Exam Flashcards
(184 cards)
1
Q
What does ADME stand for?
A
Absorption, distribution, metabolism and excretion
2
Q
What is toxicokinetics?
A
What the body does to A compound
3
Q
What is toxicodynamics?
A
What the compound does to the body.
4
Q
What does MOA stand for?
A
Mode of action (the generic key events of the adverse effect of a chemical)
5
Q
Is dose response quantitative or continous?
A
Quantitative, either dead or alive. Can be used on populations
6
Q
Is dose effect quantitative or continous?
A
Continous. Can be used on individualisme
7
Q
Explain the difference between stochastic death and individual tolerance
A
In stochastic death, death is random and death will occur even at concentration =0. In individual tolerance death will occur at different concentrations due to the difference among individuals. Given A fixed concentration and that time is infinit all individuals in stochastic death will die while only the ones below the threshold in individual tolerance will
8
Q
Name the different reactions in phase 1
A
Oxidation, hydrolysis, hydroxylation, dealkylation, deamination
9
Q
What drives phase 1 and 2?
A
Homeostasis
10
Q
What can be a side effect to phase 1 and phase 2 reactions and give an example
A
Bioactivation. Benzo-a-pyrene, an epoxy group is added which turns into two hydroxyl groups which allows the molecule to interact with dna
11
Q
What is thalidomide and what is the problem?
A
It is a medicin against morning sickness. It has no side effect on adults but infants where born with abnormal limbs. It is still used in South America to treat cancer and leprosy. Thalidomide binds to the promoter region with high density of G.
12
Q
What is ADR short for.
A
Adverse drug reaction. This is not always toxic only if the exposure exceeds the therapeutic index
13
Q
What are the different toxic responses?
A
Adaptation (cope with), stress (counteract or remove) and toxicity (damage /death)
14
Q
What is hornesis?
A
It means that low concentration of a toxicant can be beneficial for the organism. The difference between the beneficial dose and the toxic dose is called the hermetic region
15
Q
Explain molecular homology, the problem and give an example
A
It means that xenobiotics can mimic endogenous compounds. They are transported into the cells. Mercury, when it binds to glutathione and is degraded it end up with a cysteine dimer and is transported as this
16
Q
Name a tissue specific transcription factor
A
HNF1. This regulates CYPP450 and several phase 2 enzymes. HNF1 regulates the expression of the genes
17
Q
What is diethylstilbestrol?
A
A synthetic estrogen used until the 70s as a growth factor in livestock fed and to prevent miscarriage. Daughter had higher occurrence of vaginal cancer while sons had testicular damage
18
Q
What is di-(2ethylhexyl)phthalate (DEHP) and why is there a difference in the toxicity between humans and mice?
A
It is a ubiquitous plasticizer which is carcinogenic in mice but not in humans. DEHP activates PPAR alpha which is a nuclear receptor and thereby causes cancer. Mice have 10fold higher hepatic PPAR alpha than humans
19
Q
What are the main targets of xenobiotics and why?
A
Liver and kidney.
Due to high blood flow, up concentration and metabolizing enzymes making the toxic byproduct
20
Q
What size of molecules can pass the glomerular filtration barrier?
A
<50kDa
21
Q
What is OAT1 and OAT4 and what are the difference?
A
They are organic anion transporters but OAT1 is on the basolateral site while OAT4 is on the apical side. OAT needs hydrophobic and preferably negatively charged
22
Q
What is the driving mechanism of OAT1?
A
Uphill xenobiotics uptake is driven by downhill flux of alpha ketoglutarate
23
Q
What happens if OAT4 is inhibited?
A
The toxicity will increase because the xenobiotics are accumulating
24
Q
Name the three xenobiotics export pumps
A
Multi drug resistance associated p-glycoprotein.
Phospholipid export pump
Conjugated export pump
25
What causes multi drug resistance?
High efflux of the drug, increased production of the export pumps. P-glycoprotein, multidrug resistance associated protein and breast cancer resistance protein
26
Where is p-glycoprotein very important?
Brain-blood barrier
27
What is necrosis?
A proces that results in uncontrolled cell death. The process does not requirer ATP and are often a results of a misfunction or damage in the cell.
Upon necrosis the whole cell will swell up leading to membrane rupture and release of intracellular components into the extracellular space. This leads to the break down of adjacent cells and activation of the immune system
28
What is apoptosis ?
Apoptosis is tightly controlled process in which the cell is degraded and phagocytosed leading to no immune respons. Apoptosis requires ATP and function as the last option in removing a misfunctioning or damaged cell.
29
How does necrosis in a few cells progress to large areas of tissue?
When one cell has ruptured resulting in leaking of all it contents into the extracellular space, activated proteases, phospholipases and endonucleases are released and will start attacking and break down adjacent cells. The adjacent cells will eventually rupture and lead to necrosis of whole areas of tissue. When intracellular cell content leaks out and can spread to the lymphatic system, which results in activation of the immun system
30
What is apoptotic bodies ?
Upon apoptosis cell components are degraded and isolated into several vesicles consisting of the cell membrane. These vesicles are called apoptotic bodies. By forming apoptotic bodies the body insures that no leaking of intracellular materials into the extracellular space and thereby no activation of the immun system. The apoptotic bodies are taken up by macrophages for further degradation and recycling of the cell contents. .
31
What is an initiator caspase ?
It is a cascade that activates other caspases. Initiator caspases include caspase 2, 8, 9 and 10
32
What is an executioner caspase ?
Executioner/effector caspase are activated by initiator caspases and are responsible for breaking down critial cell constituents. There are execution caspase 3, 6 and 7
33
In brief: what is the main difference between the intrinsic and the extrinsic pathway of apoptosis ?
The extrinsic pathway is activated from the outside. When immune cells recognize a damaged cell or a tumor cell they can trigger apoptosis through binding of the Fas ligand to the Fas receptor on the target cell.
The intrinsic pathway is activated when something goes wrong in the cell, especially if mitochondria are damaged and their components leak out into the cytosol.
34
What is DISC ?
DISC is part of the extrinsic pathway of apoptosis. It’s the death-inducing signaling complex that is generated when FasL binds to FasR. It activates and cleaves procaspases that are part of the apoptosis pathway.
35
What is a death receptor?
The death receptor, also called FasR, is the receptor found on the surface of cell membranes. When FasL binds to FasR it activates the extrinsic pathway of apoptosis and may lead to controlled death of the cell. This pathway is often used in cancer cells.
36
What is FasL ?
FasL is the death ligand found on the surface of Natural killer cell membranes. Upon binding to the FasR of a target cell, the extrinsic pathway of apoptosis is activated.
The extrinsic pathway is often activated in tumor cells.
37
How can FasR be activated without ligand binding ?
Xenobiotic-induced cholestasis leads to high concentrations of bile-salts such as Glycochenodeoxycholate can trigger FasR activation.
High concentrations of toxic bile salts stimulates translocation of FasR to the cell membrane. High concentrations of FasR in the membrane leads to oligomerization of the receptors. This oligomerization results in activation of the apoptotic cascade.
38
How and where does phtalates like MEHP induce apoptosis in testicles?
Phtalates such as MEHP induces apoptosis by disruption of the vimentin-filaments in sertoli cells. The function of vimentin-filaments is to anchor sperm cells to sertoli cells.
The disruption leads to loss of connection between sertoli cells and spermatogonia, which results in massive release of FasL from sertoli cells. This triggers exposure of FasR on sperm cells and by interaction of FasR and FasL apoptosis of spermatogonia is induced. Ultimately this results in testicular atrophy (reduced number of sperm cells).
39
How does menadione inhibit apoptosis ?
Manidone is a quinone and upon bioactivation by P450 a semiquinone is formed, which leads to redox cycling. This redox cycling results in formation of ROS and oxidative stress. The ROS’ can form protein damages and in this case the victim protein is cys in the catalytic site of caspases.
In this way Menandione inhibits apoptosis, as caspases are essential in activation and management of apoptosis.
40
What are pro-apoptotic proteins?
Pro-apoptotic proteins increase the probability of apoptosis. An examples is Bax. Dimers of Bax forms pores in the mitochondrial outer membrane when the concentration is increased and there by release Cytochrome C, which activates the apoptotic cascade. Furthermore Bax can mediate opening of mPT (permeability transition pump).
Other examples are Bad, Bid, Bok, Bik and Bak.
41
What are anti-apoptotic proteins ?
Pro-apoptotic proteins decrease the probability of apoptosis. An examples is Bcl-2. Bcl-2 inhibits dimerization of the Bax pump and it can also inhibits opening of mPT
42
What is an apoptosome ?
Apoptosome is an heptamere of CARD domains and seven procaspase 9’. Each CARD domain is composed of one Apaf1 unit and one cytochrome c in the binding domain of Apaf1.
The function of the apoptosome is to cleave procaspase 9 thereby initiating the intrinsic apoptotic pathway.
43
How can COX-2 inhibitors induce apoptosis ?
COX-2 is responsible for the formation of prostaglandins, which has a stimulating effect on the anti-apoptotic protein Bcl-2.
When Cox-2 inhibitors are present less prostaglandins are produces and the amount of Bcl-2 decreases. Thereby COX-2 inhibitors induce apoptosis.
44
What is Apaf-1 ?
Apaf is the protein which binds Cytochrome c and forms the apoptosome.
45
What is the main differences between apoptosis and necrosis ?
```
Apoptosis
1. Cellular condensation
2. Membranes remain intact
3. Requires ATP
4. Apoptotic bodies phagocytosed
no immune response
5. Ladder-like DNA fragmentation
6. Individual cells are affected
7. No release of intracellular proteins
```
Necrosis:
1. Cellular swelling
2. Membranes are broken
3. ATP is depleted
4. Cell lysis > inflammatory reaction
5. Random DNA fragmenta.on
6. Whole areas of tissue affected
7. Exposure of intracellular compounds in extracellular fluid
46
How does phenobarbital affect liver mass ?
Phenobarbital increases the mass of the liver, as phenobarbital increases the levels of Bcl-2. Bcl-2 prevents liver cells to degrade and therefore the number of hepatocytes increases.
47
What is the common result of different mechanisms of mitochondrial toxicity?
The common result upon mitochondrial toxicity is decrease in ATP production
48
What is a protonophore?
A protonophore is a compound which is able to un-couple oxidative phosphorylation across the inner mitochondrial membrane by disrupting the proton gradient across the membrane. Protonophores are often small and weakly acidic.
An example is pentachlorophenol which shuttles protons from the intermembrane space into matrix.
49
What is carnitine?
Carnitine is a small compound which binds to fatty acids to facilitate transport of long-chain fatty acids (C14-C18) into mitochondrial matrix.
If carnitine is depleted no long chained fatty acids will enter the mitochondria and participate in ATP formation.
50
What is valproic acid?
Valproic acid is an anti-epileptic drug.
The Cyp-metabolite of Valproic acid can inhibit beta-oxidation by forming esters with CoA in the mitochondrial matrix and carnitine in cytosol. Thereby valproic acid depletes both CoA and carnitine which both are essential for beta-oxidation of fatty acids.
In addition Valproic acid is able to inhibit beta oxidation enzymes directly.
When beta-oxidation is inhibiteted this will lead to accumulation of fatty acids in the cytosol as fatty acid vesicles (microvesicular steatosis). This can lead to liver failure.
51
What is fluoroacetate ?
Fluroacetate is a natural plant toxin which is used as a rodenticide.
Fluroacetate mimics acetate and therefore it can participate in TCA cycle instead of acetate. Therefore flurocitrate is formed, by the same mechanism as citrate is formed. Flurocitrate is strongly electronegative and it inhibits Aconitase by interfering with iron in the active site and thereby TCA is blocked. This results in accumulation of citrate and decreased ATP production.
The organs which are mostly effected by this mechanism is the heart and CNS.
52
How does cadmium produce ROS in mitochondria?
Cadmium produces ROS in mitochonria as it blocks between the iron center of b566 and the semiquinone in complex III of the electron transport chain. This leads to accumulation of semiquinone which can interact with O2 forming superoxide.
This is an example of how a non-redox active metal can form ROS.
53
Why does doxorubicin accumulate in mitochondria?
Doxorubicin accumulates in the mitochondria as it has a high affinity to cardiolipin.
Cardiolipin is placed in the inner mitochondrial membrane instead of cholestrol.
54
What is the effect of doxorubicin in mitochondria?
Doxorubicin abstract electrons from complex I in ECT. This is possible as doxorubicin is a quininone, which is changed to a semi-quinone when the electron is abstracted. This results in a redox cycle when the quinone is reformed by formation of superoxide.
The redox cycle will result in GSH depletion, oxidation of mtDNA, reduction of ATP production and opening of mPT. This will ultimately result in necrosis
55
What is MOMP / mTP?
MOMP(mitochondrial outer membrane permeabilization) is a pore in the outer mitochondrial membrane which is composed of aggregates of pro-apoptotic proteins (Bax and/or Bak).
Formation of this pore results in dislocation of cytochrome C to the cytosol which will induce apoptosis.
The formation of MOMP is inhibited by anti-apoptotic proteins.
56
What induces MOMP ?
Xenobiotics and Pro-apoptotic proteins from the Bcl-2 family can induce formation of MOMP
57
What is mPTP?
mPTP (mitochondrial permeability transistion pore) is a channel in the mitochondrial membrane, that allows passage of compound < 1500 Da. It is normally closed, but can be opened by e.g. an increase of Ca2+ and oxidative stress.
58
What are the consequences of opening mPTP?
When the mPTP is opened (often by ROS) it leads to a depolarization of the inner membrane. It releases intra-mitochondrial solutes. If not closed the mitochondria will swell due to an extensive water influx caused by differences in osmolality inside and outside the mitochondria. This leads to rupture of the outer membrane.
59
What is Glycocheno-deoxycholic acid ?
Glycheno-deoxycholic acid is the glycine conjugate of the bile salt chenodeoxycholic acid. It can have toxic effects upon accumulation resulting to an increase in oxidative stress (diverts electrons from ECT), recruitment of Bax leading o MOMP and mPTP opening and thereby rupture of the mitochondria.
60
Why is mtDNA more vulnerable to damage than genomic DNA?
- It has no histones.
- There are mitochondria specific enzymes that are related to mtDNA metabolism, which are selectively targeted by xenobiotics.
- Certain xenobiotics ammumulate in the mitochondria, which increases the risk of damage.
61
What is tacrine?
Tacrine is an example of a choline esterase inhibitor used in the treatment of Alzheimer. When it enters the intermembrane space in mitochondria, it gets protonated (positive charge). Its positive charge drives it in to the matrix because of the partial positively charged environment in the intermembrane space. In the matrix it intercalates with DNA which leads to inhibition of DNA topoisomerase II, thus inhibiting DNA replication. This leads to genotoxic stress and impairment of oxidative phosphorylation which eventually results in apoptosis of the cell.
62
Where does beta-oxidation take place?
mitochondrial matrix
63
Where do protons accumulate in mitochondria and what are the charge consequences on the internal membranes?
Protons are pumped from the matrix to the intermembrane space by the electron transport chain. This leads to a partially negatively charged environment in the matrix and a partially positively charged environment in the intermembrane space, as protons themselves are positively charged. The pH in the intermembrane space is 7.2 while pH in matrix is 8.
64
Metabolism of lipids, carbohydrates and amino acids liberates a common compound. Which one? How? And where is it utilized?
Lipids, carbohydrates and amino acids all form acetyl-CoA that is a part of the TCA-cycle. In this cycle NADH is formed. NADH is an electron carrier that transfers electrons to complex 1 in ETC which leads to the production of ATP.
65
What are the common targets of toxicity in mitochondria?
The most important targets in mitochindria is the membranes (especially the inner membrane), inhibition of electron transport and inhibition of energy supply (by blocking beta oxidation or TCA cycle).
66
Why is cyanide extremely toxic ?
Cyanide inhibits complex IV by binding strongly to Fe(II) in redox active proteins. This blocks ETC which leads to a rapid depletion of cellular ATP which leads to cell deaths.
67
What are furanocoumarines?
Furanocoumarins are natural plant compounds from especially citrus fruit. (Coumarin structure combined with a furane ring). Inhibits several CYPs
68
What is MPO ?
MPO, myeloperoxidases, are enzymes characteristic of immune cells (i.e. In bone marrow) where they can convert i.e. metabolites of benzene to the corresponding semiquinones og quinones.
69
What is UGT ?
UGT, UDP-glucuronyltransferase, a family of phase-II enzymes catalyzing transfer of glucuronic acid from UDPGA to xenobiotics. In most cases this facilitates elimination of the xenobiotics due to increase in water solubility , size and addition of a negative charge.
70
What is diclofenac ?
Its a NSAID taken to reduce inflammation and pain in certain conditions. It is thought to inhibit the prostaglandin synthesis by inhibition of COX. Known to have caused the near-extiction of vultures on the Indian sub-continent.
Makes unstable acylglucuronids (phase-II)
71
What is NSAIDs ?
NonSteroidal Anti-Inflammatory Drugs.
Provides analgetic and antipyretic effects, and in higher doses, anti-inflammatory effects.
Ex: Aspirin and Ibuprofen.
Nonselective inhibitors of COX1 and COX2.
72
What is a neutrophil ?
Phagocytic cells of the immune system
73
What is glucuronidation?
Glucuronidation is the addition of glucuronic acid to a substrate (phase-II). Glucuronic acid has to be energized first (UDP-GA). Glucuronidation is often involved in xenobiotic metabolism of substances such as drugs, pollutants and endogenous compounds like bilirubin, androgen and , estrogens.
Quantitatively most important Phase II in most species.
74
What is 2-naphtylamine ?
An aromatic compound made in aniline (dye) manufacturing, can cause bladder cancer.
75
What is glutathione ?
Enzymatically synthesized tripeptide
| Antioxidant, radical scavenger, H2O2 degration, regenerator of ox. proteins
76
What is aflatoxin ?
Toxin from the mold Aspergillus flavus. Most potent chemical carcinogen (Aflatoxin B1). Bioactivation by CYP = Bind to proteins and DNA (to G:C forming adducts in guanine)
77
What is NAT ?
NAT: N-acetyl transferase. NAT catalyses the transfer of an acetyl-group from Acetyl-CoA to aromatic amines (fx Serotonin) and terminal amines (acetylation) (phase-II). In humans: NAT1 and NAT2.
78
What is SULT ?
SULT: SULfoTransferaser. A super family of cytosolic enzymes catalysing the transfer of sulphate groups from PAPS (energized sulphate) to alcohols and amines (phase-II
79
What is GST ?
GST: Glutathion-S-transferase. Superfamily of enzymes transfering glutathion to xenobiotics (phase-II). GST has high specificity for GSH and low specificity for xenobiotics. Highest occurence in liver. Induced by many xenobiotics. 45 % of caucasians lack GST-M1, a polymorphy linked to bladder and lung cancer.
80
What is a nitrenium ion ?
Extremely reactive electrophil ( N+) often formed by re-arrangement of N-hydroxy compounds
81
What is tamoxifen ?
Anti-estrogenic drug used for treatment of breast cancer.
| Can be bioactivated by sulphatation (phase-II). Unstable product re-arrange to carbonium ion (highly reactive)
82
What is suicide inhibition ?
Epoxidation of xenobiotics often lead to covalent binding to DNA. However, some of the epoxide metabolites (i.e. of certain furanocoumarines) are so reactive that they immediately form covalent bonds with the CYP enzyme that created them thereby destroying the CYP. This is called suicide inhibition.
83
What is PCB ?
Polychlorinated bisphenyles: Lipophilic and persistent xenobiotics interacting with the aryl hydrocarbon receptor (AHR)
84
What is ROS ?
Reactive oxygen species:
Superoxide (O2-)
Hydrogen peroxide (H2O2)
Hydroxyl radical (OH)-
85
What is superoxide ?
*O2-
86
Name some natural sources for superoxide in aerobic organisms
Leaks from: P450 (sER) and Oxidative phosphorylation (mitochondrias).
Also emmitet as immun-respons by MPO (myeloperoxidases).
Relates to other ROS by: O2 --> O2− --> H202
87
What is redox cycling ?
A P450 reduction of a quinone to semiquinone (of fx. BenzoQuinone or B(a)P) donates a H from NADPH, which are immidiatly transfered to surrounding O2 creating O2−, and reforming the quinone.
This circle continues untill NADPH- and GSH, depletion in the cell --> causing DNA-damage (G), protein-damage (S-S formation, carbonylation) and lipid peroxidation (fragmentation of membrane lipids).
88
What is a free radical ?
Free radicals are atoms or molecules that have one or more unpaired electrons.
89
What is NQO ?
NQO : NAD(P)H-QUINONE OXIDOREDUCTASE
Cellular protective enzyme that enables the cell to circumvent
the potentially toxic step in redox cycling which produces semiquinone.
The cell is able through NQO to reduce quinones via a 2 electron
Transfer reaction directly to hydroquinone
90
What is CCL4 ?
CCL4 : Carbon Tetrachloride
A commonly used industrial solvent
CCL4 can cause a toxic mechanism through
Lipid peroxidation. Since tha small size and
its lipophilic properties it is easily distributed
In fatty compartments.
CCL4 can cause:
Kidney toxicity
Liver toxicity
91
What is a Fenton reaction?
Mechanism producing the extremely reactive hydroxyl radical from hydrogen peroxide if redox active metals are present (i.e. Fe, Cu)
92
What is glutathion?
Glutathione is a tripeptide (Glutamate-Cystein-Glycine), one of the most important defence mechanisms against oxidative stress.
Found in cytosol, mitokondria og cell nuclei.
Largest amounts in hepatocytes (liver)
Radical scavenger (non-enzymatic)
Conversion of hydrogen peroxide to water (Glutathioneperoxidase, GSPx)
Reduction af oxidized disulfide bridges (-S-S- -SH + SH-) formed by oxidative stress
Also involved in Phase II metabolism, conjugation.
Binds metal i.e. Hg2+
93
What is SOD ?
SOD (Superoxide dismutase) the enzyme converting the superoxide anion radical to hydrogen peroxide: one of the main defence mechanisms against oxidative stress.
94
What is catalase ?
Converts H2O2 (formed by metabolism or superoxide dismutase) to water and oxygen
95
What is metallothionein ?
Small cysteine-rich protein induced by, and binding metals as Zn, Cu, Cd, Hg thereby protecting SH-groups on other proteins in the cell
96
How is DNA damaged by ROS ?
ROS can react with DNA thereby giving mutations. In particular OH reacts with G producing 8-OH-G.
97
What is primaquin ?
Primaquin: an anti-malaria drug. Two metabolites of primaquin participate in redox cycling, producing superoxide radicals. Should not be administered to persons with polymorphisms in G-6-PD resulting in very low activity of the enzyme.
98
What is the consequences of lowered activity of G6PD as seen in some human polymorphies ?
G-6-PD catalyzes the conversion of G-6-P to 6-P-Gluconolactone (pentose phosphate-pathway) leading to recovery of GSH from GSSG.
GSH is important for the defence against ROS by conveting H2O2 to H2O.
Further, xenobiotics might be more toxic in persons with defect or reduced activity of G-6-PD due to lower levels of glutathion.
G-6-PD polymorphism is an advantage in malaria infested populations since Plasmodium cannot mature normally in G-6-PD deficient cells.
99
Name three cellular antioxidants
Vit. C (ascorbic acid)
Vit. E (tocopherol)
GSH
100
What is B(a)P?
Benzoapyren Five-ring PAH (tar compound) formed by combustion of organic matter (i.e. In cigarette smoke)
Highly lipophilic and ubiquituous environmental contaminant
101
How can B(a)P produce ROS ?
By redox cycling after phase-I bioactivation:
102
How can iron be prevented from causing Fenton reactions ?
By tightly regulating the pool of free iron (LIP) in the cell:
By regulating uptake and excretion
By binding to ferritin
103
Why is benzene toxic to the bone marrow ?
For Benzene to become toxic it must be hydroxylated by CYP2E1 (mainly in the liver) and further oxidized to hydroquinone, which accumulates in the bone marrow.
The bone marrow is rich in MPO’s (myeloperoxidases) that oxidize hydroquinone to semiquinone due to the many immune cells. Further, redox cycling between semiquinone and quinone produces superoxide anion radicals (oxidative stress).
This can lead to hypoplastic and aplastic anemia (depletion of erythrocytes and leukocytes).
104
What is an electrophil ?
An electrophile is a compound with a partially positive or even positive charge (i.e. Carbonium and nitrenium ions) that accepts one pair of electrons from a nucleophile in a chemical reaction. In toxicology it is usually xenobiotics or their metabolites with an intramolecular center with a low electron density and so, are reactive.
105
Name some nucleophilic cellular targets
Water
Glutathione
Cysteine, methionine, lysine and histidine residues.
106
What is methyl isocyanate?
Methyl isocyanat er et intermediært produkt er dannes ved produktion af pesticider, Det er meget reaktivt, stærkt elektrofilt og reagerer derfor straks med nukleofiler eller med nukleofile steder på et protein. Derfor høj potentiale for at inducere toksicitet i luftvejene hvis man ved et uheld kommer til at inhalere det. (ulykken i Indien!!!!)
107
What is microcystin and why is it toxic?
Microcystins are cyclic hepapeptides syntesized by blue-green algea and inactivate protein phosphatases. The inactivation of protein phosphatases leads to liver toxicity.
108
What are the nucleophilic targets in proteins?
Sulfur-containing amino acids (like Cys and Met), and nitrogen in side chains of Lys and His.
109
What is the toxic metabolite of hexane?
n-hexane excibits neurotoxic effects. This is caused by the bioactive metabolite 2,5-hexandione which is formed by oxidation in the liver.
The neurotoxicity is caused by a specific interaction between 2,5-hexandione (or other similar γ-diketones) with neuronal target proteins, where a selective adduct is formed by a nucelophilic attack from N on lysine residues on the elctrophilic carbonyls.
110
Which base in DNA is the most frequent target for electrophilic attack?
Guanine is the primary target for electophilic attack and potential carcinogenicity.
O-6 and N-2: Disruption of normal hydrogen bridges between partner bases. This may lead to point mutations (during replication)
111
What is a DNA adduct ?
The term ”DNA adduct” is used, when a chemical is covalently bond to a piece of DNA, which can cause cancer.
Examples of chemicals who can covalently bind to DNA: hydroxylamines ,aldehydes and epoxides
112
What are organophosphates?
Organophosphates are the general name for esters of phosphoric acid, and are one of the most important biochemicals.
Organophosphates are the basis of many nerve gasses and insecticides, this biochemical are also used as plasticizers and solvents.
What are the consequences when organophosphates binds irreversible?
Organosphosphorus esters react with the active site of AchE (a serine hydroxyl group) resulting in a phosphorylated and inhibited enzyme. With some insecticides this process is irreversible and the duration and severity of toxicity are prolonged.
113
What is acetylcholin esterase ?
Acetylcholin esterase (AcChE) is a serine esterase that hydrolyzes the neurotransmitter acetylcholine (Ach)
114
How are organophosphates bioactivated?
Organophosphate (OPs) is used as insecticides. In insects it is bioactivated by P450.
115
What are parabens ?
Parabens are compounds that are used for preserving cosmetics. They have estrogenic activity and the activity increases with the length of the alkyl group. The parabens that are used most frequently are methyl-, ethyl-, propyl- and butylparaben.
116
Name a xenobiotic capable of binding to PPARα
Phthalates (ex. DEHP) are able to bind to the PPARα receptor.
117
What xenobiotics can bind to PPARγ ?
```
PPARγ receptorer: 10-100x mere tilstede i fedtvæv, end andre. PPARγ receptoren accepterer en bred vifte af ligander! Fx er mange EDC’er (Endocrine Disrupting Chemicals) i stand til at aktivere PPARγ receptoren og starte differentiering af fedtceller. Udsættelse for disse kemikalier kan være en betydende faktor i forøgelsen af diabetes og overvægt hos især unge mennesker.
Nogle af de mistænkte EDC’er:
Bisphenol A
Dioxiner
Organoklorin pesticider
Organofosfat pesticider
Phthalater
Arsenik
Nicotin
PPARγ accepterer en bred vifte af ligander!!!
```
118
What is aromatase ?
Aromatase is the CYP enzyme that catalyses the formation of estradiol from testosterone
119
What is TBT ?
TBT: Tributyltin
Can cause maskulinisation of the molluscs (bløddyr), especially snails ( ex. purpur snails)
Normally, Testosterone will be converted into estrogen by an aromatase p450. But when TBT is present, it will block the aromatase and thus not be able to convert testosterone to estrogen and thus make the molluscs more masculine. (induction of aromatase this could happen to Vinclozolin (azoles) → anti-androgenic effect) (Inhibition → accumulation of Testosterone)
120
What is TDS ?
The testicular dysgenesis syndrome:
4 different types of disorders in the male sex development
1) Increased incidence of testicular cancer
2) Falling sperm quality
3) Cryptorchidism => testicles not falling correctly down into the scrotum
4) Hypospadias => lack of coalescence of the urethra
121
What is vitellogenin ?
Vitellogenin is a yolk protein. It exist in sexually mature female fish. But it almost doesn’t in male fish, only if exposed to estrogen or xenoestrogen. This makes it a good biomarker for exposure of xenoestrogens
122
What is procloraz ?
Procloraz is an endocrine disrupter that is masculinizes females.
123
Give examples of ER agonists
Xenoestrogens (or environmental estrogens) are endocrine disrupters that can elicit an agonistic response mediated by the estrogen receptor (ER), and they comprise of a diverse and chemically unrelated group of xenobiotics. Examples of ER agonists are alkylphenols (e.g. p-nonylphenol, used in detergents and emulsifiers), bisphenol A (BPA, e.g. used to make plastics), hydroxy-PCBs (metabolites of long-banned, persistent chemicals) and DES (e.g. used in livestock feed to increase weight gain).
124
What is receptor cross-talk ?
Receptor cross-talk is interaction between two receptors such that one receptor, when activated, modulates the action of the other. This type of interaction often involves the production by the first receptor of a second messenger, which then modulates the action of the second receptor.
There exists for example interaction (cross-talk) between the AHR and the ER, where e.g. TCDD (ligand for the AHR) can regulate the ER in either a positive or a negative way. When an ER ligand is absent, the TCDD/AHR/ARNT complex directly binds to the ER and activates it. When an ER ligand is present, the TCDD/AHR/ARNT complex can impair the expression of estrogen-responsive genes – that is the activity of the ER is somehow impaired.
125
Why is lipid peroxidation a chain reaction ?
When a radical reacts with a non-radical (lipids), it always produces another radical,
which is why the process is called a "chain reaction mechanism".
126
How can lipid peroxidation be terminated ?
The radical reaction stops when two radicals react and produce a non-radical species. This happens only when the concentration of radical species is high enough for there to be a high probability of collision of two radicals.
127
What is hormesis ?
Princippet i hormese er at, små doser af et toxin beskytter(giver øget fitness). Ved stigende doser vil dosisresponskurven falde under baseline og man ville f.eks. Kunne se en udvikling i flere tumorer.
128
Give an example of
A cellular adaption
A toxic response
Et eksempel på en cellulær adaption er MT. MT induceres af og binder toksiske metaller, der vil derfor bliver produceret mere MT når cellen bliver udsat for toksiske metaller. Cellen adapterer altså til de nye betingelser.
Et toksisk respons kan defineres som en allergen reaktion, nekrose, DNA skader, lipid peroxidation. Alle disse eksempler er en konsekvens af, at nogle holder op med at fungere.
129
What is the meaning of AUC in toxicology?
What is on the x- and y-axis when plotting it?
AUC= Area under the curve, hvor meget af et stof og hvor længe er det i kroppen
X-aksen= tid , y-aksen=plasmakoncentration
130
What is the proximal tubuli?
Proksimale tubuli er en del af nefronet i nyrerne. Mange stoffer bliver reabsorberet her.
131
What is HNF-1 ?
Hepatic Nuclear Factor – 1
En kernereceptor, der er specifik for leveren!
Hvis der er noget der kan via en kernereceptor give toksicitet! Vil det specifikt være i leveren
Inducerer en byge af bl.a. P450 Phase II enzymer => mulighed for bioaktivering
132
What is DES ?
DES (Diethylstilbestrol)
Syntetisk østrogen
=> Brugt meget i 70’erne, kan give testikulær dysgenese syndrom
133
How is methylmercury transported into the brain ?
Det er et lille og lipofilt Organisk kviksølv der kan give skade på CNS. Det transporteres over blod hjerne barrieren af Methionin transporteren! Methionin transporteren kan ikke kende forskel på Organisk kviksølv eller methionin;
134
What is a polarized cell ?
| Give an example
En Celle der har en forskellig ladning i begge ender (basolateral og apikal ende)
Organisk anion –og cation transportere
Ex; Nephrocytter
135
What is OATP ?
OATP Organic anion transporting peptides
| Passive facilitated transport for både influx og efflux
136
What is cephaloridine ?
Anti-biotikum af lactam gruppen
Nyretoxisk (proximale tubuli) grundet forskel i affinitet mellem basolateral influx (OAT1) og apikal efflux (OAT4)
Opconcentreres med toxisk effekt (lipid peroxidation) pga pyridine ring.
137
What is an ABC pump ?
ATP-binding cassette protein
| Familie af proteiner involveret i aktiv transport.
138
Which carrier transports paraquat into lung cells?
Polyamin transporter
139
What is cholestasis ?
En tilstand hvor galden ikke kan komme fra leveren til tarmen!
140
What is P-glycoprotein?
P-glycoprotein er et poreformende protein, som ved brug af ATP laver efflux af xenobiotika. Det kan fjerne stoffer fra membranen før de når cytoplasma.
141
What is the most important Phase-I enzyme family?
CYP P450
142
Write the general formula for the action of the mono-oxygenase system
RH + O2 + NADPH + H+ ROH + NADP+ + H2O
143
Give an example of an aromatic hydroxylation
Benzene ring with a hydroxyl group
144
Describe how toxicokinetic factors can lead to toxicity with one example
An example are statins which is a drug used to lower the plasma concentration of cholesterol. If other drugs are taken along with statins competitive inhibition occur. The rate-limiting step is the hepatic uptake and elimination into the bile via anion exporter pump e.g. the p-glycoprotein, but if competitive inhibition occur the retention time will increase.
145
Describe how toxicodynamic factors can lead to toxicity with one example
An example is thalidomide. Thalidomide binds to promoter regions with high occurrence of G bases, which is e.g. genes responsible for the limb development. When it binds to the promoter then it blocks the transcription and the fetus lacks limbs
146
Explain how chemicals can be selectively toxic to specific tissues/organs
Homology, when a xenobiotica looks like a certain compound in the different tissues / organs. It can also be called mimicry when xenobiotics mimic an endogenous compound.
147
Give two examples (in details) of molecular homology (mimmicry)
Both methyl and inorganic mercury bind to glutathione methyl-Hg-SG and GS-Hg-SG. This are realized into the bile or bloodstream. In the lumen of the proximal tubule (kidney) they are degraded to dicysteinyl-Hg compelx and inorganic mercury is then taken up since it mimics the cysteine dimer. The methyl mercury is transported to the CNS methyl-Hg-cys and mimics methionine and is transported over the blood-brain-barrier. In the brain it can have thiol-reactive effects.
DES is a synthetic estrogen used in livestock fed to increase cattle weight and used to prevent miscarriage and treat prostate cancer. It showed no effect in adult but daughters showed vaginal cancer and sons showed testicular deformaties. DES react with the estrogenic receptor
DEHP: an ubiquitous plasticizer that can activate the nuclear receptor PPAR which is abundant in the liver. Activation of the receptor results in decrease in apoptosis, increase in cell proliferation (hastig vækst / formering), increased productionof oxidative stress. This is not characterized as carcinogenic for human but for mice, this is due to the fact that mice have tenfold higher occurrence of PPAR.
148
Give an example where toxicity arises due to polarization of a cell
Cephaloridine is a zwitterion and is transported in via the OAT1 transporter but has a low affinity towards OAT4 and is therefore accumulating making it toxic.
149
Explain the concept of chemosensitizing
Chemosensitizing is blocking efflux pumps such that the toxic substance can enter the cell and stay there. It is an advantage when giving chemo but at disadvantage otherwise
150
What happens in oxidative desulphuration?
The double bound S is replaced by a double bound O.
151
How is the P450 system organized physically?
Mainly in ER
152
Name the main Phase II reactions
Glucoronide conjugation, sulfate conjugation, gluthathion conjugation, amino acid conjugation, acylation, methylation
153
What types (Phase-I or Phase-II) are the reactions? And give examples of compounds.
i) Epoxidation.
ii) Oxidative desulphuration
iii) Glucuronidation
i) phase I - benzo(a)pyrene is bioactivated by CYP and creates an epoxide.
ii) phase I - Thiopental is converted to phenobarbital which can induce CAR and induce CYP.
iii) phase II - it can cause formation of unstable esters. e.g. diclophenac.
154
Carbon tetrachloride (CCl4) can be bioactivated by P450. What is the toxic species formed
CCl3* and CCl3-OO*.
155
Name the sources for
i. Furanocoumarines
ii. Aflatoxin
iii. Benzo(a)pyrene
i. Furanocoumarines: grapejuice
ii. Aflatoxin: Aspergillus flavus
iii. Benzo(a)pyrene: cigarette smoke
156
Furanocoumarines, Aflatoxin and Benzo(a)pyrene are all bioactivated by the same mechanism. Which one?
CYP is bioactivating it by incorporating an epoxide
157
Bioactivation of arylamines often start with a particular phase-I reaction. Which one?
N-hydroxylation followed by an N-acetylation
158
Bioactivation (by a phase-I reaction) of anilin can cause met-hemoglobin formation. How?
Aniline goes through oxidation and then redox activity between phenylhydroxylamine and nitrobenzene occur which drives the production of met-hemoglobin. Hb2+ from hemoglobin is oxidized to Hb3+ which is met-hemoglobin.
159
How are ROS formed?
Leaking by CYP, Fenton reaction, UV radiations, redox cycling.
Superoxide dismutase:
Fenton reaction:
```
Leaking by CYP, Fenton reaction, UV radiations, redox cycling.
O2+e- ->O*-
Superoxide dismutase:
2O2*- + 2H+ -> O2 + H2O2
Fenton reaction:
H2O2 + Fe2+ ->HO* + OH- + Fe3+
```
160
How do higher organisms protect themselves against ROS?
Antioxidants, glutathione, SOD, metallothionein,
161
Quinones as a chemical group is known to induce ROS. What is the process called and how does it take place?
Redoxcycling. Quiones are reduced by CYP to semiquinones, which is again reduced to hydroquinone which not toxic. However hydroquinone can be oxidized to semiquinone by CYP and oxidized to quinone and at the same time O2 is reduced to superoxide anion
162
Write up the Fenton reaction
H2O2 + Fe2+ --> *HO + OH- + Fe3+.
| Cu2+/Cu3+ can also be used
163
How can it be prevented?
H2O2 is degraded by catalase and glutathione peroxidase (GS-Px). Furthermore iron is bound to trasferritin.
164
Describe in details (with formulas) the process of lipid peroxidation
A hydroxyl radical interferes with the lipid and turns it into a lipid radical. Since this radical is unstable it is conjugated to another lipid radical. Oxygen interact with this lipid turning it into a lipid peroxyl radical. another lipid reacts with the lipid peroxyl radical adding an H to the radical whereby it self becomes a radical. The lipid peroxyl radical is now a lipi hydroperoxide and can participate in the fenton reaction, making a lipid alkoxyl radical, which splits into free radicals and lipid aldehyde. Aldehydes from lipid peroxidation is DNA- and protein reactive species
165
Describe glutathione
Tripeptide, glutamate, cysteine and glycine. Cystein has sulfhydryl groups which is an important reactive group. It has high steady state concentrations. The bond between cysteine and glutamate protecs the tripeptide from protease digestion. It is most abundant in liver cells
166
What is the toxic metabolite of benzene? and why is the bone marrow the main target?
Benzene is metabolized by CYP to p-benzohydroquinone which in the bonemarrow is oxidized to the semiquinone and quinone by MPO. The cells have very low activity of NQO which is an enzyme that can do a two-electron transfer converting the quinone to a hydroquinone. They have low activity of NQO, they have high number of rapidly dividing cells (cellular damage is easily manifested) and high MPO activity
167
How do doxorubin induces these catalase, glutathione peroxidase and metallothionein among others?
Doxorubin is a pro-oxidant, meaning that it is a induces the defense line. The proteins is upregulated.
168
Explain how protonophores can disrupt the function of mitochondria using pentachlorophenol as an example.
Protonophore e.g. pentachlorophenol can pass the outer membrane because they are small lipophilic compounds, in the intermembrane space they are protonated and can pass the inner membrane and release the proton and diffuse out again and do the same all over again. They interrupt the proton gradient and decrease the ATP production
169
Describe how compounds can inhibit
i) lipid peroxidation
ii) TCA cycle
i) valproic acid can bind to CoA and this complex inhibits the enzymes that cleaves the bond and releases acetylCoA. Valproic acids therefore deplete the amount of CoA
The TCA-cycle: Flouroacetate mimics acetate and can be converted in the TCA-cycle until the it is converted to florucitrate which inhibits and enzymes
170
Give two examples of inhibitors of the ETC.
Cadmium inhibits complex III and there will be created semiquione which can create superanion.
Cynide inhibits complex IV
171
Explain in details how doxorubicin can cause mitochondrial toxicity
Doxorubicin is a quinone and is bound in the inner membrane to cardiolipin due to high affinity. Some of cardiolipin is close to complex I and the quinone donates an elelctron to p450 and a semiquinone is created. This will fulfill the octet rule and receives an elelctron from O2 creating an superanoxide
172
What is an electrophil?
Electrophiles have a low density of electrons and are positively charged. Xenobiotics are often electrophiles.
173
Give some examples of electrophils and describe how they are formed
Soft electrophiles: quinones
Hard electrophiles: methyl isocyanate, NAPQI
They are formed when a compound is metabolically activated. It is often inactivated by small nucleophiles in the body.
174
Which biological target molecules do they form covalent bonds with?
Often amino acids with S groups (cysteine or methionein) or aminoacids with sidechains containing N. Most often DNA and proteins. CYP.
175
Describe some factors that determine which proteins are susceptible to adduct formation
- Half-life of the metabolite / compound. At short half-life they will react fast and often with the enzyme that created them e.g. CYP and inhibit the enzyme and thereby their own production. If they have a long half-life they will reach distant targets.
- Concentration of the metabolite / compound. Biliary accumulation if a compound is up-concentrated due to choleostasis. Uptake and efflux of the compounds.
Physico-chemical charecteristics. pH and exposed nucleophiles
176
Give an example of an adduct and describe the consequences.
Microcystins comes from algael blooms and is taken up into the hepatocytes by OATP. It binds to PP-1 (protein phosphatease) with a carbon-sulfur bond (NOT a covalent bond). Microcystin is located at PP-1 active site and the enzyme is inhibited
177
What are the nucleophilic targets of DNA? Describe in detail how benzo[a]pyrene makes a point mutation
The nucleophilic targets of DNA are the oxygen and nitrogen in the nucleic acids. In guanine it is most often O-6 ad N-2 which are normally involved in the hydrogen bridges with partner bases.
Benzo-a-pyrene is bioactivated and gets an epoxide which is transformed into two hydroxyl groups. A new epoxide is created and due to steric hindrance the epoxide hydrolase cannot transform the epoxide to hydroxyl groups and instead the epoxide bind covalently to N in the guanosine base. Therefore guanosine can only create two hydrogen bonds and binds to adenosine instead of cytidine.
178
Descibe the toxic mechanism of hexane
n-hexane is metabolized in the liver to 2,5-hexanedione which is ultimate toxic metabolite for inducing neuropathy. 2,5-hexanedione is a diketone and the distance between the ketones is pivotal for the toxicity. Diketones interact and form selective adducts to neuronal target proteins. A carbon in the diketone is electrophilic and are attacked by the nitrogen of lysine in the protein. This results in a pyrrolidine. The lysine is found in the fibers of neurons and when diketone binds to lysine and creates pyrole the big complex will weaken the structure and it will collaps making the signaling between neurons week.
179
Discuss in general terms the mechanisms behind species-specific toxicity for the following compounds:
DEHP
OP
Tamoxifen
- DEHP (diethylhexyl phthalate): it is a plasticizer that causes liver tumors. It is not carcinogenic in humans because we have 10 times lower amount of PPAR. DEHP is an activator and ligand of PPAR which results in decreased apoptosis, increased cell proliferation etc.
- OP (organophosphates): Organophosphates inhibits acetylcholin esterase by binding to the enzyme covalently which prevents the enzyme from cleaving acetylcholin which therefore will accumulate.
- Tamoxifen: is an antiestrogenic drug used to treat breast cancer. It creates liver tumors in rats, fewer in mice and almost none in humans. This is due to a difference in enzymes. Tamoxifen is a substrate for SULT which is a toxic pathway because it creates a carbocation which can react with DNA. Rats have no UGT which would be a clear detoxification way and have a high concentration of SULT.
180
Benzo(a)pyrene can cause toxicity by inducing oxidative stress and/or by binding directly to DNA. Describe the two pathways leading to these conditions.
In the pathway leading to oxidative stress Benzo(a)pyrene is bioactivated by CYP1A1 followed by phase II and becomes a quinone which can induce ROS. In the pathway with binding to DNA an epoxide is created by bioactivation by CAP1A1 leading to point mutations.
181
Describe the two main routes by which a cell can be triggered to enter apoptosis.
Extrinsic and intrinsic pathway.
Intrinsic: Bcl2 proteins create the megapore and cytochrome C is released from the mitochondria. Cytochrome c activates procaspase 9 to caspase 9 which activates procaspase 3 to caspase 3 which leads to apoptosis.
Extrinsic: a death receptor. A ligand binds to the receptor which activates procaspase 8 to caspase 8 and activates procaspase 3 to caspase 3.
182
Explain some differences between necrosis and apoptosis
Apoptosis: energy dependent, no swelling, no rupture, only the precise cell is affected
Necrosis: no energy is needed, swelling, rupture, big area is impacted.
183
Explain how phenobarbital and menadione can inhibit apoptosis. What are the toxicological consequences?
Phenobarbital inhibits hepatocellular apoptosis by upregulating Bcl2 and thereby stimulating antiapoptotic signals. Liver cell mass increases.
Menadione is a quinone and inhibits caspases. Menandione (Vitamin K3) is bioactivated by P450 to semiquinone. Massive oxidative stress due to redox cycling. Depletion of GSH, oxidative protein modifications etc. Release of cytochrome c from mitochondria. Normally: release of cytochrome c will lead to apoptosis. Reason: ROS oxidize cys in catalytic site of Caspase. Inactivation of apoptosis
184
Explain how the following compounds can cause toxicity
a. epoxide
b. N-hydroxy compounds
c: Quinones
d. Di-ketones
e. Acylglucuronides
a. epoxides: really reactive and bind to e.g. N in DNA bases.
b. N-hydroxy compounds: can be rearranged to a nitrenium ion which is a really reactive electrophile.
c. Quinones: can make redoxcycling and interfere with e.g. electron transport chain.
d. Di-ketones: is a metabolite of hexane which can bind to lysin in neurons and weaken them.
e. Acylglucuronides: are prone to undergo hydrolysis, intramolecular rearrangements and covalent interactions with cellular target molecules (proteins, nucleic acids) by covalent binding. Comes from e.g. carboxylic-acid containing drugs e.g. NSAID.
