BIO200H5 Study Guide - Chemotherapy, Pharmacology, Median Toxic Dose

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1 Dec 2013

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30 min is often a good time (except vitucilin no effect in 30 min) Slowly increase dose until you see effect or toxicity. Dose response - you increase dose and see response. We test in intervals because we get dose response that is very usual. Inverted u - comes out to a solution if you put a lot. Drug is binding to a protein binding site - after the point where it occupies all site, stops increasing response. Drug is binding to a protein binding site- after the point where it occupies all site, stops increasing response. Drug b is less potent because you have to go higher doses to get effect. Semi-log plot - you get the whole response curve. Fitting curves are more difficult to draw before. Take 10 rats - give different doses each time. Graph level off = rmax = all animals are having seizure. Making a statement for a-put ed50 a at the bottom.

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For Discussion (2-3 paragraphs) â¢ What was the concentration of your pre-diluted NPs? â¢ Determine the effect of NPs on brine shrimp lethality. â¢ Determine the effect of NPs on cell viability. â¢ What is (are) the control(s) in this experiment? What is its purpose? â¢ Based on your data in this lab, what is a safe concentration for brine shrimp Threshold Level of Toxicity (TTL)? â¢ Often indicator species are used to study the overall health of an ecosystem. If you were to study an ecosystem containing brine shrimp, would you use it as an indicator species? Why or Why not? Explain your reasoning. â¢ What possible sources of error were present in this experiment?

Introduction

LD50 is a commonly used term to describe acute toxicity: LD stands for Lethal Dose and 50 is used because the dose could kill 50% of the animals exposed to the chemical. For LD50 tests, the animals are given specific amounts of the chemical, orally, by injection or dermally. The value is then reported along with the method of administration and the type of animal used in the test. LD50 is typically reported in milligrams or grams per kilogram of body weight of the animal. The lower the LD50 is, the higher the toxicity of the chemical would be. This information is always provided in Safety Data Sheet (SDS) of chemicals in the âtoxicity dataâ category. A similar measure, the LC50 is usually used to describe the ecological hazards of chemicals. LC stands for lethal concentration that could kill 50% of the animals after 24-96 hours. This value is always provided in Safety Data Sheet (SDS) of chemicals in the âEcological Dataâ category. In this lab, you will be measuring LC50 or LD50 of iron oxide nanoparticles using brine shrimp. Iron oxide nanoparticles are magnetic. Application of magnetic nanoparticles ranges from magnetic storage media to medical industry where they can be used for targeted drug delivery or as contrast agents in MRI. Thus, measuring environmental and biological toxicity of these nanoparticles is crucial. To test toxicity of any such reagents on small organisms, Daphnia and brine shrimps (also known as Artemia) can be used. Artimia are found in salt lakes around the world. They are important live food for aquarium fish. In this lab, you will use brine shrimp to measure toxicity of the nanoparticles. Trypan blue is a stain used to assess cell viability. It is able to diffuse through cell membranes of dead cells but is unable to permeate live cells. This stain can be used to assess cell survival 1 Fall 2017 following exposure to chemicals, nanoparticles and experimental drugs. The ratio of live: dead cells can be used as a preliminary assessment of cytotoxicity and translated to tissue viability.

Methods Part A: Serial Dilution of NP Solution 1) Label 5 test tubes as 0, 1:1, 1:10, 1:100, 1:1000 2) Add 2mL of seawater to tube 0 3) Add 2mL of specific group stock solution to tube 1:1 and add 2mL of seawater to it. 4) Transfer 1mL of stock solution to tube 1:10 and add 9mL of seawater to this tube 5) Transfer 1mL of 1:10 solution to the tube labeled as 1:100 and add 9mL of seawater to â¨this tube 6) Transfer 1mL of 1:100 solution to the test tube labeled as 1:1000 and add 9mL of â¨seawater to this tube Part B: Preparation of test environments 1) Label 5 wells of 6-well plate as control (0), 1:1, 1:10, 1:100 and 1:1000 2) Label 3 petri dishes as: control (0), 1:1 and 1:1000 â¨NOTE: Be sure to include your initials on all plates 3) Transfer 2mL of each solution from the corresponding test tube to the respective wells on your six-well plate and each petri dish Part C: Preparing specimens for the Trypan blue cell viability test 1) Transfer 2 brine shrimp into each of the pre-labeled petri dishes containing the solution of NPs and seawater (0, 1:1 & 1:1000) and set a timer for 30 minutes. 2) Transfer a single brine shrimp into the unlabeled petri dish with a drop (~50ul) of seawater and use the dissecting microscope to make observations related to the behavior and morphology of untreated brine shrimp.â¨Make observations in lab notebook of all samples Part D: Preparation of specimens for the toxicity assay 1) 2) 3) 4) 5) Add 10 brine shrimp into each well in the 6 -well plate Make observations in lab notebook Using a dissecting microscope (if needed), count the number of brine shrimp in each well (alive shrimps).â¨Record your data in your notebook using the graphic below as a template. Close the plate and store it for next observation.â¨Observations must be done within 24h-96hâ¨Note: There will be times allocated and a sign up paper for observations Part E: Trypan blue viability test 2 Fall 2017 1) Label 3 glass slides to match the petri dishes prepared in Part C. 2) Transfer 1 brine shrimp from each petri dish onto the corresponding slide. â¨Make observations in your lab notebook 3) Add a drop (~60 Î¼l) of PBS on top of each specimen 4) Add 60 Î¼l of Trypan blue to your shrimp and incubate for 1-2 minutes at room â¨temperature. 5) Wick away excess liquid 6) Add 100 Î¼l PBS to sample and observe (while alive!), looking for blue cells, using the â¨dissecting scope â¨Make observations in your lab notebook 7) Label 3 microfuge tubes to align with each sample. 8) Transfer each brine shrimp from the slide into the corresponding microfuge tube 9) Add 50 Î¼l of PBS to the tube and use the mechanical disruption tool to homogenize the â¨sample for ~2 minutes until a cell suspension has been generated. 10) Load 30-50 Î¼l of your sample onto a new glass slide and add a cover slip. 11)Observe the sample on high power and use the scale below to qualitatively assess cytotoxicityâ 0-no blue 1-some blue 2-fairly blue 3-totally blue.

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CELL RESPIRATION LAB

Section:

Type in your answers in complete sentences, be precise and specific to earn full credit, reformat if necessary

Purpose: The purpose of this lab activity is to analyze the effect of exercise on cellular respiration

Experiment 1 Enzymes in Food

Why is cellular respiration necessary for living organisms?

Why is fermentation less effective then respiration?

List the two co-enzymes involved in cellular respiration and describe their role.

How many ATP molecules are produced in
Glycolysis	Citric Acid Cycle	Electron Transport	Fermentation

Table 1. Fermentation by Yeast

Tube	Initial Gas (mm)	Final Gas (mm)	Net Change
1% Glucose Solution
1% Sucrose Solution
1% Equal Solution
1% Splenda Solution
1% Sugar Solution

Include your hypothesis for this experiment. Be specificâ¦in other words we sure to include at least one end point you will measure to support your predictions. Natural sugar causes fermentation by producing CO2

Did you notice a difference in the rate of respiration between the sugars? Did any of the artificial sugars provide a good starting material for fermentation? Yes no artificial sugar did not provide a good starting material for fermentation as there was no change in height of gas.

Which tubes displayed evidence of anaerobic fermentation? How did you know? Due to change in gas height we can determine fermentation was occurring during to production of CO2

Hypothesize why some of the solutions were not metabolized. Research their chemical formulas.

How do the results of this experiment related to the role yeast play in baking?

Experiment 2Aerobic Respiration in Beans

How did the color of the bromothymol blue solution in each beaker change over time in each condition?

What is the mechanism driving the bromothymol blue solution color change?

What can be inferred from the color change of the bromothymol blue solution?

What evidence do you have that cellular respiration occurred in the beans? Explain your answer.

What are the controls in this experiment, and what variables do they eliminate?

If this experiment were conducted at 0 Â°C, what difference would you see in the rate of respiration? Why?

Table 2: Bromothymol Blue Color Change Over Time for Pinto Bean Trial
Time	Pre-Soaked Pinto Beans	Dry Pinto Beans	No Pinto Beans
0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hours

Table 3: Bromothymol Blue Color Change Over Time for Kidney Bean Trial
Time	Pre-Soaked Kidney Beans	Dry Kidney Beans	No Kidney Beans
0 min
30 min
60 min
90 min
120 min
150 min
180 min
24 hours

----------------------2. Physician Order: DiaBeta 5 mg p.o. Drug Label: DiaBeta tablets 2.5 mg. How many tablets will you give the patient?

------------------------3. Verapamil 2.5mg IV is ordered. The vial contains 5mg/mL. How many mL will be given per dose?

------------------------- 4. The order is for Morphine 8 mg I.M. The drug label reads morphine 10 mg/mL. How many milliliters will the patient receive?

---------------------------5. Order: Fentanyl 50mcg IM. Available is a vial containing 0.05mg/mL. How many mL will be administered?

--------------------------- 6. Dr. orders: give Â½ gm of a med. You have on the shelf 200mg/mL vials. How many mL will you give?

----------------------------7.The order reads 125 mL D5W per hour. The set delivers 10 gtt per mL. How many gtt/min will you administer?

----------------------------8. Your client weighs 75 kg. She is to receive 0.004 mg of Dopamine per Kg of body weight per minute. You are to add 400 mg of Dopamine to 500 mL normal saline. The drip factor of your tubing is 60gtt/mL, how many gtt/min will you deliver?

------------------------ 9. Order 60mg/kg/day in 2 divided doses. Available: 100mg/mL vials. Patient weighs 125lbs. How many mL will you give per dose?

-------------------------10. Order: Lopressor (Metoprolol) 100 mg po, bid. Available: Lopressor, 50 mg tablets. The client is going on a 2-week vacation & must have adequate medication to last him until he returns home. How many tablets will he need to have to cover the time of his vacation?

------------------------- 11. Order: Kanamycin 1.5 gm po q8h x 7 days. Available: Kanamycin 500 mg capsules. How many capsules will the client receive in the 7 days he is to receive this medication?

--------------------------12. Order: Heparin, 600 U (units) SQ daily. Available: 1000 U/mL. How many mL will the patient receive?

--------------------------13. Two liters of IV Lactated Ringer's is ordered to be given over 12 hours. The drop factor is 60gtt/mL. The IV has been running for 8 hours and 700 mL remain. How many gtt/min are needed so

that the IV finishes in the required time?

-------------------------14. The client is receiving a heparin infusion at 20mL/hour. The medication is provided as 25,000 units in 500mL of D5W. How many units of heparin will the client receive during a 8-

hour shift?

--------------------------15. Order: Give 500mg of Keflex po q4 hr. Available: 250 mg tablets. How manytablets will the patient receive in a 24-hour period?

---------------------------16. The order is to give a medication 0.25 g po four times a day. The medication is available as 150mg /5mL. How many tsp will be given for each dose?

----------------------------17. A bottle of Tagamet tablets is labeled 200mg/tablet. The order is for 400 mg twice a day. The patient will be on this medication for two weeks. How many tablets will be prescribed to ensure he has coverage during this period?

---------------------------18. Order: 1500 mL of NS to be administered over 12 hours. Tubing label states Drop factor: 15gtt/mL. The pump will be set to deliver how many mL per hour?

---------------------------19. Your patient is to receive 1 liter of D5W over the next 8 hours. The tubing is labeled 60 gtt/mL. How many gtt/min will you give?

--------------------20. The patient is to take tetracycline, 0.75 g PO. The medication is available in 250 mg tablets. How many tablets will the patient take per dose?

BIO200H5 Study Guide - Chemotherapy, Pharmacology, Median Toxic Dose

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