Toxicology Exam 3 Flashcards
Toxicokinetics:
how a toxic substance enters our body, moves through it and is eliminated. It deals with the absorption, distribution, metabolism, and elimination (ADME) of toxic substances
study of the movement of xenobiotics in the body
Toxicodynamics:
refers to how our body reacts to that toxic substance on a biochemical and physiological level. Its main focus is how that substance interacts with the body’s molecular and cellular components to produce a toxic response.
study of the interaction of xenobiotics with biological tissue
Oxidative stress:
when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of the body to detoxify or repair the resulting damage
Can cause DNA damage then active p450→ apoptosis, chemicals directly poison mitochondria → bad because mitochondria needs to make ATP
Toxic molecules targets DNA of molecular oxygen
Toxic molecules include:
Formation of ROS: Superoxide anion, hydrogen peroxide, hydroxyl radicals
Formation of nitrogen molecules: peroxynitrite, nitrogen dioxide
Formation of carbonate anion
Superoxide:
When does it form?
Consequences?
Superoxide is a reactive oxygen species (ROS), a free radical formed from molecular oxygen
When does it form? When molecular oxygen acquires an unpaired electron in its outer atomic orbital
Consequences: oxidative stress→damage to mitochondria, proteins, lipids, and DNA
What leads to superoxide formation?
Redox cycling (acts as a catalyst to form superoxide)
Redox cycling:
where a xenobiotic (foreign substance) undergoing repeated cycles of reduction and oxidation
Types of xenobiotics: Paraquat (lung toxicity), nitrofurantoin (lung toxicity), doxorubicin (liver/heart toxicity)
Xenobiotics transfers an electron from NADPH (when electron is removed, it becomes NADP+) to molecular oxygen (O2) through p450 reductase→makes superoxide
Consequence of redox cycling is degradation
What happens during this continuous cycle (redox) that keeps taking electrons from NADPH?
NADPH cellular levels drops
Superoxide radical metabolism happens in two pathways:
Pathway 1: nitrogen dioxide toxicity (covalent modification of target)
Pathway 2: hydroxyl radical toxicity (breakdown of lipid membranes)
Superoxide Pathway 1
Pathway 1: nitrogen dioxide toxicity (covalent modification of target)
Superoxide radical reacts with nitric oxide (NO) to form peroxynitrite (ONOO-) → reacts with CO2 to form ONOOCO2- → NO2 (nitric dioxide) & CO3-
Superoxide Pathway 2
Pathway 2: hydroxyl radical toxicity (breakdown of lipid membranes)
Step 1: superoxide radical undergoes dismutation through superoxide dismutase (SOD) → hydrogen peroxide (H2O2)
Step 2: Fenton reaction: (Fe+2) reacts with hydrogen peroxide→Fe+3, HO (hydroxyl radical) + OH-
Fe+2 is oxidized to Fe+3
Electron in hydroxyl radical comes iron
Hydroxyl radical initiated vicious cycle, which breaks down lipid membranes
- Causes membrane dysfunction/leakines
- Cell death occurs if this reaction is not stopped
What are three pathways to inactivate superoxide?
From 1st reaction of pathway 2: superoxide dismutase (SOD) → hydrogen peroxide (H2O2)
- Glutathione peroxidase
- Peroxiredoxin
- Catalase: an enzyme that plays a role in the defense against oxidative stress by facilitating the breakdown of hydrogen peroxide into water and oxygen
superoxide dismutase (SOD) → hydrogen peroxide (H2O2) → CATALASE→ water & oxygen
Four general targets of xenobiotics in dysregulated gene expression
Dysregulation of transcription factors (TF) activation (xenobiotics can act as agonist or antagonist)
Dysregulation of TF DNA binding
Alterations of mRNA transcription by RNA polymerase dysfunction
Alternation of protein translation
G protein-coupled receptor (GPCR) - General pathway
General pathway → GPCRs are coupled to G proteins (G protein coupled means its signals through proteins with ligand binding to receptor) - G proteins have 3 subunits:
Gα (alpha), β (beta), γ (gamma)
Activation: ligand binding causes exchange of GTP for GDP in the alpha subunit
Inactivation: Innate GTPase activity in the alpha subunit hydrolyzes GTP to GDP
Family of G proteins (Gx): three types of alpha subunits: s, i, q
Gs (stimulate adenylate its downstream target)
Gi (inhibits adenylate decrease inside the cell),
Gq (connects to pathway through calcium and phospho c)
G protein-coupled receptor (GPCR) signal transduction: Adenylate Cyclase Pathway:
Xenobiotic → GPCR → Adenylate cyclase → cAMP → Protein kinase A → CREB → Cell changes
Adenylate cyclase:
An enzyme that catalyzes cAMP production
AMP binds to and activates Protein Kinase A (PKA)
PKA is a protein kinase that phosphorylates many proteins to affect their function
Key target: cAMP Responsive Element Binding (CREB), transcription factor that regulates gene expression
Cholera toxin
Cholera toxin (biological made) (CTX, Vibrio Cholerea bacteria): increases adenylate cyclase activity (↑cAMP) through a GPCR coupled to Gsα
Found: GI/diarrhea
Increase in CREB
Pertussis
Pertussis toxin (PTX, Bordetella pertussis bacteria): decreases adenylate cyclase activity (↓cAMP) through a GPCR coupled to Giα
Found: respiratory/whooping cough
Decrease in CREB
Cholera/pertussis are
Cholera/pertussis are ADP ribosyltransferase enzymes
Catalyzed the covalent modifications of G alpha using NAD+ as a substrate
Know how alterations in cAMP could affect gene expression via CREB transcription factor
Relationship between cAMP and CREB: they are both directly proportional, meaning if one increases
Increase cAMP increase CREB
Decrease cAMP decrease CREB
Define homeostasis and Maintenance functions common to all cells
- Self-regulated processes
- Dynamic equilibrium of biological processes
- Disruption can result in cellular toxicity (e.g., cytotoxic responses) and disease
- Levels of homeostasis: Cell-organ-body
Maintenance functions common to all cells:
- Cellular metabolism: chemical reactions necessary to sustain life (e.g., anabolic and catabolic reactions)
- Macromolecule assembly (e.g., cytoskeleton, membranes, vesicles)
- Endocytosis/exocytosis (e.g., nutrients/wastes)
- Cellular ATP production
- Regulation of intracellular ionic environment → Ca
Functions of ATP → ATP is central to many cellular processes
Adenosine triphosphate (ATP)
It is one of 4 nucleotide bases used in the biosynthesis of DNA and RNA
It is a precursor to enzyme cofactors such as NAD+, FAD+
It participates in cytoskeleton function (e.g., molecular motors, microtubule assembly, actin polymerization)
Uptake/export of substances (e.g., transporter proteins and endocytosis/exocytosis)
It is involved in signal transduction pathways such as adenylate cyclase, protein kinases, etc.
Ion homeostasis/flux across membranes (e.g., ion pumps and transporters)
It is necessary for the maintenance of cellular Ca2+ homeostasis
Five targets of xenobiotics in mitochondria
1) Kreb cycle inhibitors (arsenite, ONOO-) or depletion of NAD+/NADH (e.g., redox cyclers)
2) Electron transport inhibitors (rotenone, cyanide, ONOO- phosphine, antimycin-A, menadione)
3) Dissipation (uncoupling) of proton gradient (2,4- dinitrophenol, pentachlorophenol, amiodarone)
4) Inhibition of ATP synthase (DDT, chlordecone, N- ethylmaleimide, p-benzoquinone)
5)Inhibition of substrate delivery (hypoxia: carbon monoxide; lung poisons; hypoglycemia)
Mitochondrial dysfunction results in ATP depletion and disruption of cell function including alterations in Ca2+ homeostasis
Calcium (Ca2+) functions:
- Key signal transducing molecule of the cytoplasm
- Regulation of enzyme activity (e.g., kinases, hydrolytic enzymes, etc.)
- Direct regulation of transcription factor function (e.g., calcium response factor, basic helix-loop-helix transcription factors, etc.)
- Regulation of cytoskeleton (e.g., anchoring of actin microfilaments to plasma membrane)
Cytoplasmic calcium concentration is maintained at a very low level relative to the extracellular environment → Ca high out and low in
Three general mechanisms regulate cytoplasmic Ca2+
1) Transport out of cells across the plasma membrane
2) Sequestration in the endoplasmic reticulum
3) Sequestration in the mitochondrial matrix via a Ca2+ uniporter (selective ion channel) in the matrix membrane
Relationship between ATP biosynthesis and Ca2+ homeostasis
Calcium homeostasis requires cellular energy (ATP) input
- Flux Ca2+ against its concentration gradient (from lower to higher concentration)
Depletion of cellular ATP can result in a bioenergetic catastrophe→rise in cytoplasmic calcium
ATP is essential for maintain low intracellular Ca (by moving calcium ions into the ER by calcium pump) →bioenergetic control
Three mechanisms to maintain Ca2+ homeostasis
Na+/Ca2+ plasma membrane exchanger
Plasma membrane Ca2+ pumps
ER membrane Ca2+ pumps
Disruption of Ca2+ homeostasis by xenobiotics:
Xenobiotics interfere with Ca+2 homeostasis through
direct inhibition pumps
plasma pore formation
release from cellular compartments like ER, mitochondria
depleting ATP through damage to mitochondria
Where is that increase Ca+2 coming from?
indirect and direct
two different mechanisms
Direct mechanism: decreased activity of ER/plasma membrane Ca2+ pumps
Indirect mechanism: decreased activity of Na+/Ca2+ exchanger due to decreased activity of sodium (Na+)/potassium (K+) ATPase
- Xenobiotic inhibition of Na+/K+ ATPase: Na+/K+ ATPase maintains the extracellular-intracellular Na+ gradient (i.e., membrane potential)
Overview: ATP and Ca
ATP maintains ionic balance: ensures proper distribution of Na+ & Ca+2
ATPase hydrolysis for energy: ATPase enzymes hydrolyze ATP & NADP+ to release energy, which is used by the Na/Ca pump to push calcium against its concentration gradient
Sodium-Calcium Exchanger Importance: maintains homeostasis by utilizing energy from the sodium gradient to pump calcium out of the cell - trying to fix the excess of Ca because we already have a lot of Ca (inside cell) so more calcium→catastrophe
- This stabilizes it!
ER & Ca+2 Signaling: ER is where proteins are synthesized and P450 enzymes are located, important for calcium signaling & maintaining proper gradient of calcium
Mitochondria damage impact: damage to mitochondria, reducing ATP as an energy source, can disrupt the Na/Ca exchanger→ increased intracellular Ca+2 levels
- This can activate enzymes inappropriately & interfere with normal cellular functions that rely on calcium signaling
Contractions gradient of Ca and sodium gradient know that. Sodium and Ca high outside and low inside.
Proton gradient:
A low proton concentration inside the cell, coupled with a high proton concentration outside the cell→creates a high proton gradient
This gradient facilitates the flow of calcium INTO the cell, emphasizing the importance of ATP in maintaining proper calcium balance
Cellular dysfunction:
cell death → Ultimate consequence of altered ATP/Ca2+ regulation
Mitochondrial dysfunction is a common mechanism of toxicity
Two major cell death pathways:
Necrosis: “loud” passive process (not programmed)
Apoptosis: “quiet” programed process has direct and indirect
Necrosis: “loud” passive process (not programmed)
Necrosis: role of mitochondria
Increase in inner mitochondrial membrane permeability formed by aggregated membrane proteins leads to mitochondrial leakiness to ions, swelling, complete cessation of ATP synthesis and then necrotic cell death
Necrosis: characteristic morphological changes
- Cell swelling, membrane blebbing, and cell lysis
- Release of cellular constituents into the interstitial fluid
- Tissue inflammatory response
- Infiltration of inflammatory white blood cells (i.e., neutrophils and macrophages)
- Activation of inflammatory cells to release reactive oxygen species and degradative enzymes (e.g., proteases)
- Further injury to tissue
Apoptosis: “quiet” programed process - Indirect mitochondrial dysfunction
Genomic DNA damage (e.g., double stranded breaks)
Induced by ionizing radiation or alkylating xenobiotics → Stabilization of the transcription factor, p53 → P53-induced expression of pro-apoptotic proteins of the Bcl-family of proteins, including Bax → Bax translocation to the mitochondria → Induces release of Cyt c to initiate the caspase cascade
Indirect: P53 is a tumor suppressor protein→induces release of Cyt C→caspase cascade→promotes apoptosis→essential for maintaining tissue integrity & to eliminate damaged cells
Apoptosis: “quiet” programed process - Direct mitochondrial dysfunction
Loss of mitochondrial cytochrome C ensures block of ATP synthesis
Cyt c complexes with Apaf-1 to activate caspase-9 → Caspase-9 is an initiator protease that activates effector caspases (e.g., caspase 3) → Effector caspases activate a specific DNase leading to cleavage of DNA between nucleosomes → Cleavage of structural proteins, such as actin and lamins facilitates cell disassembly
Direct: Cytochrome c is released from the mitochondria→ initiates apoptosis→leads to activation of caspases→essential for maintaining tissue integrity & to eliminate damaged cells
Apoptosis: characteristic morphological changes
- Condensation of nuclear and cytoplasmic material
- Cell shrinkage without lysis of the plasma membrane
- Cellular fragmentation into membrane-bound (apoptotic) bodies
- Externalization of phosphatidylserine on the cell surface
- Removal of apoptotic cell fragments by phagocytosis
- Lack of tissue inflammatory response
Affect qualitative or quantitative changes in gene expression:
Qualitative: gain/loss of function of genes
Quantitative: changes in expression level of genes
Genotype and Phenotype
Genotype: DNA sequence of cells (e.g., genes, alleles)
Phenotype: traits coded by genes (e.g., cellular functions, eye color)
Genetic code:
Protein sequence determines gene function
Codon sequence determines protein sequence
codon - protein - genetic
Genotoxic Xenobiotics vs Non-Genotoxic Xenobiotics
Genotoxic Xenobiotics: Change DNA sequence
Non-Genotoxic Xenobiotics: do not change DNA sequence
Genotoxic Xenobiotics: Change DNA sequence
Four Types of Genotoxic Mechanisms
Gene Mutation
Gene Amplification
Chromosomal Aberration
Aneuploidy
Gene Mutation
Mutation: any change in DNA sequence
Mutation Classifications:
1)Point mutations
A.Missense: amino acid substitution (may change protein function)
B.Nonsense: causes protein truncation (shorting)
2) Frameshift mutations
A. Insertions
B. Deletions
General consequences of gene mutations on gene products:
1) Silence gene: no functional product (e.g., loss of function)
2) Modification of the function of the gene product (e.g., gain of function)
3) Alteration in level of expression of the gene product (e.g., alteration in transcription factor binding site in promoter region of gene)
Gene Amplification:
duplication of entire gene
DNA is responsible for building and maintaining your human structure. Genes are segments of your DNA, which give you physical characteristics that make you unique.
Chromosomal aberration:
Chromosomal aberration: alterations in chromosome structure/morphology
1) Large fragment deletions in chromosomes
2) Sister chromatid exchange: exchanges of large fragments between sister chromosomes
Aneuploidy:
Aneuploidy: Deviation from normal number of chromosomes (increase or decrease)
Ex: Trisomy syndromes: 21 Down’s syndrome (3 copies of chromosome 21) and Klinefelter’s (XXY) syndrome
Genotoxic Xenobiotics:
Mutagens: xenobiotics that causes a DNA mutation
Clastogen: xenobiotics that cause chromosomal breaks
Aneugens: xenobiotics that cause aneuploidy
Non-Genotoxic mechanisms:
Xenobiotics may alter DNA function without damaging DNA directly via epigenetic mechanisms
Epigenetic changes are modifications to the genome that do not alter the sequence of the DNA → “Transmission of alternative states of gene activity”
- Affect function of regulatory (promoter) regions of genes
- Can alter the responsiveness of the gene promoter to transcription factors
- Can turn genes off or on, or modify their expression level
Two types of chromatin:
Euchromatin: lightly packed regions of DNA that are typically accessible to transcription (areas of gene expression)
Heterochromatin: tightly package regions of DNA that are inaccessible to transcription
Epigenetic mechanisms: DNA methylation
Decreases transcription factor accessibility
↑ (hyper) methylation: favors heterochromatin and gene inactivation
↓ (hypo) methylation: favors euchromatin and gene activation
Durable change but may also be reversible
Can be inherited from parents
Epigenetic mechanisms: Histone acetylation
Epigenetic mechanisms: Histone acetylation
Increase Acetylation: favors euchromatin and gene activation
Decrease Acetylation: favors heterochromatin and gene inactivation
Consequences of genetic toxicity
Cancer
Developmental/Birth defects
Toxicant
Toxin
Xenobiotic
Toxicant: poisonous substance of human origin (ex: drugs)
Toxin: poisonous substance of biological origin
Xenobiotic: any substance, harmful or not (e.g., poison or drug), that is foreign to a given biological system
