced production of heme) Anemia (↓RBCs, heme, hemoglobin, fatigue, impaired immune and cognitive function  Benefits of Phytate? Higher intakes/exposure to phytate may enhance the activity of natural killer (NK) cells and inhibit tumor growth (active area of research)…must be balanced with potential impact on mineral absorption Phytate e.g., effect on NK cells Microbial phytase Phytate metabolites Effects are context dependent Liberates bound minerals in the GIT, Anti-cancer effects sadly largest microbiota is in the colon limiting the benefits for mineral absorption Oxalates or Oxalic Acid Chelating agent for metal cations → binds calcium, iron, sodium, potassium, magnesium (anti-nutrient) ↓ absorption and bioavailability of minerals ORAL Exposure/Adverse Effects: nausea, severe gastroenteritis and vomiting, shock and convulsions if exposed at higher concentrations. Oxalic acid can bind calcium from the blood to form calcium oxalate, which can precipitate in the kidney tubules, joints and brain. This is the most common component in kidney stones. Chronic oxalic acid exposure can have long-term effects on bone health and bone integrity/structure. Common Dietary Sources of Oxalic acid or Oxalates: Spinach family; brassica family of vegetables; rhubarb 1-1.5 g oxalates/ 100g of fresh vegetable sources (wet weight or fresh weight)  Oxalates or Oxalic Acid Dietary sources AND can be produced in the body Industrial Uses (synthesized exogenously/extracted) cleaning agent for minerals and metals accumulated on surfaces, especially for the removal of rust (iron complexing agent) Beekeepers use as a mitacide against the parasitic varroa mite TOXICITY (from dietary sources and industrial uses/exposures) fetal defects If inhaled → inflammation of respiratory tract; respiratory distress Mitochondrial dysfunction (impaired ATP synthesis) Kidney precipitates (kidney stones) can cause kidney failure Lowest lethal dose = 600 mg/kg Lethal oral dose = 15-30g (1500g or 1.5kg of raw vegetable sources)…lower intake levels will cause toxicity symptoms…what about supplements?  Other Anti-Nutrients Avidin (found in raw egg whites) Binds biotin (vitamin B7) and prevents its absorption Over time can lead to biotin deficiency (thinning hair, scaly rash, depression, lethargy) Biotin is important in metabolism, functions as a cofactor in TCA cycle reactions, lipogenesis, gluconeogenesis, etc. Amylase Inhibitors which prevent the action of the enzyme amylase (breaks the glycosidic bonds of starches and other complex carbohydrates), preventing the release and absorption of monosaccharides Found in beans (legumes) and cereal grains (wheat) Lipase Inhibitors (e.g., tetrahydrolipstatin), which interfere with lipases that catalyze hydrolysis of triglycerides Found in some plants e.g. panax ginseng (also known as Asian or Chinese ginseng) Saponins Legumes (soybeans, beans, peas, lentils, etc.) are the main saponin containing foods Lower levels found in asparagus, spinach, Allium species (e.g., onion, garlic) → GIVE A BITTER TASTE & plants are less palatable to predators Content reduced by soaking/washing prior to consumption (if you discard the soak water) Structure = highly amphipathic with a hydrophobic aglycone backbone and hydrophilic sugar molecules…this gives saponins a foaming and emulsifying properties. Saponins are a diverse family of compounds https://upload.wikimedia.org/wikipedia/commons/thumb/5/58/Solanine_chemical_structure.png/220px-Solanine_chemical_structure.png Fat soluble Water soluble Saponins General Mechanism of Anti-nutrient Action (there are many Saponins) Bind nutrients directly and prevent their absorption Inhibit function of digestive enzymes → decreasing protein digestion and absorption (protease inhibitor) Toxicity in humans is rare; impaired nutrient absorption across a spectrum of nutrients is possible due to saponins amphipathic nature Not recommended for dogs (found in low levels in some dog foods) → causes diarrhea, vomiting, cramping and pain. Regular contact with saponins can cause skin irritation, such as red bumps, flaky patches and hair loss Saponins, an anti-nutrient that’s not all bad (?) Active area of research, beneficial “dose” is controversial 3 types of saponins (triterpenoid, steroid and steroidal glycoalkaloids) once synthesized are further modified by plant P450 and UGT enzymes to form specific types Saponin metabolite biology gets very complicated and is beyond the level of our course Saponin Health Benefits (active area of research in cell culture and animal studies), where they have been shown to decrease: Blood lipid concentrations Cancer risk (decreasing tumor cell proliferation and increasing apoptosis) Blood glucose response Dental carries Platelet aggregation Remember: the dose makes the poison  Thiaminases Variable resistance to denaturation by heat Image result for thiaminases" Thiaminase Thiamin e.g. niacin or Pyrimidine Thiazole pyridoxine Thiamine Pyrophosphokinase Thiamine Pyrophosphate (TPP) Biologically active form Thiaminases destroy thiamin (vitamin B1) and are a family of enzymes expressed in: Bacteria: express the enzymes Thiaminase II or Thiamin Hydrolase, which breaks down free thiamin, but not (TPP, the bioactive form of B1) Fungi → Thiaminase II Plants → Thiaminase I Ruminants → Thiaminase I Fish (fresh and salt water species) and shellfish species → Thiaminase I Thiaminases can accumulate up the food chain  Thiaminases Plants and ruminants express the enzyme thiaminase I (which degrades all forms of thiamin) Thiaminases from plant sources are heat stable Some phytochemicals can interfere with the absorption of thiamin OR lead to the degradation of thiamine by non- thiaminase-mediated mechanisms (e.g. higher thiamine degradation at neutral (pH 7) versus acidic conditions (pH 5.5): Flavonoids (quercitin/rutin) found in onions, apples, berries, etc. (many dietary sources) Polyphenols and catechol derivatives (e.g. found in tea and other dietary sources) Caffeic acid Chlorogenic acid Tannic acid Thiamin break down can be counteracted with appropriate levels of vitamin C (ascorbic acid) or citric acid RECOMMENDATION Delay the consumption of tea or other tannin-containing products after a meal consume foods high in ascorbic acid (vitamin C) along with the meals heat products containing thiaminase enzymes BEFORE consumption (can help degrade some enzyme activity but is less effective for plant thiaminase sources)  Thiaminases → Thiamin Deficiency Thiamin (B1) in the form of thiamine pyrophosphate (TPP) is required for the function of pyruvate dehydrogenase (converts pyruvate → acetyl CoA) Deficiency syndrome = BeriBeri Inability to metabolize energy → muscle wasting, fatigue and nerve damage (↓ cognitive function) Deficiency risk increases with frequent consumption of thiaminase containing foods Deficiency of thiamin is also common in chronic alcohol consumers = Wernicke-Korsakoff syndrome Individuals typically have low thiamine intakes, but have increased need for thiamine to support metabolism of alcohol as a substrate for energy production Problem: Alcohol decreases thiamine absorption, chronic alcohol intakes can lead to malabsorption of most nutrients, including vitamin B1.