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NST 110
| Question | Answer |
|---|---|
| AhR | –Aryl Hydrocarbon Receptor – ligand-based txn factor –Involved in genes such as CYP450 |
| ARNT | –Aryl hydrocarbon Regulation Nuclear Translator –Forms heterodimer with AhR |
| AhR chaperone complex | Hsp90, XAP2, p23 |
| XRE/DRE | Xenobiotic/Dioxin Response Element |
| Ligand binding to AhR | –Chaperone complex dissociates –AhR forms heterodimer with ARNT –Binds to XRE/DRE in CYP promoter region |
| Identifying genes triggered by XRE/DRE (EXPERIMENT) | Transfect reporter gene into cells Treat cells with X/D compound –Measure reporter gene activity –Delete genes and measure changes in reporter genes |
| Identifying XRE/DRE (EXPERIMENT) | –Transfect/measure/etc again –Delete and mutate to identify response element –Use affinity chromatography to purify proteins bound to RE |
| Identifying proteins that bind to response element (EXPERIMENT) | Gel Shift assay: 1. Radio-label response element (probe) 2. Incubate with nuclear extracts 3. Run on SDS-PAGE – probe-bound proteins will move slower 4. Use affinity chromatography to purify the probe-bound proteins |
| AhR and Benzo[a]pyrene (SEE GRAPH IN NOTES) | AhR mediates genes (Cyp1A1/B1) involved in BaP carcinogenesis – not bioactivated in AhR-null mice (SEE GRAPH IN NOTES)OT |
| Other effects of AhR-KO | Reduced liver size and fertility |
| Synthetic ligands of AhR | TCDD, TCDF, 3MC, ICZ |
| Endogenous ligands of AhR | Lipoxin A4, Tryptamine, Indirubin, Indigo, Bilirubin, ITE, FICZ |
| Antioxidant Response | Activation of Phase II enzymes – used to protect against oxidative stress/agents |
| AREs | Antioxidant Response Elements – upstream regulatory sequences present on responsive gene |
| Nrf2 | –Basic Leucine Zipper TF (bZIP) –Binds to ARE and recruits txn machinery |
| Maf TF family | Heterodimerize with Nrf2 before binding AREs |
| Nrf2 functions in the oxidative response (not likely tested) | Increase txn of Phase II enzymes (inactivate oxidants, detox electrophiles) Stimulate GSH activity and regen Stimulates NADPH synth Enhances toxic drug export via MDR Stimulates damaged protein recog/repair/removal |
| BHT (EXPERIMENT) | Butylated Hydroxytoluene – example of lung-injuring compound Nrf2 protects against. See graphs and pictures for details |
| Keap1 | Cytosolic repressor protein – binds to Nrf2, retains it in cytoplasm, and promotes its degradation. |
| Keap1 mechanism | Inducers oxidize Keap1 thiol groups to dithiols, causing release of Nrf2. Nrf2 is then phosphorylated and brought into nucleus. |
| NHR | Nuclear (Hormone) Receptor – Proteins inside cells which sense hormones and other lipophilic molecules |
| NHR Domains | –DBD – DNA-binding domain (most conserved.) Uses 2 zinc fingers –LBD – Ligand-binding domain. Binds specific lipophilic molecules –AF1 and AF2 (N and C terminals) usually act as transactivation domains (contact txn factors) |
| Type I Nuclear Receptors | –Primarily Hormones: Most (not Estrogen) are located in cytoplasm –Bound to Hsp90 complex; dissociates upon ligand binding and enters nucleus Binds to inverted HREs as a homodimer |
| Type II Nuclear Receptors | –Located in nucleus – sits at HRE as a heterodimer with RXR –Without ligands, NR-RXR recruit txn corepressors –Binding of ligands releases corepressors; recruits coactivators |
| RXR | 9-cix retinoic acid receptor – heterodimerizes with Type II receptors |
| Type III and IV Nuclear Receptors (Not tested) | Type III: Bind as homodimers to non-inverted repeats Type IV: Bind as mono/dimers but only to half HREs |
| Agonists | Compounds that bind to a receptor and trigger the same response as the endogenous ligand May have an effect on de/sensitization |
| Antagonists | Compounds that block ligand/agonist binding by binding to the same site more competitively |
| SRMs and SERMs | –Selective (Estrogen) Receptor Modulators - synthetic, may trigger different (ant)agonist responses in different tissues –Can be useful medicinally (i.e. breast cancer) |
| SERM testing (EXPERIMENT) | In cells, SERMs are tested against estrodiol (E2, natural) to observe how many genes (esp. common genes) are activated between the two |
| Point of looking at SERMs | –Ligand effects are dependent on cell/gene/tissue context. –Gene expression can be affected by combinations of ligands, tissues and cofactors. |
| CAR | Constitutive Androstane Receptor - example of a xenobiotic sensor |
| CAR-KO mice (EXPERIMENT - JUST READ THE DAMN SLIDES) | Used to study the effects of CAR on a variety of internal and external factors |
| BrdU | 5-bromo-3-deoxyUridine - An analog of tryptamine. Can be incorporated into DNA and then substituted, allowing detection via antibodies. |
| Organophosphate Nerve Agents | Sarin, Tabun, VX - irreversibly inhibit active site of acetylcholinesterase |
| Acute Toxicity of OP nerve agents | –Muscarinic hyperstimulation - Oversecretion (vomiting, salivating, etc), bronchoconstriction, reduced heart rate –Nicotinic hyperstimulation - convulsions/tremors |
| Lethality of OP nerve agents | Hyperstimulation of nicotinic ACh receptors leads to hypostimulation - reduced neurotransmission, muscular/respiratory paralysis, death |
| OP Antidotes | –Atropine - muscarinic ACh receptor antagonist. No effect on nicotinic –2-PAM - regenerates AChE 2-PAM is a poor blood-brain barrier crosser so both are often taken together |
| Short term effects of CB1 binding (Just write these out a few times as a web) | 1. Hyperpolarization, decreased neurotransmission 2. Lowered neurotransmitter release 3. Inhibited motor and memory function |
| Long term effects of CB1 binding (Just write these out a few times as a web) | Impairment of memory: 1. Lowered glutamate impairs short and long term memory formation 2. Lowered cAMP inactivates PKA and CREB, impairs longterm memory |
| Rimonabant | Cb1 antagonist (sort of) - Potential uses in weight loss and diabetes treatment, but was discontinued due to depressive/suicidal side effects |
| Opiates | Bind opiate receptors to produce effects such as analgesia, CNS inhibition, and an intense "high" |
| μ opioid receptor (Draw out effects as a web) | –Bound by morphine and heroin (morphine diacetate) –Mediate neuronal inhibition by suppressing Glu neurons |
| Addiction | Characterized by compulsive drug use despite negative consequences - follows a chronic course of action |
| Tolerance | Need for more drug to produce the same effects - often caused by desensitization of primary and/or dopaminergic receptors |
| Dependence | An adapted physiological state which compensates for excessive stimulation by a drug |
| Withdrawal | Removal of the withdrawal state - may have physical and mental components |
| Dopamine modulation, role in addiction | Most drugs inhibit GABA neurons, leading to disinhibition of dopaminergic neurons. This associates the drug with satisfaction and happiness |
| VTA | Ventral Tegmental Area (midbrain) - where most drugs raise dopamine levels. |
| NAc | Nucleus Accumbens - Area of brain containing limbic and corticol regions (motivation, behavior). μ-opioid dopaminergic receptors here are often affected, leading to strong connections/addiction. |
| Dopamine Receptors | 5 different types - N1 involved in drug effects. |
| Short term effects of D1 stimulation | Opens Ca channels, closes K channels, and induces Glu receptors to move to surface - immediate connection between substance and reward |
| Long term effects of D1 stimulation | Stimulates gene expression through CREB protein - strengthens long term connection through synaptic plasticity and long term potentiation |
| Parkinson's disease (probably not tested) | Characterized by loss of dopamine neurons in substantia nigra - leads to loss of muscle control due to excessive contraction |
| External causes of Parkinson's (probably not tested) | Molecules like MPTP or MPP+ (similar to dopamine) are taken up in dopaminergic receptors; proceed to degenerate neurons |
| Toxicity factors of metals in humans | 1. Interactions with essential metals 2. Formation of metal-protein complexes 3. Age and stage of development 4. Lifestyle factors 5. Chemical form or speciation 6. Immune status of host |
| Sources of Cadmium (Cd) | –Byproduct of zinc/lead mining and smelting –Used in electroplating, pigments, and batteries |
| How Cd generally gets into humans | Primarily through food - plants readily take it up through soil, which comes from contaminated water and industrial sludge. Bioaccumulate heavily in shellfish as well. |
| Other sources of Cd | May be present in workplace air - due to use in industry, or cigarettes |
| Absorption of Cd | –5-8% in GI tract –15-30% in respiration –Up to 50% in smoking cigarettes |
| Other factors in Cd absorption | Low Ca stimulates synthesis of Ca binding proteins, which can bind Cd - can also use vacant Ca channels Low Fe allows Cd to use Fe transporters as well |
| Cd excretion | Successfully excreted Cd comes out in urine |
| Primary sites for distribution of Cd | Liver and kidney - transported by binding to red blood cells and plasma protein (particularly albumin) |
| MT | Metallothionein - forms mostly nontoxic complex with Cd. Induced by presence of Cd in liver. |
| Fate of Cd-MT complex | –If stored in liver, likely excreted. –If stored in kidney: 1. May be taken up into lysosomes - will be de-complexed, may be excreted or re-complexed 2. May be taken up into renal cells by megalin/cubulin endocytosis - can cause cell death |
| Study of MT effects (EXPERIMENT) | Wild type and MT-OEx cells are both treated with various compounds - IC50 for Cd is tenfold larger, while other compounds (Hg) are not significantly affected |
| MTF-1 | Metal-responsive Transcription Factor 1 - Recognizes metal response elements in the promoters of genes like MT |
| Mechanism of MT induction by Cd | 1. Cd competes with Zn to bind metal response elements, promoting MT expression 2. Cd also induces MT expression via MTF-1 independent pathway |
| Acute Toxicity of Cd | –Ingestion will cause nausua, vomiting, abdominal pain –Inhalation will cause flu-like symptoms (cadmium blues) which may last up to a week |
| Chronic Toxicity of Cd | –Many effects - particularly skeletal –Blocks Vit-D metabolism, which inhibits Ca absorption - screws up cell function and skeletal composition |
| Specific Case of Skeletal Effects | –Itai-itai byo - Characterized by deformities and severe pain in joints –First Cd poisoning known to world - Japan, 1950 |
| Cd-induced Carcinogenesis | –Can displace redox active Fe/Cu from binding proteins and inhibit IDPm - affects mitochondrial functions –Also inhibits DNA repair systems |
| DNA Repair Systems (inactivated by Cd and other things) | –Mismatch repair - Corrects mistakes; wrong bases or mismatches –Nucleotide Excision repair - Removes lesions, which may block txn/replication –Base Excision Repair - Repair damage to bases, ssDNA breaks, mutations |
| Three states of Mercury | –Hg0 (metallic) –Hg+ (mercurous) –Hg2 (mercuric) |
| Distribution of Mercury | Mainly present in atmospheric mercury vapor - can be rescued from MM |
| Methylmercury (MM) bioaccumulation | –Mercury is methylated in aqueous microorganisms and can bioaccumulate sharply in aquatic food chains –Tuna is generally at the top of these food chains and contains heavy MM |
| Commercial uses of mercury (which may easily release vapor) | –Cathode in electrolysis of brine –Dentistry –Scientific devices –Gold mining process |
| Absorption of Hg | –Metallic (liquid) mercury - Very little in GI –Quickly vaporizes and is absorbed, then oxidized to mercuric (Hg2) –Mercuric - ~15% GI absorption from food –MM - absorbed 90-95% in GI |
| MM Toxicity | –MM forms a complex with Cys - resembles Met –Transported into brain by LAT1 and LAT2 –Forms MM-OH and OH radicals, which damage everything (esp. membranes) –Very neurotoxic, especially to mothers/fetuses |
| Acute Toxicity of Hg vapor | Corrosive bronchitis, pneumonitis - can lead to tremors and death over time |
| Chronic Toxicity of Hg vapor | Many symptoms - Including dramatic personality and mood changes |
| Toxicity of mercuric and metallic mercury in the body | –Concentrate in kidneys - associated with chronic kidney failure and GI toxicity |
| "Antidote" to Hg Toxicity | –Selenium has been seen to reduce Hg toxicity in nature - sea animals, birds –May redistribute, compete for binding sites, or form inert complexes |
| Physicochemical properties that determine environmental toxicity - may affect mobility | –Lipophilicity and Vapor Pressure |
| Hazard vs. Risk | –Hazard refers to the nature of the toxicity of the chemical when exposed –Risk is the probability of a chemical causing harm (Basically Hazard*Exposure) |
Created by:
yazaria
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