toxicology

Name of the Student
Professor’s Name
Toxicology-Coursework
10th January 2018
TUTORIAL 1
Compound A is a carboxylic acid with a pKa of 5.0. The protonated form of A has a partition coefficient of 1.0. If A is ingested, indicate
a) its most likely site of absorption: stomach
b) the effect of achlorhydria on the absorption: absorption is reduced
c) the effect of administration of the compound immediately before and immediately after a substantial meal: before a meal, it is increased, but after a meal, it is reduced.
2. Why is insulin ineffective if given orally?
Since insulin is a polypeptide hormone, it can be digested by proteases in the gastrointestinal tract. Hence, insulin is not administered orally; rather, it is administered subcutaneously.
3.What is meant by the following terms:
a)ADME
xenobiotic
systemic
topical?
ADME refers to absorption, distribution, metabolism, and excretion of a drug or a toxicant. It is an abbreviation for pharmacodynamics.
Xenobiotic is referred as any compound that is foreign to the body. It can either be a drug or a toxicant or a nutrient or a pollutant.
Systemic refers to any intervention that reaches different systems of the body through the arteries and veins.
Topical refers any intervention that is applied to the superficial surface of the body. Such applications are usually made to the dermal surface.
Work cited
Biochemical Toxicology Introduction Week1.pptTUTORIAL 2
Patient C is a type II (NIDDM) diabetic whose condition is controlled by the routine administration of tolbutamide.

She collapses into a comatose state following antibiotic treatment. Chemical analysis reveals an abnormally high level of tolbutamide in the blood. Explain the sequence of events.
Administration of antibiotic replaced tolbutamide that was bound to plasma albumin and tolbutamide was released into the blood leading to the high onset of hypoglycemia.
Indicate the primary routes of xenobiotic excretion in mammals.
Xenobiotic compounds are primarily excreted in a water-soluble form. The primary routes of Xenobiotic excretion are; kidneys (excreted in urine), liver (excreted with feces), and lungs (as volatile end products of metabolism).
Distinguish between phase I and phase II metabolism.
Answer: Phase 1 reactions include hydroxylation, oxidation, reduction, and epoxidation. The main role of phase 1 reaction is to deactivate a xenobiotic or to convert into a more polar form. On the other hand, Phase 2 reactions convert the end products of phase 1 reaction into a more polar form. Moreover, phase 2 reactions are also important if phase 1 reactions fail to convert a xenobiotic into a hydrophilic form. However, the objective of both phase 1 and phase 2 reactions is to eliminate the toxic end products of metabolism by converting them into water-soluble form so that they can be easily eliminated out of the mammalian body. Phase 2 reactions are referred as conjugation reactions because they form conjugates with end products of phase 1 metabolism. The major phase 2 reactions include glucuronidation, sulphation, acetylation, and acylation.
4. Compound B has an acid dissociation constant of 2.5 x 104. Its molecular weight is 240 daltons, and its plasma half-life (t1/2) is 24 hours. On excretion, it appears predominantly in the feces, both as itself and as the glucuronide conjugate. [Neither B nor its metabolites bind to plasma proteins].
Indicate the following;
a) its most probable site of absorption
b) its most likely route of excretion
c) why the t1/2 is so long.
Ka= 2.5 x 104
pKa drug =
-log10(2.5 x 104) =3.6
pH=3.6 hence a weak acid
in the stomach (Ph=2) then BH is likely absorbed in the stomach
The likely route of excretion is via the liver into the bile and out through feces
Due to enterohepatic circulation, as it is recycled.
Work cited
Biochemical Toxicology Distribution Excretion Metabolism Week 2.ppt TUTORIAL 3
Nitrobenzene was administered orally to two groups of rats at a dose of 225mg.kg1. Group A had been previously treated with an antibiotic which significantly lowered the bacterial population of the gut. Group B had not been pre-treated. Metabolites were detected in the urine as follows:
% original dose group A Group B
3nitrophenyl sulphate 11.0% 11.0%
4nitrophenyl sulphate 22.0% 22.0%
4hydroxyacetanilide 0.9% 16.0%
Explain these results regarding the metabolism of nitrobenzene.

phase I = hydrolyzed to 4-nitrophenol and 3- nitrophenol
phase II = 3-nitrophenyl sulphate, 4-nitrophenyl sulphate.
4-nitrophenyle sulphate —-reduction 4-hydroxynitric-
4hydroxyacetanilide

Gut flora is killed by the antibiotic, so less metabolite is formed in pre-treated group A cats.
2. Compound C was radio-labeled and incubated under aerobic conditions with human hepatocytes. The incubation was stopped by the addition of ethanol. Following centrifugation, the ethanol was removed and saved as fraction 1. The pellet was extracted with diethyl ether, and this solvent extract was saved as fraction 2.
The pellet was further extracted with 0.9% w/v sodium chloride; this extract was saved as fraction 3. This fraction was incubated with ßglucuronidase then further extracted with ethanol (fraction 4) and diethyl ether (fraction 5).
The radioactivity in each fraction was recorded and expressed as a percentage of the original administered compound C.
Fraction% original radioactivity
1 -Ethanol 20
2 -diethyl ether 32
3 -NaCl 39
4 -Ethanol 39
5 -diethyl ether 0
-What is the function of ßglucuronidase? Breaks glucuronide conjugates
-How might the identities of the metabolites be established? Chromatography/ MS
-Describe as a far as possible the metabolism of C. C metabolized in the liver by CYP2D6 an isoform of cyt P450.
-What is the likely explanation for any residual radioactivity in the pellet?
Some of it (9%) may be still bound to micro-molecules of hepatocytes.
Work cited
Biochemical Toxicology Metabolism Week3.pptBiochemical Toxicology Factors Modifying Toxic Effects Week 5.pptxTUTORIAL 4
1.In rats, the major metabolites of 1,4-dichlorobenzene (Mr=147) are 2,5-dichlorophenyl sulphate and 2,5-dichlorophenyl glucuronide.
Explain
how these metabolites may arise from the parent compound
1, 4-dichlorobenzene is first metabolized by CYP2E1 isozyme and the products are either sulphated through PAPS (phosphoadenosine-phosphosulphate) and UDP-glucuronide.
The likely route of excretion of the metabolites:
Major routes: kidneys (78 to 85%) and liver (2 to 5%) through urine and feces respectively.
How this metabolism in cats, pigs, and rats differs.
Cats are deficiency of Glucuronosyltransferase so unable to do metabolismPigs are deficiency of Sulfotransferase so unable to do metabolismRats are Fine as they can perfume both phases I and Phase II
2.1, 3-Dinitrobenzene was administered orally to rabbits. The major metabolites detectable in the urine were 2,4-dinitrophenol, 1-3-diaminobenzene, 3-nitroaniline and 2-amino 4-nitrophenol. 30% of the dose was excreted as glucuronides and 6% as sulfates. Prior treatment of the rabbits with antibiotics did not alter the amounts of 3-nitroaniline or 1, 3-diaminobenzene excreted.
Give detail of the likely metabolic pathways involved and possible sites of metabolism of 1, 3-dinitrobenzene in the rabbit.
It is likely to take place in the liver. Metabolism of 1, 3-dinitrobenzene is primarily by nitro-reduction.
The end products are dichlorophenols which are further conjugated to 2,5-dichlorophenyl sulphate and 2,5-dichlorophenyl glucuronide.
3.Account for the following scenario and include details of the biochemistry of this accidental poisoning.
“On arriving at a house, paramedics discovered an elderly woman lying unconscious on the floor next to a dead cat. She was admitted to hospital but died several days later from liver failure. The following evidence was submitted at the inquest: the house was well-ventilated, and the weather was warm at the time that she was found; the cat showed evidence of a non-fatal respiratory infection; the woman was apparently poorly nourished; traces of tissue-bound N-acetyl parabenzoquinoneimine were found in the livers of both the woman and the cat; her pet dog had been found unharmed.”
The woman suffered from high fever or flue same for the cat and the dog. She subsequently administered high dose of PAR and she gave some to her pets. Due to her malnutrition, she had low PAR metabolism and NAPQI accumulated to a high level and due to her old age her GSH was too low to cape will high NPQI hence she eventually fainted.
For her cat due to the lack of glucuronide could not metabolise PAR hence she died from liver damage while the dog which had glucuronide metabolised PAR and excreted normal.
4.Describe how the following modify the toxic actions of xenobiotics.
a)Diet: decreases the absorption rate of xenobiotics except if fatty in which case it increases the absorption of lipid-soluble xenobiotics. Inadequate nutrition may lead to lowered plasma albumin concentrations while excess nutrients compete for plasma albumin binding sites leading to increasing in plasma xenobiotics in both cases. High protein, indole containing vegetables, charcoal grilled meat, vitamin E and C increase the rate of metabolism. Diet affects the pH of urine hence affecting the excretion of drugs.
b)chemical interactions; Drugs can influence xenobiotic metabolism by competing with the substrate (xenobiotic) binding sites of cytochrome P450 enzyme system
c)genetic variability; the acetylator, an inherited genotype determines the rate of drug metabolism. Individuals either have the FF (fast) or Ff (fast) or ff (slow).
Work cited
Biochemical Toxicology Factors Modifying Toxic Effects Week 5.pptxTUTORIAL 5
Review the decision-making process involved in drug safety testing. Choose a test involved in this process and describe how it may be undertaken.
The Median Lethal Dose Test or LD50 Test:
This test aims at establishing the medium lethal dosage of a substance or a drug.
Rats and mouse are chosen as test specimen due to their availability, and the substance is administered orally to mimic human administration. Three doses are chosen; a dose to kill 50%, a dose to kill between 10-50% and another to kill between 50-90%. An average of seven doses are used with increasing concentration gaps of 1.2 -2 expressed in mg/kg or mg/m2. The minimum sample number is 50 specimens, and after 7-14 days the results are collected and used to access the lethality. A graph is plotted from the results and the concentration that corresponds to the 50% lethal mark obtained from the graph.
2.Give a critical account of the use of the Ames test in the detection of potential carcinogenic activity.
The Ames test is used to detect mutagens. Typically, the tests use Salmonella typhimurium. It detects reverse mutations in S. typhimurium by measuring the number of histidine-dependent (his-) mutant organisms reverting to histidine independent wild-type (his+). His- cells are incubated in a medium that contains insufficient histidine to permit visible growth. If a toxicant is then added that is capable of inducing reverse mutation, some cells will revert to his+ and can grow to form colonies when plated onto agar. The more mutagenic the toxicant, the greater the number of colonies. However, even without a genotoxic agent, there will be a low background number of spontaneous mutations. Different strains of S. typhimurium mutants are used where each strain detects a different type of mutation including frame shift, and base pair.
Explain the design and implementation of a long-term carcinogenicity study.
Long-term study design involves lengthy exposure of the specimen to the test substance, more than 70% of the specimen’s lifespan. Results should, however, be collected continuously for evaluation purposes and to determine the effects of the drug at the various levels of growth.
X is a nitroaromatic compound which is extensively metabolized by anaerobic bacteria within the mammalian gut, and 95% of it is converted to acetanilide which is excreted in the urine.
Using the standard Ames test, the following data were found for X:
Number of his+revertants per plate
Concentration of X
(μmol dm-3) -S9
(x±SD) +S9
(x±SD)
0 50±10 52±12
10 45±12 47±13
50 47±12 53±10
100 48±10 51±11
150 59±15 48±12
Comment critically on the protocol used (standard Ames test) and show how it may be used to assess the genotoxicity of metabolites of X. Detailed statistical analysis is not required.
The protocol is appropriate because it helps to identify whether a chemical compound is carcinogenic or mutagenic and whether its carcinogenic/mutagenic potential is dose-dependent. The test assay reflects that X is a weak mutagen because even after considering the standard deviations the number of His+ colonies is not double than that of the number of His- colonies.
Work cited
Biochemical Toxicology Measures of Toxicity Week 6.pptxTutorial 6 – Free Radicals in Disease
What does ROS stand for? List as many examples of ROS as you can.
The term ROS stands for Reactive Oxygen Species. Examples of ROS are hydrogen peroxide, superoxide anion, singlet oxygen, and hydroxyl radicals.
2.Define oxidant or oxidative stress.
Oxidative stress is defined as the stress or dysfunctions that stems from ROS or free radical-mediated damage.
3.Write out the Haber-Weiss equation. Explain its relationship to the Fenton reaction.
The Haber-Weiss reaction is the basis of formation of free radicals or ROS from hydrogen peroxide and molecular oxygen. The reaction is catalyzed by superoxide dismutase or peroxidase and occurs in two steps. In the first reaction, the ferric ions of the enzymes are converted to ferrous ion by reacting with superoxide anion. In the second reaction (called Fenton’s Reaction), the ferrous ions of the enzyme convert hydrogen peroxide into hydroxyl radicals, hydroxyl ion, and revival of the ferric state of the enzyme making the process a cycle.
Fe3+ + O2- = Fe2+ + O2HABER WEISS
Fe2+ + H2O2 = Fe3+ + OH- + OH. FENTON
——————-
Net Reaction; H2O2 + O2 = OH- + OH. + O2
4.Give examples of types of ROS damage.
Due to their high reactivity, ROS damage lipids, DNA, proteins, and RNA leading to aging.
5.Give examples of diseases or disorders where ROS are implicated.
Parkinsonism and Alzheimer’s disease.
6.Give an example of a biomarker of ROS damage in each of the following cases,
a) DNA: 8-hydroxy-2-deoxyguanosine
b) lipid: fatty acid peroxy radical
c) protein: carbonylated proteins
7.Explain the role of mitochondria in ROS- mediated damage.
The mitochondria are the primary source of ROS in the body. Since this ROS can cause damage to the body and the mitochondria, they have a mechanism for reducing the production and mitigating the produced. Superoxide dismutase removes the 02 formed, Catalase (CAT) removes the H2O2 formed while the glutathione peroxidase (GSH Px) = GSH removes H2O2 and lipids peroxides.
8. What is an antioxidant?
Antioxidants is a substance scavenge free radicals and prevent oxidative stress by neutralizing such radicals.
9.What is the relationship between antioxidants and ROS?
Antioxidants are compounds that scavenge free radicals and ROS. Hence, if the number of antioxidants increases within a cell then the amount of ROS would decrease and vice versa.
Work cited
AS6005 Reactive Oxygen Species RM-Lecture week 4.pptx Tutorial 7 – Eicosanoids and Disease
What are eicosanoids? –give examples of main groups
Eicosanoids are derivatives of phospholipids that are produced from arachidonic acid or other PUFA (polyunsaturated fatty acids). The major enzymes that take part in the formation of eicosanoids include cyclooxygenases (COX-1 and COX-2), lipoxygenase, and glutathione-s-transferase. The common eicosanoids are leukotrienes, prostaglandins, and thromboxane.
2.Explain the origin of the word ‘prostaglandin.’
The compound was first isolated in seminal fluid; hence, it was named after the prostate gland
3.Why are eicosanoids considered not to be true hormones?
Because they are not synthesized within a cell
Draw the structure of 5,8,11,14,17eicosapentaenoic acid and explain why it is a ω-3 fatty acid.
O
90170035560009632953556000963295133350009632951333500096329513335000
45593001365250037541201365250030575251365250024257001365250018599151365250050495206413500484251064135004559300641350042221156413500403669564135003754120641350034925006413500336232564135003057525641350027959056413500267652564135002425700641350022840956413500213169564135001859915641350016313156413500141351064135001166495641350096329564135007169156413500
HO
They are so called because such molecules bear a double bond on the third carbon atom from the end of the carbon skeleton (carbon chain).
5.Which series of prostaglandins derives from 8,11,14eicosatrienoic acid?
Leukotrienes
6.Explain, concerning the early biochemical processes, the anti-inflammatory action of corticosteroids.
Corticosteroids reduce the expression of interleukin-2 genes which leads to low levels of inflammatory mediators.
7.Complete the following:
The action of ________cox-1/cox-2_______ on arachidonate initiates the pathway leading to series 2 prostaglandins, ___prostacyclins___________and thromboxanes.
The action of _________lipoxygenase_____ on arachidonate initiates the pathway leading to the unstable epoxide __leukotriene____________ A4
8.Explain the role of cytochrome P450 in inflammatory states.
The enzyme produces epoxyeicosatrienoic acids (EETs) which inhibit inflammation
9.What is the evidence that the anti-inflammatory action of flavonoids is not simply antioxidant activity?
Flavonoids inhibit cyclooxygenase and lipoxygenase activity in the neutrophils rather than inhibiting oxidation.

Tutorial 8-Micronutrient Tutorial
1.What groups of elements or compounds would you include as micronutrients?
These are nutrients that are required in very small amounts (in microgram quantity), but they are highly essential for the different physiological functions of the body. Some examples of micronutrient are iron, copper, molybdenum, selenium, and zinc.
2.How do trace elements differ from trace metals?
Trace elements are chemical elements as per their existence in the periodic table and are required in extremely low concentrations by our body. On the contrary, trace metals are the metallic subsets of such elements that are usually present in low amounts in our body. Insufficient concentrations of trace metals may lead to the genesis of different diseases. Some examples of trace elements are iron, copper, molybdenum, selenium, and zinc.
3.Define and give examples of acute phase proteins.
These proteins refer to a class of proteins whose plasma concentrations either increases or decreases in response to an inflammatory response within the body. They are further subdivided into positive and negative acute phase proteins. Positive acute phase proteins are those whose plasma concentrations increase in response to inflammatory response, while negative acute phase proteins are those whose plasma concentrations decrease in response to inflammatory response. The acute phase proteins are often an integral part of the innate immune system. For example, various proteins of the complement system such as the C-reactive protein are recognized as acute phase proteins. Some other examples of acute phase proteins include haptoglobin, ferritin, and ceruloplasmin.
4.In diagram 1 explain what is happening to iron:

The plasma concentrations of iron are reduced because after surgery the free iron is utilized to synthesize red blood corpuscles. This is because surgery is often associated with blood loss. However, the iron: transferrin ratio initially falls in the plasma because iron is delivered from transferrin to the RBCs. However, after iron is transferred from transferrin, the iron: transferrin ratio is maintained at a steady state.
5.What is HPN and what micronutrients may be affected by this form of nutrition?
HPN refers to home parenteral nutrition. In HPN, the nutrients are administered through injectables and not through the oral route. Such nutritional regimes lack micronutrients because of metabolic limitations. The major micronutrients that are affected include iodine, chromium, and zinc.
6.a) Explain how selenium functions as a micronutrient.
Selenium is an essential trace element that is used to synthesize antioxidant enzymes in our body. Selenium acts in a coordinated manner with vitamin C and vitamin B6 to prevent oxidative stress. It also has anti-carcinogenic activity.
b) Describe a disorder associated with selenium deficiency.
One such disorder that is associated with selenium deficiency is Keshan disease or Keshan syndrome. In this disorder, the major symptoms are myocardial necrosis and decreased contractility of the myocardium. The disease stems from oxidative injuries to the myocardium.
7.Explain how cigarette smoking is believed to affect plasma ascorbate turnover.
Ascorbate or vitamin C is a scavenger of free radicals. Cigarette smoke contains not only free radicals but also generates free radicals within the body. Since ascorbate is used to scavenge the high titer of free radicals within the body, its turnover increases. This means ascorbate levels are not maintained in the blood, and the body is prone to oxidative stress.
Tutorial 9 – Nucleotide Metabolism and Disease
1.Please fill in the gaps:
Lesch-Nyhan syndrome is caused by a hereditary deficiency of —————hypoxanthine-guanine phosphoribosyl transferase (HGPRT)———–. This is a vital enzyme of the purine —————-biosynthesis———reactions. Lack of HGPRT would lead to accumulation of hypoxanthine except that it is removed by purine —–xanthine oxidoreductase——————— to yield uric acid. In Lesch-Nyhan sufferers there can be a —————–three to four-fold increase in uric acid. In addition the high levels of ———-uric acid—————-which build-up as a result of diminished enzyme activity stimulate a high rate of —xanthine———————– purine biosynthesis.
2.In the purine ring structure, NAME
a) TWO metabolites which are the origin of the nitrogen atoms
aspartic acid (N1) and glutamine(N3)
b) TWO metabolites which are the origin of the carbon atoms.
Glycine (C4/C5) and carbon dioxide (C6)
3.Give THREE roles for 5
PRPP (phosphoribosyl pyrophosphate) is the major substrate for three enzymes of purine metabolism pathway.
PRPP aminotransferase for de-novo synthesis of purines
Acts as a key substrate for rate regulation step
Substrate for HPRT, hence, helps in salvage pathway

4.Fill in the missing information:
Pharmaceutical
Site of Action
Consequences
Ribavirin Mimic nucleoside and competes for purine ribonucleotides Lowering of [GMP]
Oxipurinol Xanthine oxidase
(XO) Inhibits xanthine oxidase and lower uric acid levels
Desferrioxamine
(Deferoxamine) Chelation of iron Lowering of [deoxynucleoside and nucleotide]
Hydroxyurea Ribonucleotide reductase Reduce dCTP and dGTP
6-thioguanine HGPRTase TGMP,TGTP, IMP
Cyclopentenyl cytosine CTP synthetase Lowering of [CTP]
Nolatrexed Thymidylate synthase Lowering of [dTMP]
5-Formyltetrahydrofolate 1-carbon metabolism supporting dTMP production Increase ADP levels, phosphate, and methyltetrahydrofolate
NRTIs Reverse transcriptase Lowering proviral DNA synthesis
Dihydrofolate Dihydrofolatereductase Tetrahydrofolic acid
Tutorial 10 – Cell signaling
1. Name the common types of membrane-bound receptors.
The common types of membrane-bound receptors are ionotropic and metabotropic receptors. For example, nicotinic and muscarinic receptors are ionotropic receptors. Such receptors are either ligand-gated or voltage-gated. On the contrary, G-proteins are examples of metabotropic receptors.
2. Where else can you find receptors?
Receptors can be found in the cytoplasm and nucleus of a cell apart from the plasma membrane.
3. These two receptor classes differ in their cellular location. What properties must the signaling compound possess to bind to the nonmembrane-bound receptors?
The first messenger (signaling compound) should be liposoluble to access nonmembrane- bound receptors.
4. G-PCR consists of a receptor protein and another GDP bound protein. How does this cause a signal cascade?
When the first messenger interacts with the G-protein, its alpha subunit gets phosphorylated and detaches from the beta and gamma subunits as G alpha-GTP. The G alpha-GTP complex can either stimulate membrane-bound adenylate cyclase or phospholipase C. If adenylate cyclase is activated, then cyclic AMP (cAMP) is produced which stimulates protein kinase A to mediate the cellular effects. On the contrary, stimulation of phospholipase C would lead to the genesis of diacylglycerol (DAG) and IP3 (inositol triphosphate) from PIP2. DAG can stimulate protein kinase C to mediate its cellular effects, while IP3 extrudes calcium ions from the endoplasmic reticulum to mediate further effects.
5. What effects does phosphorylation have on proteins?
Phosphorylation can either activate or deactivate proteins.
6. Protein tyrosine kinase receptors.
On binding of EGF ligand to EGF receptors, the receptor dimerizes, and autophosphorylates. How does this lead to signal transduction?
Once they bind to such dimerized receptors, they deploy Janus tyrosine kinase to phosphorylate themselves (receptors). After such phosphorylation, the receptor adds phosphate groups to STAT (signal transducers and activators of transcription) proteins and activates them. The activated STAT proteins access the nucleus and modulate expression of various genes that promote the growth of cells.
Tutorial 11: Forensic Toxicology
a) What is the UM phenotype?
UM, phenotype refers to an individual who is ultra-rapid metabolizers (UM). Such individuals require a higher dosage of a drug because these individuals exhibit higher rates of drug metabolism.
b) Explain why a nursing mother with a UM phenotype for CYP2D6 should not ingest codeine.
Because codeine is an inducer of CYP2D6 which can convert different compounds into its active metabolites, and such metabolites may enter the baby during nursing. Moreover, it can form morphine that can have narcotic effects on the child.
How can P/M ratios help distinguish between acute and chronic drug ingestion?
P/M ratio signifies parent drug to metabolite concentrations. In acute toxicity, the P/M ratio is elevated, while during chronic toxicity the ratio is lowered.
Why is the detection of ethanol in post-mortem samples not proof of ante-mortem ingestion?
This is because microbes can form ethanol even after the death of an individual.
Which blood sample is considered to be least susceptible to post-mortem changes?
Femoral vein
Which sample and which marker should be used to determine alcohol ingestion in the weeks before sampling?
Blood and Ethyl glucuronide
In entomotoxicology which material is likely to make the best sample and why?
Insect (carrion-feeding insects) samples are the best because they feed on intoxicated tissues; hence, they can portray drug overdose in an individual. Moreover, insect samples are easily homogenized, and mass spectrophotometers can analyze them.
What is the three-stage process of treating cyanide poisoning?
Supplement with amyl nitrite through inhalation
Intravenous administration of amyl nitrite
Inject sodium thiosulfate rhodanese
What makes carbon monoxide poisoning so deadly?
Besides having 200 times affinity for haemoglobin compares to oxygen, Carbon monoxide is odorless hence highly undetectable. Victims suffocate without knowledge of what is the cause most of the times.
Work cited
Biochemical Toxicology-Forensic aspects with small molecules week 9.pptxPRACTICAL1
Metabolism of Metronidazole by Intestinal Flora
Introduction
Often compounds exhibit toxicity only when they have been metabolized. In other words, it is one or more of the metabolites which are responsible for the toxicity. For many of these compounds the mixed function oxidase system incorporating cytochrome P450 is of prime importance with the bulk of the metabolism taking place in the liver. However, for other compounds, substantial metabolism may occur elsewhere in the body and by systems other than cyt. P450 dependent ones.
In this practical, the metabolism of metronidazole will be studied. Metronidazole is a nitroimidazole drug which has been used for many years to treat anaerobic infections such as that caused by Trichomonas vaginalis.
This experiment aims to investigate the type of metabolism undergone by metronidazole in the mammalian gut. To achieve this, the drug has been incubated with the contents of a rat caecum (part of the intestine). The incubation mixture has been divided; each separate volume has had a different treatment and samples have been removed from these volumes at varying time intervals for analysis.
Method
Each group is provided with a series of tubes. Into each of these tubes has been placed a sample of caecal contents which has been incubated with metronidazole in the presence of nitrogen.
Each series of tubes represents a different experiment as follows:
Caecal contents plus .001moldm3 metronidazole sampled at 0, 10, 20, 30 and 40 minutes.
B As A but oxygenated at 10 minutes.
C As A but with .01moldm3 sodium nitrite added.
D As A but with .01moldm3 sodium nitrate added.
E As A but bubbled with carbon monoxide, not nitrogen.
F As A but using caecal contents taken from animals pre-treated with Penicillin G for four days previously.
G Not an experimental series but instead a standard curve of metronidazole containing 0, 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mmoldm3.
Take each sample in your series and treat as follows:
Add 0.5cm3 of the sample to 1.5cm3 of ethyl acetate in stoppered tubes.
Add 0.2cm3 of 0.1moldm3 potassium hydroxide to each tube, whirl mix for 1 minute and centrifuge for 5 min at 1000 rpm.
Remove 0.5cm3 of the upper ethyl acetate layer and add to 2cm3 of 0.1moldm3 hydrochloric acids, whirl mix for 1 minute and centrifuge for 5 min at 1000 rpm.
Carefully remove 1.5cm3 of the lower aqueous layer and add to 2cm3 of 1moldm3 potassium hydroxide.
Measure the absorbance of the extracted sample at 320nm against a water blank.
Record the results and display them for the benefit of the whole class.
Treatment of results
Convert each of your values from absorbance into [metronidazole] using the calibration curve derived from series G.
TIME (minutes) Practical 1 Absorbance result
0 0.509
10 0.39
20 0.187
30 0.063
40 0.061
Plot your results as [metronidazole] (on a log scale) versus time (on a linear scale).

3.Comment on the resulting plots observed for A.
The absorbance of metronidazole reduced because gut microbes reduced metronidazole over time.
4.Comment on the difference of A from plots BF.
In the absence of oxygen, metronidazole is rapidly reduced
In the presence of sodium nitrite, the anaerobic metabolism of metronidazole is inhibited.
In the presence of sodium nitrate, the anaerobic metabolism of metronidazole is not inhibited.
In the presence of carbon monoxide, the anaerobic metabolism of metronidazole is not inhibited.
Pre-treatment with penicillin G almost stopped anaerobic metabolism of metronidazole.
Explain as far as possible the results seen in 3. and 4.
In the absence of oxygen, metronidazole is rapidly reduced: During anaerobic condition, the anaerobic bacteria in the gut disrupt the imidazole ring of metronidazole
In the presence of sodium nitrite, the anaerobic metabolism of metronidazole is inhibited: extracellular nitrite inhibited ferredoxin of anaerobic bacteria.
In the presence of sodium nitrate, the anaerobic metabolism of metronidazole is not inhibited: extracellular nitrate did inhibit ferredoxin of anaerobic bacteria.
In the presence of carbon monoxide, the anaerobic metabolism of metronidazole is not inhibited: CO promote anaerobic conditions
Pre-treatment with penicillin G almost stopped anaerobic metabolism of metronidazole: penicillin G is a substrate for ferredoxin and competes with metronidazole.
6.Critically evaluate the experimental method and the analytical technique.
The experiment was framed to assess the metabolism of metronidazole under anaerobic conditions. The analytical technique simulated in-vivo environments; hence, the findings were predictable and reliable
Work cited
Asquith J C et al. British Journal of Radiology (1974) 47 474
Searle A J and Willson R L. Xenobiotica (1976) 6,8 457464.
PRACTICAL2
Lipid Peroxidation Experiments with Liposomes
Introduction
Polyunsaturated fatty acids (PUFAs) are susceptible to peroxidation. Such lipid peroxidation is comprised of three stages; initiation, propagation, and termination. Initiation involves the abstraction of an allylic hydrogen atom from a PUFA leaving behind a lipid radical. Such abstraction can readily be carried out by reactive oxygen species (ROS) such as hydroxyl radicals. Following the abstraction, chemical rearrangement propagation occurs. During the next stage (propagation) the modified PUFA becomes available for oxygen addition, and the newly formed radicals (lipid, lipid peroxyl, and lipid alkoxy radicals) attack (abstract hydrogen from) PUFAs. This has the effect of amplifying the damage to the lipids. Eventually, the concentration of radicals becomes so great that they preferentially react with each other rather than attack PUFAs. When two radicals react, they form a non-radical product which cannot attack PUFA thus the process has reached the termination stage.
Reactive oxygen species can be generated chemically, by ionizing radiation or by light-mediated reactions. In this series of experiments, ROS are produced by a ferric-ascorbate couple. The substrate is a soybean liposomes preparation which is rich in PUFAs. The peroxidation is determined by measuring the yield of malondialdehyde (aldehydes and ketones are by-products of lipid peroxidation). Malondialdehyde reacts when heated with thiobarbituric acid to produce a pink-colored compound which can be assayed spectrophotometrically at 532nm.
Your group will carry out either experiment A or experiment B.
Method
EXPT. A Volume of addition to tubes (cm3)
Tube number Buffer Liposomes Cysteine FeSO4
1,2,3 1.8 2.0 0.1 0.1
4,5,6 3.8 0.0 0.1 0.1
7,8,9 2.8 1.0 0.1 0.1
10,11,12 1.9 2.0 0.0 0.1
EXPT. B Volume of addition to tubes (cm3)
Tube number Buffer Liposomes Cysteine FeSO4
1,2,3 3.3 0.5 0.1 0.1
4,5,6 2.3 1.5 0.1 0.1
7,8,9 1.3 2.5 0.1 0.1
10,11,12 1.9 2.0 0.1 0.0
1.Prepare the tubes by adding buffer, liposomes, and cysteine as indicated in your table. DO NOT ADD the FeSO4 until you are ready to start the reaction.
2.Mix the contents well.
3.Start the reaction with the addition of FeSO4.
NOTE: START THEM AT TIMED INTERVALS.
4.Mix the tubes very well to ensure that oxygen dissolves into the solution.
5.Incubate the tubes at 37ºC for 30 minutes in a water bath.
6.Stop the reaction by adding 1ml of BHT.
NOTE: STOP THEM AT EXACTLY 30 min. INCUBATION.
7.Add 1ml of 1%TBA followed by 1ml of 10% HCl.
8.Heat the tubes in a boiling water bath for 20 minutes (cover the tubes to reduce evaporation). IT IS IMPORTANT THAT THE WATER IS BOILING.
9.Cool the tubes on ice for 5 minutes.
10.Add 5ml of CHCl3 to each tube mix thoroughly then centrifuge at 1600g for 10 minutes.
11.Read the absorbance of the aqueous top layer at 532nm and record the values.
12.Record your results and make them available for the whole class at the end of the session.
Practical 2            
             
             
EXPT. A Volume of addition to tubes (cm3)          
Tube number Buffer Liposomes Cysteine FeSO4 Absorbance Raw Mean Absorbance
1 1.8 2 0.1 0.1 0.24  
2 1.8 2 0.1 0.1 0.22  
3 1.8 2 0.1 0.1 0.22 0.225
4 3.8 0 0.1 0.1 0.04  
5 3.8 0 0.1 0.1 0.05  
6 3.8 0 0.1 0.1 0.05 0.046
7 2.8 1 0.1 0.1 0.16  
8 2.8 1 0.1 0.1 0.13  
9 2.8 1 0.1 0.1 0.15 0.143
10 1.9 2 0 0.1 0.14  
11 1.9 2 0 0.1 0.13  
12 1.9 2 0 0.1 0.12 0.126
             
             
EXPT. B Volume of addition to tubes (cm3)          
Tube number Buffer Liposomes Cysteine FeSO4 Absorbance Raw Mean Absorbance
1 3.3 0.5 0.1 0.1 0.14  
2 3.3 0.5 0.1 0.1 0.12  
3 3.3 0.5 0.1 0.1 0.12 0.125
4 2.3 1.5 0.1 0.1 0.18  
5 2.3 1.5 0.1 0.1 0.18  
6 2.3 1.5 0.1 0.1 0.19 0.181
7 1.3 2.5 0.1 0.1 0.2  
8 1.3 2.5 0.1 0.1 0.28  
9 1.3 2.5 0.1 0.1 0.32 0.265
10 1.9 2 0.1 0 0.2  
11 1.9 2 0.1 0 0.14  
12 1.9 2 0.1 0 0.08 0.138
             
Stock solutions
Buffer:0.2M sodium phosphate buffer pH7.4
Liposomes:2mg/ml working stock
Cysteine:25mM cysteine in deionised water
FeSO4:2.5mM ferrous sulphate in deionised water
BHT:20mg/ml butylated hydroxytoluene in methanol
TBA:1%w/v thiobarbituric acid
HCl:10%w/v hydrochloric acid
CHCl3 :Chloroform
Answer the following questions for yourself – similar questions may appear in your end-of-module exam:
1.Which components are necessary to stimulate lipid peroxidation?
Free radicals and reactive oxygen species
2.How would you test for the involvement of oxygen?
By the Haber-Weiss Reaction
3.What combination of components gives the maximum lipid peroxidation? Explain this.
Hydroxyl radicals and superoxide anion because they can withdraw H+ ions from fatty acids and convert the same into fatty acid peroxy radicals
4.Which ROS may be involved? How could you test for these?
5.What could you use to inhibit lipid peroxidation?
Antioxidants like vitamin E or steroids like methylprednisolone
6.What relevance does this experiment have to any physiological/pathological situation?
Lipid peroxidation is the major cause of hemiplegia and paraplegia after acute spinal cord injury.
Work cited
Caillet, S et al. Fenton Reaction applied for screening natural antioxidants Food Chemistry (2007) 100 542-552
Chen, QI et al. Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo PNAS (2007) 104, 21 8749–8754
Lykkesfeldt, J. Malondialdehyde as biomarker of oxidative damage to lipids caused by smoking Clin Chimica Acta (2007) 380 50-58
Tsuda. Antioxidative Activity of the Anthocyanin Pigments Cyanidin 3-O-β-D-Glucoside and Cyanidin J.Agric.Food Chem (1994) 42 2407-2410