Endpoints of Toxicity - Assignment
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Added on 2021-06-14
Endpoints of Toxicity - Assignment
Added on 2021-06-14
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2Question 1List 6 different cellular enzymes that can be used to assess different endpoints oftoxicity. a reaction scheme and add references to substantiate your statements.The cells are able to respond very fast to toxic materials through changing ofmorphologies, biochemical processes and altered growth behaviours. The biochemicalchanges are involved in changing the normal functions of the cells and are thus significant inendpoint toxicity tests. The enzymes are commonly used in various cellular biochemicalroles, leading to their increased use in cytotoxic tests. Lactate dehydrogenase (LDH)- This is an intracellular enzyme which catalyses thereversible conversion of lactate into pyruvate. Therefore, when there is a health relatedcondition, the LDH concentrations in serum becomes high. An example is upon theadministration of some therapeutic agents which are nephrotoxic or hepatotoxic, hence theLDH can also be used in monitoring organ and cell toxicity. In the LDH assay, the L-lactateand NAD+ are converted to pyruvate and NADH. The formed pyruvate thus reacts with thehydrazine compound to form a coloured product 1. Thus the concentration of the LDH inevery serum sample is measured by measuring the absorbance of the final solution at 510nm. Alanine aminotransferase (ALT)- this assay is based on ALT enzyme which dependson pyridoxal phosphate to catalyse the reversible transfer of amino groups from amino acidalanine to alpha ketoglutatrate, to form glutamate and pyruvate. This enzyme is commonly
3found in the liver and to a less extent in other tissues. When injury to hepatocytes occurs, theconcentrations of ALT in the serum becomes high, and hence a biomarker for hepatocytestoxicity 2. The assay is conducted to detect pyruvate in a reaction that coverts a colourless to acoloured product whose absorbance is measured at 570nm. Creatinine kinase- Creatinine kinase is applied in the enzymatic assay of creatinekinase in patient serum. This enzyme is applied in the diagnosis of diseases that are linked toheart, central nervous system and the skeletal muscles 3. When this enzyme is present inblood, it catalyses the transfer of a high energy phosphate from creatine phosphate to ADP toform ATP. In the muscles, such a reaction is important because the resulting ATP is used inpresence of the enzyme hexokinase to convert glucose to glucose-6-phosphate to make moreenergy in glycolysis. At the same time, this reaction involves the reduction of NADP+ toNADPH. Therefore, the activity of creatine kinase is measured by testing the rate offormation of NADPH which is monitored at 340nm. These reactions take place in presence ofN-acetyl-L-cysteine which is the enzyme activator. Caspase: this enzyme assay is used in the detection of cell apoptosis, due to theactivation of the caspase enzymes by the cells 4. These enzymes are activated so that they cancatalyse the cleavage of protein substrates in the cells causing the destruction of the cell wall,and thus cell death. The caspase assay there facilitates the detection of these enzymes inliving cells on real time basis. The caspase type 3 and 7 cleaves the PARP protein to form85kDa and 25kDa fragments .in this assay, antibodies against the 85kDa fragment are used asmarkers for apoptotic cells. Hexokinase: this enzyme activity is involved in the first step of glycolysis to convertglucose to glucose-6-phosphate 5. At the same time, glucose-6-phosphate is oxidized byglucose-6-phosphate dehydrogenase to produce NADH, a product that can reduce acolourless molecule to form a coloured product, whose absorbance can be measured at
4450nm. The hexokinase enzyme is very common in many cells and thus critical for glucosemetabolism. Too low levels of hexokinase enzyme are associated with diseases like musculardystrophy, while too low levels could be due to tumours. However, with early detection, it ispossible to diagnose, predict and treat the underlying disease. Beta-hydroxy-butrate: this enzyme assay is used to determine the presence of ketonebodies in blood. The ketone bodies are formed when the levels of glucose are low especiallyduring starvation and fasting. Additionally, the ketones like Beta-hydroxy-butyrate can rise indiabetics and alcoholics 6. Thus, the use of Beta-hydroxy-butrate is used in determination ofthe Beta-hydroxy-butyrate levels through a coupled enzymatic reaction, whereby theabsorbance is measured at 450nm, which is proportional to the amount of Beta-hydroxy-butrate present in a sample. In this assay, Beta-hydroxy-butyrate, which is produced by thehepatocytes and released into the tissues as a source of energy.Lactase enzyme is critical for the metabolism of a disaccharide known as lactose. Thisis measured using colorimetric and fluorometric methods. The presence or absence of lactosein blood or urine by hydrolysing lactose by the enzyme lactase 7. Lack of lactase in cells leadsto fructose intolerance. Question 2 Discuss in detail how a colony formation assay can be used to measure DNArepair in response to ionizing radiation and how this differs compared to the standardcolony formation assay that assesses toxicity of a drug. Also, list at least 5 advantages, 5disadvantages and possible artefacts associated with this assay.The ionizing radiations are associated with negative effects to the human cells such ascell necrosis, programmed cell death, autophagy and multinucleation among other negativeeffects. These processes make the cells lose their colony forming abilities during the
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