NUTR*4510 Toxicology, Nutrition & Food Unit 4 PDF

NUTR*4510 Toxicology, Nutrition & Food Unit 4: Chemical Carcinogens in Foods First – Neurotoxicity Perspective (then we can discuss cancer) Adverse effect or damage caused to the structure and/or function of any the following: Brain Central Nervous System Peripheral Nervous Sy...

NUTR*4510 Toxicology, Nutrition & Food Unit 4: Chemical Carcinogens in Foods First – Neurotoxicity Perspective (then we can discuss cancer) Adverse effect or damage caused to the structure and/or function of any the following: Brain Central Nervous System Peripheral Nervous System May be permanent or reversible Lipophilic xenobiotics accumulate in lipid-rich regions of the body e.g. brain and/or spinal column Can result in problems with ANY of the following: Cognition Nerve transmission, action potentials, synaptic function Neurotransmitter function Sensory function/perception Motor function Behaviour & emotions  Some Possible Neurotoxicity Phenotypic Manifestations or Characteristics do not memorize. Just gives you an idea of the extent of effects Confusion Poor concentration Memory loss Personality changes Behavioural changes (mood, activity, etc.) Pain (headache or localized elsewhere in the body) Numbness Hyperesthesia (excessive physical sensitivity of the skin) Paraesthesia (tingling feeling or “pins and needles”) Loss of sensation Muscle weakness Loss of coordination, ataxia Difficulty with speech or communication, swallowing Paralysis Involuntary muscle jerks or spasms Seizures Dizziness or vertigo Changes in Sensory function – visual, auditory, taste, smell, touch) Changes to levels of consciousness Respiratory distress (rapid or slowed breathing; shallow breaths; asphyxia) Coma Cardiovascular effects= tachycardia, bradycardia, hypotension, pulmonary edema, etc. Gastrointestinal effects = abdominal pain, diarrhea, vomiting Table: Grading of Common Neurotoxicity Symptoms Common neurotoxicity manifestations: Encephalopathy → damage or malfunction in the brain resulting in an altered mental state. May be accompanied by physical changes difficulty swallowing Dysphasia → impaired speech…could be impaired ability to verbalize speech and/or impaired comprehension of speech Somnolence → feeling sleepy or drowsy Seizure → uncontrolled electric disturbance in the brain, can manifest as any of the following: Change in behaviour Change in movement/spasms Change in consciousness  Let’s Pause and Review some Basic Information about Cancer @ high rate = morphology as the tumor grows larger it will eventually develop a distinct border In Cancer → abnormal cell morphology (structure) and abnormal cell growth (proliferation). rapid uncontrolled cell division  What is Cancer? Group of more than 100 different diseases (different types of cancer in different tissues) Characterized by abnormal cell growth and division Cell growth and proliferation is controlled and highly regulated…when this is lost and cells keep growing and dividing → form a mass of tissue (tumor) abnormal non cancerous growth Tumors can be either benign or malignant cancerous Precancerous conditions (premalignant cells) – abnormal cells that may develop into cancer if they aren’t treated. polyps in the colon Some cells will undergo apoptosis During subsequent rounds of cell division, these cells may pass on the genetic changes and the new cells gradually become more abnormal and eventually become malignant (but this is a long process)  Precancerous (premalignant) Definitions Hyperplasia – an abnormal increase in the number of cells Atypia (atypical) – cells look slightly abnormal under the microscope Metaplasia – cells look normal under the microscope, but are not the type of cell normally found in that tissue or are Dysplasia – cells develop abnormally – in both appearance (phenotypically) and exhibit abnormal organization COMMON PRECANCEROUS CONDITION as these cells develop into a tumor over time eg. ACE -----> grows into a tumor over time EXPERIMENTALLY →Cell culture/cell lines, animal tissues OR biopsy taken from human subjects viewed under the microscope in place. Has not moved, meaning we do not have metastasis cancerous tumor www.wikipedia.com  Metastasis The process of cancer cells spreading to other parts of the body → form new tumors 3 ways: Invasion (direct extension) → the tumor grows into surrounding tissues or structures Through the bloodstream (hematogenous spread) → cancer cells break away from the tumor and enter the bloodstream to travel to a new location secondary organ. Through the lymphatic system → cancer cells travel to other locations by traveling through the lymph vessels to any organ or lymphoid increased angiogenesis https://wirtzlab.johnshopkins.edu/research/cancer- metastasis/ https://www.technologynetworks.com/cancer- research/videos/metastasis-how-cancer-spreads-322917  In Cancer: - Increased cell proliferation due to the increased cell division - Decreased or blocked cell apoptosis cancer cells become non-responsive to the pro-apoptotic signals → results in long-lived rapidly dividing transformed cells contributing to a growing tumor (increasing in size) that ultimately spreads to other locations/tissues in the body (metastasis) or cancer skip G0 and just keep dividing tumor cells will secrete many different growth factors anti-growth live longer Hossain et al., Front Biosci, 2  Examples of Types DNA Damage one of the most point mutations, common DNA lesions G→T is common resulting from ROS. but not only one Affecting Guanine and results in a mismatched pairing of nucleotides Hindi et al., 2021, Cell. Mol. Life Sci Examples of Types DNA Damage Adduct = a segment of DNA bound to a structure (e.g., reactive intermediate)  DNA Repair Mechanisms Importantly, we also have DNA REPAIR MECHANISMS to try and fix damage to the DNA when it occurs: - Base Excision Repair ** most common - Nucleotide Excision Repair - Mismatch Excision Repair - Double Strand Break Repair Base Excision Repair (BER) (in brief) Primary DNA repair mechanism, fix common forms of DNA damage Used for: ROS, single-strand breaks, or alkylating agents (that induce cross-links in the DNA) Enzymes involved: 1. DNA glycolase → recognizes the damage 2. An abasic site (apurinic/apyrimidinic, or AP site) is recognized by AP endonuclease (makes a single strand break) 3. DNA polymerase adds the correct base complementary to the undamaged strand 4. DNA ligase seals the nick in the DNA backbone, completing the repair process Examples DNA Damage or Mutations Leading to the Development of Cancer Mutations leading to impaired function in DNA repair mechanisms → therefore, cells can’t fix/correct any DNA damage E.g. the genes encoding the enzymes involved in DNA repair are damaged! Leading to non-functional DNA glycolase Mutations in tumor suppressor genes → these genes normally function to slow down cell division, help repair damaged DNA and/or induce cell apoptosis…if they’re not functioning properly cells grow out of control. Mutations in cell cycle genes → results in cells continuing to divide/proliferate Mutations in apoptosis genes → results in cells that CANNOT undergo apoptosis and are long-living Mutations in genes that promote cell proliferation → results in a gain of function and INCREASED cell proliferation Chemical Carcinogenesis Process (also called the Cancer Process) HCA, PAH, NA (strong inducers of this process) Ethanol is a weak inducer of this process DNA damage - Repair incomplete or incorrect - DNA replication locks error into new strand Mutation - Mutation may increase expression/function of a gene that favours cell division, or decrease expression/function of a gene that downregulates cell division Initiation ) - Irreversible Promotion - Various stimuli that encourage cell division allow accumulation of mutated/initiated clones (copies of the transformed cell) Progression ) - Accumulation of additional mutations, favoured by rapid cell division (PERMANENT CHANGES) = tumour with distinct boundaries Invasion - Accumulation of additional mutations, favoured by rapid cell division - Angiogenesis, expression of proteases, loss of requirement of cell-cell contact… = Malignant, invasive tumour Metastasis The Cancer Process & Proliferative Growth  do not need to memorize this process. More so understand how we can look at things under a microscope to see that we can see cancer. Problem that we might not be able to take samples of the right area. Experimentally → collect a tissue sample (biopsy from animals or humans) and examine under the microscope https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/carcinogenesis In this remainder of this unit we’ll discuss some examples of dietary carcinogens…from the perspective of xenobiotic metabolism Mycotoxins Polycyclic aromatic hydrocarbons (PAH) Heterocyclic amines (HCA) Nitrosamines Mycotoxins Mycotoxins = toxic metabolites made by molds/fungi -Likely act as growth regulators and defense molecules (analogous to phytoalexins in plants…we will discuss these later in the course!) Molds grow on a variety of different crops and foodstuffs including cereals, nuts, spices, dried fruits, apples and coffee beans, often under warm and humid conditions. Mold growth can occur either before or after crop harvest, during storage, on/in the food itself often under warm, damp and humid conditions. Most mycotoxins are chemically stable and survive food processing (stable at temperatures >160 °C. Exposure to mycotoxins can happen either directly by eating infected food or indirectly from animals that are fed contaminated feed, in particular, from milk. Mycotoxins can cause a variety of adverse health effects and pose a serious health threat to both humans and livestock. Aflatoxin is the most potent. The adverse health effects of mycotoxins range from acute poisoning to long-term effects such as immune deficiency and cancer. Short term aflatoxin exposure example: turkeys exposed to 100-200 parts per billion of aflatoxin exhibited signs of immune deficiency and liver damage within 2 weeks. Approximately 5 billion people exposed to aflatoxins annually  General Paradigm for Mycotoxin Effects Form DNA adducts = chemical or X binds directly o the DNA - causes DNA strand breaks, mutations and/or misreading of the DNA - can be used experimentally/clinically as a biomarker for exposure to a particular toxin or xenobiotic. - Genotoxic → damage DNA Mycotoxins also bind lysine residues in proteins → alters protein function and can have diverse possible biological outcomes impacted as a consequence Can lead to cell apoptosis BUT if widespread within a tissue it can adversely affect tissue function leads to strand breakages Altered signalling -Bind and damage pathways guanine/cytosine (GC)- rich regions of DNA, leads to altered protein function Metabolite-lysine adduct Phase II reaction urine undergoing a P2 reaction is what we want. Since it can get excreted Mycotoxins Food product(s) Mycotoxin Commonly Contaminated Toxicity Aflatoxins Peanuts, grain, berries, -Hepatocarcinogen corn, tree nuts, spices, Produced by Aspergillus oilseeds species need to know these yellow=major sources. CYP3A4, CYP2A6 and Ones we should know CYP1A1/2, CYP2E1 Fumonisin Corn and several other -Neurotoxic grains (rice, wheat, barley, -Hepatotoxic Produced by strains of maize, rye, oat, and millet) -Nephrotoxic Fusarium species Possible carcinogen CYP1A, 4A, 2B, 2C11, High levels of this toxin → Esophageal & liver cancer 2E, and 3A1 occur when hot, dry in humans weather is followed by a period of high humidity Ochratoxin Grains (wheat, rye barley), -Nephrotoxic coffee, spices, grapes, produced by Aspergillus beer, wine -Probably carcinogenic and Penicillium molds CYPIA1/IA2, 2B1 and 3A1/3A2 Fusariotoxins: Wheat, corn, oats barley - GIT inflammation and produced by Fusarium and other grains under bleeding → prolonged can ↓ species normal weather conditions, nutritional status e.g. but especially in cool, wet - Severe gastroenteritis Zearalenone, conditions - Highly toxic if inhaled → Deoxynivalenol dyspnea - Skin blisters with topical CYPIA and CYP3A exposure families **All have similar acute effects: nausea, vomiting, abdominal pain, etc.  Aflatoxin Exposures (Similar for Other Mycotoxins) Larger concern in hot/humid climates B1, G1 and G2 are as an FYI Different forms of aflatoxin (AFB1, AFM1, B1, G1 and G2) → result in toxicity AFB1 – found in contaminated crops AFM1 – found in milk of animals that consumed AFB1 in their food Broad implications - nausea, vomiting, Common for increasing abdominal pain, Outcome susceptibility convulsions, and to other other signs of conditions: acute liver injury. e.g. Infections or Chronic diseases https://www.nature.com/articles/s41598-018-35246-1  Aflatoxin Metabolism Also P450 BOTH AFB1 and AFM1 metabolism in will form an 8,9- the lung epoxide reactive (inhaled intermediate spores) & skin (direct contact) Aflatoxin (parent compound) GSH/GST Excretion Aflatoxin B1-8,9-epoxide + others (urine) Highly toxic reactive intermediate Bind lysine residues in proteins → alters their function we empathize liver damage though that it can affect the kidney as well we make aflatoxin B1-8,9-epoxide quickly since so many CYP metabolize AFB1 and AFM1 Mycotoxins Food product(s) Mycotoxin Commonly Contaminated Toxicity Aflatoxins Peanuts, grain, berries, -Hepatocarcinogen corn, tree nuts, spices, Produced by Aspergillus oilseeds species CYP3A4, CYP2A6 and CYP1A1/2, CYP2E1 Fumonisin Corn and several other -Neurotoxic grains (rice, wheat, barley, -Hepatotoxic Produced by strains of maize, rye, oat, and millet) -Nephrotoxic Fusarium species Possible carcinogen CYP1A, 4A, 2B, 2C11, High levels of this toxin → Esophageal & liver cancer 2E, and 3A1 occur when hot, dry in humans weather is followed by a period of high humidity Ochratoxin Grains (wheat, rye barley), -Nephrotoxic coffee, spices, grapes, produced by Aspergillus beer, wine -Probably carcinogenic and Penicillium molds CYPIA1/IA2, 2B1 and 3A1/3A2 Fusariotoxins: Wheat, corn, oats barley - GIT inflammation and produced by Fusarium and other grains under bleeding → prolonged can ↓ species normal weather conditions, nutritional status e.g. but especially in cool, wet - Severe gastroenteritis Zearalenone, conditions - Highly toxic if inhaled → Deoxynivalenol dyspnea - Skin blisters with topical CYPIA and CYP3A exposure families **All have similar acute effects: nausea, vomiting, abdominal pain, etc. https://drjockers.com/barbeque/  Smoking Well-done red meat CYP species Tissue location Inducers (e.g.) Nuclear receptor Substrates (e.g.) CYP1A1 Lung, GIT PAH, HCA, Aryl hydrocarbon PAH, HCA, PCBs, dioxins receptor (AhR) Aflatoxin CYP1A2 Liver *NOT PCBs, dioxins Polycyclic Aromatic Hydrocarbons (PAH) - Polycyclic aromatic hydrocarbons (PAHs) are a class of > 100 chemical compounds composed of up to 6 benzene rings fused together such that any 2 adjacent benzene rings share two carbon bonds - E.g. Benzo(a)pyrene (BaP): PAH = large lipophilic Oral and inhaled compounds. Know BaP as an exposures example - Formed from burning carbon-containing compounds (e.g. food fat or carbohydrates, wood chips, tobacco, etc.) at > 250 C - Carcinogenic (for many types of GI cancers): - Induce expression of the CYP1A family via AhR - Metabolized by CYP1A family to reactive electrophile (mainly CYP1A1/CYP1A2, also CYP 1b family to a lesser extent) ***Phytochemicals compete with BaP for CYP activation and AhR binding - Indole-3-carbinol (I3C) an example of an isothiocyanate (ITC) - Curcumin - Quercetin - Flavones → includes luteolin (celery, green peppers) Dietary influences on phase 1 metabolism Non-nutrient-based: 1. Polycyclic aromatic hydrocarbons (PAH) and heterocyclic amines (HCA) in BBQ foods and tobacco Recall - Bind AhR and induce from CYP1A1 Unit 2gene expression (↑ and CYP1A2 protein by extension) - “vicious cycle” with regular consumption→ more CYP1A1/2 = more bioactivation and cancer risk 2. Phytochemicals [e.g. isothiocyanates (ITCs)] - 1000s of X with variable effects: a) Bind AhR and block the large induction of CYP1A1 and CYP1A2 caused by PAH, HCA, PCBs and dioxins (i.e. function as AhR antagonist via competitive inhibition) - Phytochemicals are often present in diet at higher concentrations than other X - Phytochemicals only mildly induce CYP1A1 and CYP1A2 (i.e. weak AhR agonist) b) Bind and inhibit CYP activity (i.e. CYP antagonist)  Examples of PAH BaP be familiar with it's structure Many possible different types of PAH’s can be formed …all of them share a ring structure. We COULD study each one individually and assess it’s cancer risk and metabolism but we will only focus on BaP  BaP Metabolism B AhR A - (stable, lipophilic Increased CYP1A1 and CYP1A1/2 CYP1A2 protein in SER parent compound) risky C Reactive Epoxide Electrophile know that a reactive Reactive Epoxide electrophile. Do not need to know it's name Nucleophile hydratase Diol fast D GSH/GST Some E Phase II mercapturic acid CYP1A1/2 conjugates in urine “bay region” fast Reactive F UDPGA/ Diol-Epoxide UDPGT Electrophile Phase II GSH/GST → Ultimate BaP -7,8-diol-9,10 epoxide urine Phase II carcinogen “bay region” steric hindrance Highly reactive limits Phase II Difficulty accessing the “bay enzyme access DNA adducts!! region” of the molecule → - Mutations LIMITED PHASE II - Initiation BaP Metabolism A. BaP is a stable secondary carcinogen – i.e. it does not directly damage DNA B. BaP binds to and activates the AhR to induce CYP1A family gene expression - More CYP1A1 and CYP1A2 protein expressed in the SER ready for phase I reactions C. BaP is metabolized by the CYP1A family to reactive epoxide electrophile - BaP is lipophilic and may accumulate in lipid-rich tissues (e.g. subcutaneous adipose tissue, developing fetal brain), so this initial metabolic step prevents accumulation and potential neurotoxicity, but is risky since it produces a reactive electrophile D. Reactive epoxide electrophile may be conjugated to GSH via GST to a mercapturic acid conjugate for excretion in the urine; however, GSH availability is likely limited due to other dietary X metabolism, and the reactive epoxide electrophile is short-lived… E. Reactive epoxide electrophile is quickly metabolized by an epoxide hydratase to a nucleophile diol intermediate - Reduces the size of the epoxide electrophile pool F. Nucleophile diol intermediate may be conjugated to UDPGA via UDPGT for excretion via urine; however, nucleophile diol intermediate is short-lived… G. Nucleophile diol is quickly metabolized by CYP1A1/1A2 to reactive diol epoxide electrophile, the primary carcinogen which damages DNA → cancer - The “bay region” of reactive diol epoxide electrophile creates stearic hindrance, meaning it cannot be conjugated to GSH via GST for excretion easily the glutathione conjugation reaction will be slow  BaP Metabolism with Phytochemicals B AhR A Antagonism by risky Phytochemicals - (stable, lipophilic CYP1A1/2 (e.g. I3C) parent compound) Limit induction of CYP1A1/2 protein prevents accumulation of C expression in SER reactive epoxide electrophile because phase I reactions Reactive Epoxide are SLOWER Electrophile Reactive Epoxide Nucleophile hydratase Diol fast D GSH/GST Some E Phase II mercapturic acid CYP1A1/2 conjugates in urine “bay region” fast Reactive F UDPGA/ Diol-Epoxide UDPGT Electrophile Phase II GSH/GST BaP -7,8-diol-9,10 epoxide → Ultimate urine Phase II carcinogen “bay region” Highly reactive steric hindrance Difficulty accessing the “bay DNA adducts!! region” of the molecule → - Mutations LIMITED PHASE II - Initiation BaP Metabolism with Phytochemicals A. BaP is a stable secondary carcinogen – i.e. it does not directly damage DNA B. BaP binds to and activates the AhR to induce CYP1A family gene expression - If available, fruit/vegetable phytochemicals (e.g. I3C) may competitively inhibit BaP for AhR binding C. BaP is metabolized by the CYP1A family to reactive epoxide electrophile - BaP is lipophilic and may accumulate in lipid-rich tissues (e.g. subcutaneous adipose tissue, developing fetal brain), so this initial metabolic step prevents accumulation, but is risky since it produces a reactive electrophile - If available, fruit/vegetable phytochemicals (e.g. I3C) may competitively inhibit BaP for CYP1A binding, or may directly inhibit CYP1A activity D. Reactive epoxide electrophile may be conjugated to GSH via GST to a mercapturic acid conjugate for excretion via urine; however, GSH availability is likely limited due to other dietary X metabolism, and the reactive epoxide electrophile is short-lived… E. Reactive epoxide electrophile is quickly metabolized by an epoxide hydratase to a nucleophile diol intermediate - Reduces the size of the epoxide electrophile pool F. Nucleophile diol intermediate may be conjugated to UDPGA via UDPGT for excretion via urine; however, nucleophile diol intermediate is short-lived… G. Nucleophile diol is quickly metabolized to reactive diol epoxide electrophile, the primary carcinogen which damages DNA → cancer - The “bay region” of reactive diol epoxide electrophile creates stearic hindrance, meaning it cannot be conjugated to GSH via GST for excretion  Grilled Meats + Phytochemicals → can we simplify this? Yes We Can! https://drjockers.com/barbeque/  PAH HCA FORM → from on cooked meats PAH → produced from incomplete combustion of organic material…can form on meats but also vegetables/fruits, carbohydrates Heterocyclic Amines (HCA) - Heterocyclic amines (HCAs) are a class of > 20 chemical compounds containing at least one heterocyclic ring (i.e., contains atoms of at least two different elements), as well as at least one amine (nitrogen-containing) group, which is typically part of the ring - E.g., 2-Amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) – most abundant HCA in cooked meat - E.g., 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) – most carcinogenic HCA - Formed from creatine (a non-protein amino acid found in muscle tissue), and other amino acids and sugars in dry cooking conditions (125 - 250 C) - Carcinogenic Effects of HCA: - Induce expression of the CYP1A family via AhR - Metabolized by CYP1A family to reactive electrophile (primary route of phase I metabolism) - Also phase I metabolism by CYP1B1 and other CYPs (these reactions occur to a lesser degree)  FORMATION OF HCAs + sugar + amino acids +heat recognize that we could have more, but focus on the two examples she giving us Heterocyclic amines (HCAs) form during Maillard browning reactions, specifically when muscle-rich foods (e.g.,pork, beef, chicken, or fish) are cooked at temperatures above 100 °C. Knize & Felton, 2005, Nutrition Reviews As we increase time meat is cooked = more Phip content know the starting points to make Phip = protein, sugar and creatinine +  - Grilling, roasting, baking and frying meats PhIP Metabolism - Dried meat fractions, gravy, bouillons, dried soups - Jerky PhIP 2 AhR Metabolism: PhIP “other PhIP occupy AhR 1 P450s” = ↑ CYP1A1 activity = ↑ phase I reaction CYP1B1 CYP1A1/2 C-hydroxylation activity 3 N-hydroxylation HO nucleophile H (REACTIVE Nucleophile Reactive N INTERMEDIATE) HO Intermediate (phase I product) UDPGA 4 UDPGT UDPGA O-hydroxylation by UDPGT NAT1/2 5 URINE Highly reactive electrophile GSH GST (1° carcinogen; reactive 6 intermediate) - Aggressively causes DNA damage Remember, there are NAT1 and NAT2 polymorphisms resulting in fast and slow enzyme activity PhIP Metabolism 1. PhIP may be metabolized by various CYPs (including CYP1B1) to a nucleophile, then conjugated to UDPGA vis UDPGT for excretion via urine; however, abundance of PhIP in diet and limited availability of UDPGA due to metabolism of other dietary X forces PhIP metabolism through the CYP1A family 2. PhIP binds and activates the AhR to induce CYP1A family gene expression - If available, fruit/vegetable phytochemicals (e.g. I3C) may competitively inhibit PhIP for AhR binding 3. PhIP is metabolized by the CYP1A family to a nucleophile reactive intermediate - If available, fruit/vegetable phytochemicals (e.g. I3C) may competitively inhibit PhIP for CYP1A binding, or may directly inhibit CYP1A activity 4. Nucleophile may be conjugated to UDPGA vis UDPGT for excretion via urine; however, limited availability of UDPGA due to metabolism of other dietary X 5. Nucleophile is acetylated by N-acetyltransferase (NAT) 1 or NAT2 to a reactive electrophile, the primary carcinogen which damages DNA → cancer 6. Phase II reaction (GSH/GST) to form a mercapturic acid conjugate that is excreted in the urine…remember this requires the final reaction with the “other NAT” (or NAT)  What happens when we consume vegetables PhIP Metabolism AND grilled meat together???? 2 AhR Metabolism: PhIP “other Vegetables compete with 1 P450s” PhIP for binding to AhR CYP1B1 C-hydroxylation 3 =CYP1A1/2 = ↓ CYP1A1 activity = ↓ PhIP phase I activity N-hydroxylation HO nucleophile H (REACTIVE Nucleophile Reactive N INTERMEDIATE) HO Intermediate (phase I product) UDPGA 4 UDPGT UDPGA O-hydroxylation by UDPGT NAT1/2 5 URINE Highly reactive electrophile GSH GST (1° carcinogen) 6 - Aggressively causes DNA damage Remember, there are NAT1 and NAT2 polymorphisms resulting in fast and slow enzyme activity Vegetables (phytochemicals) and grilled meat (PhIP) competitively compete to occupy AhR 1. In the absence of phytochemicals, PhIP will occupy AhR CYP1A1/2 activity increases Rxn 3 speeds up The Phase I product IS STILL A REACTIVE INTERMEDIATE…it can undergo a phase II glucuronidation reaction BUT it will also quickly undergo reaction 5 (acetylation reaction) producing a highly reactive 1° carcinogen 2. When phytochemicals are present there is competitive inhibition at the AhR (it’s blocked and PhIP cannot bind). Instead the phytochemical occupies the AhR Less induction of CYP1A1/2 expression Rxn 3 slows down → slower PhIP phase I reactions Lower production of the nucleophile reactive intermediate allows more product to enter rxn 4 and less entering rxn 5 Rxn 4 safely removes PhIP metabolites in urine and reduces the amount of the highly reactive electrophile product produced in rxn 5 (reduced cancer risk when PhIP and phytochemicals are consumed together Remember → there are BOTH HCAs and PAH’s in cigarette smoke Fast NAT2 acetylators will catalyze the reaction converting the PhIP-derived Nucleophile Reactive Intermediate into the highly reactive electrophile (1° carcinogen) quickly  Honey BBQ = high sugar Tumeric-garlic = contains phytochemicals (antagonists ?) Teriyaki = high sugar + some phytochemicals from garlic and ginger HAAs = HCAs - Sugar increases HCA formation via the Maillard reaction PhIP HCAs: MeIQ  Maillard Reaction “non-enzymatic browning reaction” Maillard reaction products = MELANOIDINS and can form on many different foods Maillard Reaction: a chemical reaction between amino acids and sugars in/on food that occurs when heat is introduced to produce new flavours, aromas, and colours. Reducing heat+time melanoidins New Protein Sugar flavour/aroma/colour Proteins must have a NH2 side chain – ex: Lys, Arg Must be reducing sugars (have a free aldehyde) → all monosaccharides and some disaccharides (lactose) Heat required in dry conditions: 280°F - 330°F Flavours, aromas, and colours produced depend on heat, time and pH which result in many different final reactions The Maillard Reaction is responsible for many colors and flavors in foods: The browning of various meats like steak, when seared and grilled. The browning and umami taste in fried onions. Toast. The darkened crust of baked goods like pretzels and bread. The golden-brown color of French fries. Malted barley, found in malt whiskey or beer. Dried or condensed milk. Roasted coffee. Dulce de leche. Maple syrup. Black garlic Etc… Maillard Reaction Biochemistry is More Complicated… also known as HMF flavours  Form some Maillard products during cooking but also harmful chemicals including benzopyrene (a PAH), HMF, heterocyclic amines, acrylamide, etc.) https://i2.wp.com/www.compoundchem.com/wp-content/uploads/2015/01/Food-Chemistry-Maillard-Reaction.png?ssl=1  HMF= 5-Hydroxymethylfurfural Potentially carcinogenic to humans Flavouring agent in foods Formed in the Maillard reaction and in caramelization reactions (dehydration of sugars under acidic conditions) Produced during the cooking of sugar-containing /carbohydrate rich foods higher the temperature, higher the concentration of HMF formed HMF has been identified and measured in many food products such as dried fruits, jams, coffee, caramel, fruit juices, baked goods (cookies, breads, breakfast cereals) and honey. HMF concentration is used a marker for the quality of processed foods (lower levels of HMF indicated higher quality processed foods) Intakes range 30-150 mg/person/day Used as an indicator for excess heat-treatment/processing of foods (e.g. honey, vinegars, some alcohol products, juices, etc.) Standard for honey quality Fresh honey < 15mg/kg HMF content and levels should not be higher than 40mg/kg HMF.  Simplified Metabolism of HMF Ester also needs PAPS Parent compound HMF is metabolised in human body mainly to i) 5-hydroxymethylfurfuroic acid (HMFA) ii) 5-sulfoxymethylfurfural (SMF) → the sulfate is removed leaving behind an ester that is genotoxic and mutagenic Damages DNA/RNA, causes mutations and damages proteins Acrylamide asparagine 248 F … Neurotoxic in animals and humans Reproductive toxicant → germ-cell mutagen Carcinogen in rodents , human carcinogen data is more controversial International Agency for Research on Cancer (IARC) classifies acrylamide as “probably carcinogenic to humans” → based on evidence from animal studies Neurotoxic Effects of Acrylamide Alteration of neurotransmitter levels Direct inhibition of neurotransmission Nerve damage Exposure in the human diet is low BUT concerns about accumulated neurotoxic effects Sub-chronic low-level work exposure may bring on ataxia, gait abnormalities, skeletal muscle weakness, skin abnormalities, and numbness of hands and feet…mimics fibromyalgia symptoms Reproductive effects → causes reduced sperm counts and litter sizes in rodents Acrylamide Metabolism (simplified) Binds to hemoglobin (Hb) → Hb-adduct Can bind to proteins (enzymes, serum albumin) Bind to DNA (form DNA adducts) far more biologically active compared to the parent compound CYP 2E1 CYP 1A2 GSH/GST Reactive intermediate believed to mediate many Excretion of the adverse effects of acrylamide - Rapidly absorbed and distributed around the body - Crosses placenta and is found in breast milk - acrylamide-Hb and glycidamide-Hb are biomarkers for exposure. Polymorphisms for low CYP2E1 expression and exposed to AA will have higher acrylamide-Hb levels. - Ratio of Acrylamide-Hb : Glycidamide-Hb is used to infer efficiency of xenobiotic metabolism, can be a biomarker for risk (e.g. neurotoxicity) - SNPs for low expression of GST (GSTM1 null, GSTT1 null variants) have higher levels of glycidamide-Hb after exposure  Paracetamol = acetaminophen....we’ll discuss this drug later!! Think about interactions with other xenobiotics (that use the same CYP enzymes for a P1 reaction as acrylamide) Could fibromyalgia symptoms be caused by acrylamide exposure OR worsened by similar symptoms by acrylamide in this example? Acrylamide Content in Foods I’m sorry!!! do not need to know the numbers. Juts know that Potato chips and French fries have a high level of exposure. But know that there are other courses. https://www.choicesmagazine.org/2004-1/2004-1-03.htm How to Reduce Acrylamide Content in Foods lower temperature than usually used for frying foods Remember: acrylamide forms in foods when asparagine is heated with sugars such as glucose. Formed as a by-product of cooking → baking, frying, grilling, and toasting, or any cooking method in which temperatures are greater than 120°C or 248°F. the higher the heat (and cook time) the more acrylamide forms  REVIEW: Cytochrome P450 (CYP) enzymes - Induction of gene expression Smoking Processed meat CYP species Tissue location Inducers (e.g.) Nuclear receptor Substrates (e.g.) CYP1A1 Lung, GIT PAH, HCA, Aryl hydrocarbon PAH, HCA, PCBs, dioxins receptor (AhR) Aflatoxin CYP1A2 Liver *NOT PCBs, dioxins CYP2A,B,C,D Liver Barbiturates, Constitutive Many subfamilies (other tissues too) Phenobarbital androstane pharmaceuticals receptor (CAR) CYP2E1 Liver, lung, GIT, Ketones, None - Protein Acetaminophen, skin Ethanol stabilization Alcohol, Nitrosamines, Aflatoxin CYP3A subfamily Liver, GIT Anabolic steroids Steroid hormone Endogenous (makes up to 60% xenobiotic steroids, of liver CYPs) receptor (SXR) Pharmaceuticals Tobacco xenobiotics - REVIEW - Tobacco plants produce nicotine as an insecticide - Stored in the tobacco leaf - A neurotoxin - has neuroexcitatory effects - One of the most addictive compounds characterized - Nitrogen (in atmosphere and fertilizers) taken up by plants – increases growth rate - Converted to ammonia (NH3) then nitrate (NO3-) for storage in leaf - Nitrate converted to nitrite (NO2-) during processing Nicotine + Nitrite Heating, fermentation (curing) Chewing, enzymes in saliva Nicotine-derived nitrosamine ketone (NNK) - Tobacco-specific nitrosamine - Stable, secondary carcinogen Phase I metabolism by CYP2E1, CYP2A6, and others Reactive, primary carcinogens Phase II metabolism Excreted DNA damage… Mutations… Cancer (oral, lung)  Nitrosamines - Formed by reaction of secondary or tertiary amines (portions of amino acids, i.e., protein) with a nitrosating agent, e.g., nitrite (added during food processing) - Nitrates → older use as a preservative, converted to nitrite by bacteria - Enhanced as a result of the direct-fire drying process: oxides of nitrogen in the drying air will nitrosate amines in the food being dried - Rich in processed meats (primarily cooked bacon); beer; some cheeses; non- fat dry milk; and sometimes fish - Dimethylnitrosamine (DMN) is most common in foods Amine + Nitrite (from protein) (added during processing) - Preservative to - Carcinogen in prevent bacteria in rodents Nitrosamines, e.g. DMN growth (stable) - Add salty flavour - Associated - Oxidizes with ↑risk of hemoglobin,→ esophageal and gives meat pink gastric cancer in colour humans CYP2E1 Unstable intermediate GSH/ GST Carbonium ion Urine DNA damage (reactive electrophile) Dimethylnitrosamine (DMN) Hepatotoxin → leads to liver fibrosis → liver tumors Mutagen → methylates nucleotide bases in DNA (methylguanine (above) and methyladenine are most common) Carcinogen (in rodents @ dose of 25 mg/kg body weight