Pathology

1.Describe tests that should be carried out to help evaluate the safety of a newly developed drug.
100 marks
The common measures that are used to evaluate the safety of a newly developed drug in laboratory settings are LD50 and therapeutic index.
Lethal Dose 50 or Median lethal dose is a test designed to determine the lethal status of various compounds. The LD50 value indicates the concentration of the drug that would kill half of a population that consumed it. It is used to determine the right amounts of prescription and avoid adverse effects of overdosing from newly developed drugs.
Fixed Dose Procedure (OECD 420; 1992). This test relies on observing toxicity at a minimum fixed dose level to test for the acute toxicity of a drug. From the observation, if the drug only less than 90% survive, it is classified as very toxic. For the minimum concentration of 5mg/kg, If more than 90% survive but there exist signs of toxicity, it is classified as toxic, and if more than 90% survive and no signs of toxicity, it is moved to a higher concentration using multiples of 10. This test is aimed at finding the right dosage of a newly developed drug and prevent toxic or harmful level consumption.
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.
100 marks
3.a) Give an account of the LD50 test. 60 marks
Lethal Dose 50 or Median lethal dose is the concentration of a toxic substance that kills 50% of the experimental animals. The Median lethal dose is estimated from three applications (concentrations) of a toxicant. The first concentration is the one that kills 10% to 50% of the experimental animals. The second concentration is the one that kills 50% to 90% of the experimental animals. The final concentration is the one that kills 50% of the experimental animals which is considered as the median lethal dose of that toxicant. The experiment aims to determine the toxicity of a drug or a toxicant.
b) Explain its limitations concerning the use of animals and how some of these may be overcome by more recent tests.
40 marks
The major limitations of the LD50 test are as follows;
The LD50 values significantly vary with genetic polymorphisms in a study population. Hence, such measures of drug toxicity are often unreliable and non-reproducible.
The LD50 test is not compliant with the ethical guidelines that govern experimentation with animal samples.
Require a large sample of animals with minimum number being 50 per study.
To overcome the above challenges, the 3R approach which insists on refinement replacement, and reduction has been employed together with other test strategies including the Fixed Dose Procedure, Acute toxic class, and the up and down procedure.
4.a) Give an account of the mechanisms of drug absorption.
Drug absorption in the various tissues and organs in the body is through four main mechanisms. Passive diffusion is the absorption by a semi-permeable membrane dependent on concentration gradient and lipid solubility. Carrier-mediated transport involves the formation of complex molecules which diffuse across membranes while active transport moves the toxicant against the concentration gradient using ATP. Filtration, on the other hand, is the absorption of toxicants of small particles through capillary pores as a result of osmotic and hydrostatic pressure. Cell engulfing absorbs both liquids and solids. Solid absorption is referred to as phagocytosis while liquid absorption is referred to as pinocytosis.
80 marks
b) Explain where in the body a weakly acid drug is likely to be rapidly absorbed.
20 marks
In the stomach. A weak acid will be protonated and uncharged therefore it will be well absorbed across the membranes between the stomach and the splanchnic blood system.
5.a) Using the experiment on the metabolism of metronidazole as your source of information, account for the following:
.i) In the absence of oxygen metronidazole is rapidly reduced.
Under such conditions, the enzymes of anaerobic bacteria (that inhabit the gut) disrupt the imidazole ring of metronidazole to produce N-(2-hydroxyethyl)-oxamic acid.
In the presence of sodium nitrite, the anaerobic metabolism of metronidazole is inhibited.
This is because extracellular nitrite prevented the reduction of ferredoxin (which is the prosthetic group of enzymes of anaerobic bacteria). As a result, ferredoxin cannot donate electrons to the imidazole ring of metronidazole. Hence, the anaerobic metabolism of metronidazole is prevented under such conditions.
In the presence of sodium nitrate, the anaerobic metabolism of metronidazole is not inhibited.
This is because extracellular nitrate did not prevent the reduction of ferredoxin (which is the prosthetic group of enzymes of anaerobic bacteria). As a result, ferredoxin can donate electrons to the imidazole ring of metronidazole. Hence, the anaerobic metabolism of metronidazole is not prevented under such conditions.
In the presence of carbon monoxide, the anaerobic metabolism of metronidazole is not inhibited.
This is because the presence of carbon monoxide leads to the genesis of an anaerobic milieu. To recall, metronidazole is rapidly reduced under anaerobic conditions because under such conditions; it receives an electron from ferredoxin.
Pre-treatment of rats with Penicillin G almost completely stops the anaerobic metabolism of metronidazole.
This is because penicillin G competes with metronidazole for the electrons of ferredoxin.
The final stage of the sample treatment requires 0.1 M potassium hydroxide
10 marks each
It removed traces of carbon dioxide from the experimental medium.
b) Critically evaluate the analytical method used for this experiment and suggest how it might be improved.
40 marks
The method highlighted the metabolism of metronidazole under anaerobic conditions (by gut bacteria). The experiment could have been improved by incorporating competitors of metronidazole in the media or by studying the rate of metabolism of metronidazole under aerobic milieu.

7.Explain each of the following observations:
a) 30 minutes after a rat receives a single dose of a lead salt; the lead concentration is 50 times higher in its liver rat than its plasma;
15 marks
The lead salt underwent extensive metabolism in the liver and was trapped for excretion from re-entering back to the bloodstream.
b) in a starving person a significant concentration of DDT was measured in their plasma that was not detectable when they were well fed;
15 marks
The presence of food competed with DDT for the active sites of the CYP450 enzyme.
c) after injection of amaranth into a pregnant rat the fetal concentration of this food dye is only 0.03-0.06% of that found in the mother;
15 marks
The placental barrier does impede the transfer of toxicantsd) methylmercury exerts its toxicity in the brain whereas mercuric chloride is nephrotoxic;
15 marks
Methylmercury is lipid soluble hence enters the brain rapidly unlike mercuric chloride which is less lipid soluble hence exert its effect on the kidney.
e) Malathion is selectively toxic to insects;
20 marks
This is because the neurotoxic effects of the drug are dependent on its rate of metabolism within an organism. The metabolism of Malathion is mediated by oxidative desulphuration and enterase. In case of the former reaction, Maloxon is formed. On the contrary, enterase causes the formation of Malathion Diacid. Maloxon is more toxic than Malathion Diacid.
f) administration of Metronidazole increases the unpleasant side-effects of ethanol intoxication.
20 marks
In the presence of metronidazole, the rate of ethanol metabolism is reduced. This is because both compounds compete for the active site of the same CYP450 enzyme.
8.Explain the use of the following in treating various forms of poisoning and intoxication:
a) Fuller’s Earth;
b) Prussian Blue;
c) Hyperbaric oxygen;
d) Diaminoalkanes;
e) Metronidazole.

100 marks
Fuller’s earth is routinely used to minimize exposure levels to Paraquat. Such compounds minimize the absorption of paraquat into the bloodstream.
Prussian blue is clinically indicated for mitigating the harmful effects of heavy metal poisoning. The compound is used as the sequestrating agent for heavy metals because it can incorporate metal cations through ion-exchange mechanisms.
Hyperbaric oxygen therapy (HBOT) is used to manage decompression sickness, carbon monoxide (CO) poisoning, and gas gangrene.
Diaminoalkanes prevent paraquat poisoning through inhibiting penetration through the alveolar cells.
Metronidazole is used to eliminate bacterial and protozoan intoxication.
9.Discuss the treatments for paraquat poisoning and indicate their effectiveness.
mks
Paraquat poisoning is treated in several ways with the aim to Prevent gastrointestinal absorption, accelerate elimination from lung, Inhibit, penetration into alveolar cells, Remove PQ from plasma, and Lower oxygen free radical concentrations. The above is achieved through the use of various techniques and drugs. Gastrointestinal absorption is prevented by inducing emesis, gastric leverage, and inducing diarrhea. This method is effective for it eliminates the toxicant from the body fast especially diarrhea. Elimination of the lung is accelerated using cyclophosphamide which causes ROS hence the paraquat is chemically altered before it can enter the bloodstream. Penetration into the alveolar cells is inhibited through administration of diaminoalkanes effectively keeping it out of the bloodstream, in plate cases where the toxicant has already found its way into the blood plasma, it is removed using hemodialysis and haemoperfusion to effectively prevent more damage to the body. Finally, lowering oxygen free radicals which increase the affinity for the toxicant is achieved through administration of antioxidants such vitamin C and E effectively reducing its rate of uptake.
10.Describe the roles of iron in the production of Reactive Oxygen Species (ROS).
100 marks
Iron plays a significant role in the formation of ROS. When stress conditions alter the Fe homeostasis, there increases the concentration of “free” iron in the blood. Iron catalyzes redox reactions by donating an electron to shift from Fe2+ to Fe3+. In the process, Iron converts H2O2 into hydroxyl radical which is extremely reactive, and itself is converted back to its original Fe2+ form by O2.-. The above cycle is called the Fenton Haber Weiss cycle and repeats it, again and again, creating more hydroxyl radicals.
11.a) Give an account of the formation of reactive oxygen species (ROS).
60 marks
ROS are generated as a result of the Haber Weiss reaction. The reaction generates hydroxyl free radicals from hydrogen peroxide by a two-step process. These reactions are catalyzed by peroxidases and superoxide dismutase. These enzymes contain ferric ion as their prosthetic groups. However, the equilibrium between the ferric and ferrous state determine the catalytic activity of these enzymes. In the first reaction, the Fe3+ ion is converted to the Fe2+ ion after the enzyme reacts with the superoxide anion. In the second step (which is also known as the Fenton’s Reaction), the Fe2+ ions are reconverted into Fe3+ ions after the enzyme reacts with hydrogen peroxide. As a result, the Haber Weiss reaction generates hydroxyl radicals and molecular oxygen.
1st Step: Fe3+ + .O2- = Fe2+ + O2
2nd Step: Fe2+ +H2O2= Fe3+ + OH- + .OH
Overall reaction: H2O2+ .O2- = OH- + .OH +O2
b) Briefly describe mechanisms by which cells protect themselves from ROS-mediated damage.
40 marks
There are different mechanisms by which cells and tissues try to protect themselves from the ROS-mediated damage. Antioxidants are molecules that are capable of scavenging free radicals and combat ROS-mediated damage. As a result, the free radicals either lose their lone pair of electrons or accept positive charges to become neutralized. Hence, antioxidants are implicated to combat oxidative stress and other ROS-mediated injuries. Since antioxidants can scavenge free radicals, their concentrations are inversely proportional to each other. Hence, if the concentration of antioxidant molecules increases then the concentration of ROS would decrease and vice-versa. The mitochondrion plays a key role in expediting and mitigating ROS-mediated damage. Such mechanisms stem from the opening and closing of the mPTP pores that are located within the mitochondrial membrane. The transient opening of these pores helps to eliminate ROS molecules out of the mitochondria. However, if the mPTP pores are opened for prolonged periods, the influx of the ROS molecules into the mitochondria steeply increases. Increased concentration of ROS molecules within the mitochondria leads to mitochondrial damage. Such phenomenon is referred as ROS-mediated- ROS influx. Hence, the opening and closing of mPTP pores within the mitochondria play a major role in ensuring homeostasis of ROS molecules within cells and tissues. Catalase is another enzyme that helps to mitigate the production of ROS. Catalase acts on hydrogen peroxide and converts it into molecular oxygen and water. Hence, catalase plays a major role in maintaining the turnover of hydrogen peroxide within a cell.
12.Critically evaluate the following statement,
“Cytochrome P450 is essential for the detoxification of xenobiotics”.
100 marks
Drug metabolism occurs through a two-phase process. The first step of drug metabolism is referred as “Phase 1” reactions. These reactions primarily take place in the liver and are mediated by the cytochrome P450 monooxygenase (CYP450) enzyme system. The major types of phase 1 reaction include oxidation, reduction, hydroxylation, and epoxidation. A Phase 1 reaction converts a drug either to its active or its inactive form. If a drug is activated through the Phase 1 reaction, such compound is referred as a prodrug. The major objective of Phase 1 reactions is to convert a drug from a lipophilic to a hydrophilic form. Cytochrome P450 monooxygenase is the major enzyme that plays a key role in mediating Phase-1 reactions. CYP450 exist in different isoforms, and each one of them is highly reaction-specific. The enzyme is so named because it can add one atom of oxygen from molecular oxygen to the drug or prodrug. As a result, Phase 1 reactions convert a drug or a prodrug into a hydroxylated compound. Although the hydroxylated form of the drug or prodrug is less lipophilic, however; it is converted into a more hydrophilic form through the Phase 2 reactions. Iron is the major prosthetic group of the CYP450 enzyme. In the CYP450 enzyme, iron is present in the Fe++ state. This valence state of iron helps to uptake molecular oxygen from the environment into the enzyme. However, the ferrous state of the CYP450 is highly reactive and is readily converted into the ferric state. However, the ferric state of the CYP450 enzyme cannot react with molecular oxygen. Hence, iron must exist in the ferrous state in the CYP450 enzyme. NADPH acts to reduce the ferric state of iron in the CYP450 enzyme to its ferrous state. As a result, the hexose monophosphate shunt (HMP) is more active in cells and tissues that are involved in Phase 1 reactions. To recall, the hexose monophosphate pathway is a metabolic pathway that generates reducing equivalents such as NADPH. Finally, the NADPH-CYPP450 reductase reduces the Fe3+ state of CYP450 to its Fe2+ state by transferring hydrogen atoms from NADPH to CYPP450.
13.Critically evaluate the following statement,
“The processes of Phase I and Phase II drug metabolism have been exploited to reduce the toxicity of xenobiotics.”
100 marks
Drug metabolism occurs through a two-phase process. The first step of drug metabolism is referred as “Phase 1” reactions. These reactions primarily take place in the liver and are mediated by the cytochrome P450 monooxygenase (CYP450) enzyme system. The major types of phase 1 reaction include oxidation, reduction, hydroxylation, and epoxidation. A Phase 1 reaction converts a drug either to its active or its inactive form. Likewise, a toxicant is also converted to its active or its inactive form by Phase 1 reactions. Phase-1 reactions convert a toxicant from a more lipophilic to a less lipophilic form. Although the hydroxylated form of the toxicant is less lipophilic, however; it is converted into a more hydrophilic form through Phase 2 reactions. As a result, the toxicant can dissolve in water and get s readily eliminated through the kidneys or liver. Phase-2 reactions convert the products of phase-1 reaction into a more hydrophilic form. The main objective of such reactions is to eliminate the toxic end products of xenobiotic metabolism from the body. The major Phase-2 reactions that take part in toxicant metabolism include acetylation, sulfation, and glucuronidation. These reactions are commonly referred as conjugation reactions because they form conjugates with the end products of Phase-1 reactions. These conjugates are highly soluble in water, therefore; they are easily eliminated through the liver and kidneys.
14. a) Give an account of the metabolism of paracetamol.
60 marks
Paracetamol is predominantly metabolized in the liver and exhibits a plasma half-life of 1.5 hours to 2.5 hours. Paracetamol is mainly excreted as glucuronide (55%) and sulfated (30%) conjugates. However, approximately 4% of the drug is excreted as toxic metabolites (such as conjugates with mercapturic acid and cysteine). The rate of metabolism of Paracetamol is dependent on age and dose. However, the renal clearance of the drug depends on the rate of flow of urine.
b) Explain how N-acetylcysteine works as an antidote to paracetamol poisoning.
40 marks
Paracetamol or acetaminophen is usually a safe drug and is commonly prescribed for its anti-inflammatory and antipyretic properties. The drug is normally metabolized as glucuronide (55%) and sulfated (30%) conjugates. However, increased consumption of paracetamol forms N-acetyl-p-benzoquinone imine. Usually, a low concentration of this compound is not harmful to the body. This is because the compound can form conjugates with glutathione and gets eliminated from the body. However, if the concentration of N-acetyl-p-benzoquinone imine increases significantly, it fails to conjugate with glutathione. As a result, N-acetyl-p-benzoquinone accumulates and interacts with different enzymes in the liver. Such mechanisms lead to hepatic dysfunctions including acute hepatic failure and death. N-acetylcysteine (either in its intravenous or its oral form) is recommended for mitigating the harmful effects of paracetamol poisoning. N-acetylcysteine maintains the titer of glutathione in the liver. As a result, N-acetylcysteine mitigates the harmful effects of N-acetyl-p-benzoquinone on the liver.
Questions: Component number 2
) Give an account of the basic Ames test.
40 marks
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 frameshift, and base pair.
b) Show how the Ames test can be modified to detect:
Volatile mutagensthe test compound is dissolved in DSMO before exposing them to the bacteria.
Anaerobic mutagensImplement fluctuation test.
Compounds that need to be metabolized to become mutagenic
Treat the same with CYP450 enzyme first and then expose the compounds to the Ames test.
2.Explain the use of the following in treating various forms of poisoning and intoxication:
Antabuse
It is used to manage alcohol abuse because Antabuse increases the sensitivity of an individual to alcohol intoxication.
Prussian BluePrussian blue is clinically indicated for mitigating the harmful effects of heavy metal poisoning. The compound is used as the sequestrating agent for heavy metals because it can incorporate metal cations through ion-exchange mechanisms.
Hyperbaric oxygenUsed to manage decompression sickness, carbon monoxide poisoning (CO), and gas gangrene.
Diaminoalkanesprevent paraquat poisoning through inhibiting penetration through the alveolar cells.
Sodium thiosulphate
Sodium thiosulphate is used to mitigate cyanide poisoning. It donates thiosulphate to cyanide and converts it into thiocyanate. On the contrary, thiocyanate is less toxic than cyanide.
3.Explain each of the following observations:
in a starving person, a significant concentration of DDT was measured in their plasma that was not detectable when they were well fed.

a patient receiving both tolbutamide and a sulphonamide drug suddenly collapses and enters a coma.
the toxicity of diethylstilboestrol increases by a factor of 130 in rats with ligated bile ducts compared with control rats.
weakly basic drugs are more readily excreted in acid urine.
malathion is selectively toxic to insects.
100 marks (20 each)

a. The presence of food competed with DDT for the active sites of the CYP450 enzyme.
b. Both drugs compete for the active sites of the same CYP450 enzyme, and the metabolism of both the drugs are markedly reduced.
c. The placental barrier does impede the transfer of toxicants

in acid urine, weakly basic drugs will be excreted since they will be ionised as RNH3+
This is because the neurotoxic effects of the drug are dependent on its rate of metabolism within an organism. The metabolism of Malathion is mediated by oxidative desulphuration and enterase. In case of the former reaction, Maloxon is formed. On the contrary, enterase causes the formation of Malathion Diacid. Maloxon is more toxic than Malathion Diacid.
4.a) Give an account of the mechanisms of drug absorption in humans.
80 marks
Drug absorption in the various tissues and organs in the body is through four main mechanisms. Passive diffusion is the absorption by a semi-permeable membrane dependent on concentration gradient and lipid solubility. Carrier-mediated transport involves the formation of complex molecules which diffuse across membranes while active transport moves the toxicant against the concentration gradient using ATP. Filtration, on the other hand, is the absorption of toxicants of small particles through capillary pores as a result of osmotic and hydrostatic pressure. Cell engulfing absorbs both liquids and solids. Solid absorption is referred to as phagocytosis while liquid absorption is referred to as pinocytosis.
b) Explain why a weakly acid drug is likely to be rapidly absorbed in the stomach.
20 marks
In the stomach. A weak acid will be protonated and uncharged therefore it will be well absorbed across the membranes between the stomach and the splanchnic blood system.
5.a) Using the experiment on the metabolism of metronidazole as your source of information, account for the following:
i) In the absence of oxygen, metronidazole is rapidly reduced.
Under such conditions, the enzymes of anaerobic bacteria (that inhabit the gut) disrupt the imidazole ring of metronidazole to produce N-(2-hydroxyethyl)-oxamic acid.
In the presence of sodium nitrite, the anaerobic metabolism of metronidazole is inhibited.
This is because extracellular nitrite prevented the reduction of ferredoxin (which is the prosthetic group of enzymes of anaerobic bacteria). As a result, ferredoxin cannot donate electrons to the imidazole ring of metronidazole. Hence, the anaerobic metabolism of metronidazole is prevented under such conditions.
In the presence of sodium nitrate, the anaerobic metabolism of metronidazole is not inhibited.
This is because extracellular nitrate did not prevent the reduction of ferredoxin (which is the prosthetic group of enzymes of anaerobic bacteria). As a result, ferredoxin can donate electrons to the imidazole ring of metronidazole. Hence, the anaerobic metabolism of metronidazole is not prevented under such conditions.
In the presence of carbon monoxide, the anaerobic metabolism of metronidazole is not inhibited.
This is because the presence of carbon monoxide leads to the genesis of an anaerobic milieu. To recall, metronidazole is rapidly reduced under anaerobic conditions because under such conditions it receives an electron from ferredoxin.
Pre-treatment of rats with Penicillin G almost completely stops the anaerobic metabolism of metronidazole.
This is because penicillin G competes with metronidazole for the electrons of ferredoxin.
The final stage of the sample treatment requires 0.1 M potassium hydroxide
It removed traces of carbon dioxide from the experimental medium.
b) Critically evaluate the analytical method used for this experiment and suggest how it might be improved.
The method was used to study the metabolism of metronidazole under anaerobic conditions (by gut bacteria). The experiment could have been improved by incorporating competitors of metronidazole in the media or by studying the rate of metabolism of metronidazole under an aerobic milieu.
6 a) Describe how reactive oxygen species (ROS) may arise in biological systems.
70 marksROS are generated as a result of the Haber Weiss reaction. The reaction generates hydroxyl free radicals from hydrogen peroxide by a two-step process. These reactions are catalyzed by peroxidases and superoxide dismutase. These enzymes contain ferric ion as their prosthetic groups. However, the equilibrium between the ferric and ferrous state determine the catalytic activity of these enzymes. In the first reaction, the Fe3+ ion is converted to the Fe2+ ion after the enzyme reacts with the superoxide anion. In the second step (which is also known as the Fenton’s Reaction), the Fe2+ ions are reconverted into Fe3+ ions after the enzyme reacts with hydrogen peroxide. As a result, the Haber Weiss reaction generates hydroxyl radicals and molecular oxygen.
1st Step: Fe3+ + .O2- = Fe2+ + O2
2nd Step: Fe2+ +H2O2= Fe3+ + OH- + .OH
Overall reaction: H2O2+ .O2- = OH- + .OH +O2
b) Account for why one single antioxidant enzyme (e.g., superoxide dismutase) cannot work alone to counter the action of ROS.
30 marks
This is because antioxidants convert hydrogen peroxide into hydroxyl radicals. Hence, a second enzyme called catalase is required to convert the hydroxyl radicals into water and molecular oxygen.
Summary of the Lectures
Biochemical Measures of Toxicity
Different measures are used to estimate the toxicity of a compound. Toxicity can be classified by the time of onset of toxic symptoms, the duration of exposure to the toxic compound, the dose of the toxic compound, and the severity of symptoms that stem from such compounds. Hence, toxicity can be classified as either acute or chronic if the toxic effects are visible immediately or if such effects are detected over prolonged time periods respectively. Based on the duration of toxic exposure or manifestation, toxicity can be classified as short-term or long-term. Short-term toxicity refers to the toxic effects that are manifested in an experimental animal due to exposure to such substances for five times per week. On the contrary, long-term toxicity refers to the toxic effects that are manifested in an experimental animal due to exposure to such substances over 18 to 24 months. However, the duration of long-term toxic effects varies from one individual to another. According to the dose of exposure, LD50 (median lethal dose) and the therapeutic index is the most popular measure for assessing the toxicity.
Lethal Dose 50 or Median lethal dose is that dose of a toxic substance that kills 50% of the experimental animals. Lethal Dose 50 or Median lethal dose is that dose of a toxic substance that kills 50% of the experimental animals. Hence, such dose would be detrimental to 50% of the experimental population, however; the rest 50% of the experimental population would be unaffected by such dose. The Median lethal dose is estimated from three applications (concentrations) of a toxicant. The first concentration is the one that kills 10% to 50% of the experimental animals. The second concentration is the one that kills 50% to 90% of the experimental animals. The final concentration is the one that kills 50% of the experimental animals which is considered as the median lethal dose of that toxicant.
The therapeutic index (TI): is defined as the ratio between LD50 and ED50. To recall, ED50 is defined as the dose that is required to cure or produce signs of cure in 50% of the experimental animals. A toxicant with a high TI is considered less toxic compared to their counterparts that exhibit low TIs. This is because a toxic compound with a high TI signifies that either its LD50 is higher or its ED50 is lower. A high LD50 implicates that the toxic compound is safer than their counterparts which exhibit low LD50s. Hence, the individual would not experience marked adverse effects even if the dose of the toxicant is escalated.
Toxicant Metabolism
Toxicant metabolism occurs through a two-phase process. The first step of toxicant metabolism is referred as “Phase 1” reactions. These reactions primarily take place in the liver and are mediated by the cytochrome P450 monooxygenase (CYP450) enzyme system. The major types of phase 1 reaction include oxidation, reduction, hydroxylation, and epoxidation. A Phase 1 reaction converts a toxicant either to its active or its inactive form. The major objective of Phase 1 reactions is to convert a toxicant from a lipophilic to a hydrophilic form. Cytochrome P450 monooxygenase is the major enzyme that plays a key role in mediating Phase-1 reactions. CYP450 exist in different isoforms, and each one of them is highly reaction-specific. Iron is the major prosthetic group of the CYP450 enzyme. In the CYP450 enzyme, iron is present in the Fe++ state. This valence state of iron helps to uptake molecular oxygen from the environment into the enzyme. However, the ferrous state of the CYP450 is highly reactive and is readily converted into the ferric state. However, the ferric state of the CYP450 enzyme cannot react with molecular oxygen. Hence, iron must exist in the ferrous state in the CYP450 enzyme. NADPH acts to reduce the ferric state of iron in the CYP450 enzyme to its ferrous state. As a result, the hexose monophosphate shunt (HMP) is more active in cells and tissues that are involved in Phase 1 reactions. To recall, the hexose monophosphate pathway is a metabolic pathway that generates reducing equivalents such as NADPH. Finally, the NADPH-CYPP450 reductase reduces the Fe3+ state of the CYP450 to its Fe2+ state by transferring hydrogen atoms from NADPH to CYPP450. Although the hydroxylated form of the toxicant is less lipophilic, however; it is converted into a more hydrophilic form through Phase 2 reactions. As a result, the toxicant can dissolve in water and gets readily eliminated through the kidneys or liver. Phase-2 reactions convert the products of phase-1 reaction into a more hydrophilic form. The main objective of such reactions is to eliminate the toxic end products of xenobiotic metabolism from the body. The major Phase-2 reactions that take part in toxicant metabolism include acetylation, sulfation, and glucuronidation. These reactions are commonly referred as conjugation reactions because they form conjugates with the end products of Phase-1 reactions. These conjugates are highly soluble in water, therefore; they are easily eliminated through the liver and kidneys.
Factors Modifying Metabolism of Toxicants
Although the dose and duration of exposure to a toxicant are the major factors that influence the metabolism of toxicants, however; there are other factors that may affect toxicant metabolism. In fact, such factors can affect any component of the ADME (Absorption, Distribution, Metabolism, and Excretion) process. For example, age can reduce gastric motility and the number of enterocytes within the gut epithelium. Hence, toxicant absorption decreases with the increase in age of an individual. Likewise, the body fat of an individual also increases with an increase in age. On the contrary, the water volume is reduced with the increase in age of an individual. As a result, the plasma half-life of different toxicants is increased with the increase in age of an individual. On the contrary, an increase in age reduces the metabolic capacity of the liver. Such changes are attributed to a low number of hepatocytes, the rate of enzyme activity (primarily the CYP450 enzymes), and the rate of hepatic circulation. Hence, age can influence the kinetics of both Phase-1 and Phase-2 reactions. As a result, the excretion of different toxicants is reduced. Likewise, nutrition can also influence different aspects of toxicant metabolism. For example, different nutrients can act as physical barriers to toxicants. Moreover, they can interact with different toxicants and reduce their metabolism. Likewise, inadequate nutrition can influence the distribution of toxicants. For example, impaired nutritional status could lower the plasma albumin levels in an individual. On the other hand, reduced plasma albumin concentration predisposes the risk of toxicant-mediated adverse effects. Likewise, different nutrients interact with CYP450 and reduce their activity on toxicants. As a result, the rate of toxicant metabolism is reduced under such situations.
Genetic variation also affects the toxicant metabolism. Some species only undergo a single metabolism phase while others undergo two. An example is a cat. The mode of metabolism is important, especially when choosing a specimen to use as a test sample for human drugs. It is important that the species exhibit similar metabolism characteristics as humans.
Free Radicals and Antioxidants
Reactive oxygen species (ROS) are free radicals of an atom or molecule that contain unpaired electrons in their outermost shells. These compounds are highly reactive because they tend to become neutralized by accepting electrons or donating the lone pair of electrons from and to the neighboring atoms or molecules. The most common reactive oxygen species include hydrogen peroxide, hydroxyl radical, superoxide anion, and singlet oxygen. ROS are responsible for the genesis of oxidative stress within a cell. ROS are generated as a result of the Haber Weiss reaction. The reaction generates hydroxyl free radicals from hydrogen peroxide by a two-step process. These reactions are catalyzed by peroxidases and superoxide dismutase. These enzymes contain ferric ion as their prosthetic groups. However, the equilibrium between the ferric and ferrous state determine the catalytic activity of these enzymes. In the first reaction, the Fe3+ ion is converted to the Fe2+ ion after the enzyme reacts with the superoxide anion. In the second step (which is also known as the Fenton’s Reaction), the Fe2+ ions are reconverted into Fe3+ ions after the enzyme reacts with hydrogen peroxide. As a result, the Haber Weiss reaction generates hydroxyl radicals and molecular oxygen.
1st Step: Fe3+ + .O2- = Fe2+ + O2
2nd Step: Fe2+ +H2O2= Fe3+ + OH- + .OH
Overall reaction: H2O2+ .O2- = OH- + .OH +O2
In acute spinal cord injury (ASCI) the amount of reactive oxygen species is significantly increased. The spinal cord has a high content of lipid (fatty acids) and is prone to lipid peroxidation. This is because the ROS molecules can withdraw positive ions (H+) from the neighboring fatty acids and convert the same into fatty acid peroxy- radicals. The fatty acid peroxy- radicals could further withdraw positive (H+) ions from the neighboring fatty acids and convert the same to fatty acid peroxy- radical. As a result, the chain of damage continues from one fatty acid molecule to another. This process is referred as lipid peroxidation and leads to the loss of integrity of the spinal cord.
There are different mechanisms by which cells and tissues try to protect themselves from the ROS-mediated damage. Antioxidants play a major role in minimizing ROS-mediated damage. Antioxidants are those molecules that are capable of scavenging free radicals. As a result, the free radicals either lose their lone pair of electrons or accept positive charges to become neutralized. Hence, antioxidants are implicated to combat oxidative stress and other ROS-mediated injuries. Since antioxidants can scavenge free radicals, their concentrations are inversely proportional to each other. Hence, if the concentration of antioxidant molecules increases then the concentration of ROS would decrease and vice-versa. The mitochondrion plays a key role in expediting and mitigating ROS-mediated damage. Such mechanisms stem from the opening and closing of the mPTP pores that are located within the mitochondrial membrane. The transient opening of these pores helps to eliminate ROS molecules out of the mitochondria. However, if the mPTP pores are opened for prolonged periods, the influx of the ROS molecules into the mitochondria steeply increases. Increased concentration of ROS molecules within the mitochondria leads to mitochondrial damage. Such phenomenon is referred as ROS-mediated- ROS influx. Hence, the opening and closing of mPTP pores within the mitochondria play a major role in ensuring homeostasis of ROS molecules within cells and tissues. Catalase is another enzyme that helps to mitigate the production of ROS. Catalase acts on hydrogen peroxide and converts it into molecular oxygen and water. Hence, catalase plays a major role in maintaining the turnover of hydrogen peroxide within a cell.
Forensic Toxicology
Toxicant metabolism might dramatically vary from one population to another. Hence, such attributes are useful in the field of forensic science. The field of genomics that is attributed to the study of variations in drug or toxicant metabolism as a function of the genomic constituent of an individual is referred as pharmacogenomics. Apart from the genomic constituents of an individual, the pathophysiological, physiological, and environmental attributes also influence toxicant metabolism. The major variations that are witnessed at the level enzymes that are associated with drug or toxicant metabolism (for example, CYP450 enzyme system), at the level of the transporters and receptors of such drugs and toxicants, and at the level of genes that act as biomarkers. The heterogeneity in such attributes of drug metabolism from one individual to another help to assess the identity of an individual or race. The genetic variations are evident from the different isomeric forms of the CYP450 enzyme. Moreover, the isoforms of the CYP450 enzyme are differentially expressed across various populations. As a result, the CYP450 enzyme is a unique target in the field of forensic sciences. Based on the metabolic profile of toxicants, populations are classified into three different phenotypes; the ultra-rapid metabolizers, the extensive metabolizers, and the poor metabolizers. In ultra-rapid metabolizers, two or more active genes encode for a certain isomeric form of the CYP450 enzyme, while the “extensive metabolizers” have two functional genes for a specific drug metabolizing enzyme. On the contrary, poor metabolizers either lack or have a mutated functional gene that is associated with drug metabolism. Hence, the features of toxicant metabolism extensively vary between such populations. For example, individuals who are designated as poor metabolizers would exhibit a higher incidence of adverse events compared to their other two counterparts. Such attributes stem from a delay in the conjugation reactions. As a result, toxicants are not eliminated from the individual through the kidneys and liver in a time-bound manner. Such issues increase the risk of drug toxicity and drug-related adverse effects. The field of forensic toxicology is widely used to identify individuals who are accused of criminal behavior. For example, the kinetics of ethanol in blood is often used to measure the level of intoxication in an individual. For example, a ratio of a parent drug to its metabolite is a useful estimate to assess whether the intake was acute or chronic. Hence, genotyping is often implemented in the field of forensic science.
Absorption of Toxicants
There are different routes through which toxicants are absorbed into the blood. Absorption is the first step of pharmacodynamics or the ADME (absorption, distribution, metabolism, and excretion) process. The major routes of toxicants absorption include the oral, sublingual, intramuscular, subcutaneous, intraarticular, intranasal, sublingual, intramuscular, and intravenous routes. In the stomach, a weak acid (e.g., benzoic acid) will be protonated and uncharged therefore it will be well absorbed across the membranes between the stomach and the splanchnic blood system. Mouth and the rectum are insignificant sites of absorption.
Toxicants, which are injected directly into the bloodstream (through the intravenous route) are absorbed more than their counterparts that are administered through other routes. The absorption of a toxicant is measured from its bioavailability. Bioavailability is the amount of toxicant that is present in the plasma after its absorption through any one of the routes. However, relative bioavailability is often estimated to express the relative absorption of a toxicant. Relative Bioavailability is the ratio between the concentrations of a toxicant that is present in the plasma after oral and intravenous administrations.
For absorption to effectively occur, the toxicant must pass through several membranes to the destination tissues and organs. This movement occurs through the following mechanisms; passive diffusion, carrier-mediated transport, filtration through membrane pores, and engulfing by pores. Passive diffusion is dependent on the concentration gradient and the lipid solubility of the toxicant while active transport is dependent on energy in the form of the ATP to move the toxicant against the concentration gradient. Absorption through filtration through the capillary pores as a result of osmotic and hydrostatic pressure also occurs for particles smaller than albumin filtration through membrane pores. Engulfing by cells occur in two modes; phagocytosis for solid toxicants and pinocytosis for liquid toxicants.
Distribution and excretion of Toxicants
Apart from toxicant metabolism, distribution and excretion are key attributes that govern the pharmacodynamics of toxicants. Distribution of toxicants within body fluids and tissues depend on different factors. The major factors that influence the volume distribution of toxicants include its protein binding profile, half-life, and the presence of receptors on the cells. However, the distribution of toxicants is initially dependent on their route of absorption. Hence, toxicants that are administered directly into the bloodstream (through the intravenous) are distributed earlier than their counterparts that are administered through oral or dermal routes. However, sublingual administration enhances the rate of distribution of the toxicants. Protein binding influences the distribution of toxicants. Toxicants that exhibit higher protein binding are distributed earlier than their counterparts that exhibit less protein binding. High protein binding also increases the half-life of toxicants. Hence, toxicants with a long half-life are more toxic than their counterparts who exhibit shorter half-lives. Protein-bound toxicants are less soluble in plasma an takes more time to reach the cells or tissues. Hence, toxicants which exhibit more protein binding are less toxic to their counterparts that exhibit more protein binding.
Toxicants are eliminated from the body primarily through two routes; hepatic and renal. Hence, the rate of toxicant excretion is lowered in individuals those who exhibit hepatic or kidney dysfunctions. Different measures are used to estimate the functional status of the liver and kidneys. Clearance is a popular measure that helps to assess the functional of the kidneys. Clearance is the ratio between the concentrations of a toxicant in the urine and the plasma. Excretion of toxicants is delayed in individuals those who exhibit a lower clearance and vice-versa. Creatinine or Inulin Clearance tests are commonly used to assess the functional status o the kidneys. On the other hand, the common liver function tests that are used to assess hepatic function include the SGPT and the SGOT assay. These enzymes are synthesized in the hepatocytes and are elevated in individuals who exhibit hepatic dysfunctions.
Biochemical toxicology: Gas and Small molecules
Different types of gases and chemical molecules can have toxic effects on the body. Cyanide is chemical and exhibits its toxic effects by inhibiting the electron transport chain. It interacts with the ferric ions in cytochrome a3 and prevents the transfer of electrons to molecular oxygen. This reaction marks the terminal step of electron transport and helps to generate free energy. The free energy is used to synthesize ATP by the process called oxidative phosphorylation. Hence, cyanide toxicity will eventually inhibit oxidative phosphorylation. Moreover, cyanide can interfere with other heme-iron complexes and inhibit their functions. The symptoms, occurring in quick succession, are salivation, giddiness, headache, palpitation, difficulty in breathing and unconsciousness. Cyanide toxicity is treated by inhalation of amyl nitrite, intravenous administration of sodium nitrite, and injection of sodium thiosulfate. The objective of such therapy is to convert cyanide into thiocyanate ions. Thiocyanate ions are non-toxic and are easily eliminated from the body. Although cell death occurs, whole organism death is due to respiratory arrest. The gas has a faint smell and poses the danger of poisoning without the victims noticing.
Likewise, carbon monoxide is a gaseous toxicant. Its binding capacity with hemoglobin is 200 times more than oxygen. As a result, hemoglobin fails to get saturated with oxygen and the individual exhibits hypoxia. Moreover, carbon monoxide could also bind with other heme-containing proteins such as cytochrome aa3, and inhibits electron transport chain oxidative phosphorylation. Hyperbaric oxygen therapy is used to manage carbon monoxide poisoning. Carbon monoxide poisoning is the most dangerous gas poisoning and the number one poison cause of death in the United States. The lethal characteristics of the gas can be attributed to the fact that it is odorless, colorless and non-irritative hence undetectable yet it is commonly produced by incomplete combustion of fuels
Biochemical Toxicity of Acetaminophen
Paracetamol or acetaminophen is usually a safe drug and is commonly prescribed for its anti-inflammatory and antipyretic properties. It is a commonly used pain killer with more than 320 million sales in the United Kingdom alone annually. Although it is known to be safe for use within prescription, PAR accounts for 50% of self-poisoning in hospital admission and is the leading cause of acute liver failure. The toxicity starts to appear in a ‘normal’ 70 kg person at about 15.8 g.The mean systematic bioavailability of Paracetamol is approximately 75%. The drug is extensively metabolized in the liver and exhibits a plasma half-life of 1.5 hours to 2.5 hours. Paracetamol is mainly excreted as glucuronide (55%) and sulfated (30%) conjugates. However, increased consumption of paracetamol leads to the formation of a minor metabolite called N-acetyl-p-benzoquinone imine. PAR is metabolized in the liver with 85-95% undergoing glucuronidation and sulphation to conjugates that are mainly eliminated in the urine. About 2-5% is eliminated unchanged in the urine. A further 5-10% is oxidized by cytochrome P450 to the reactive electrophile N-acetyl-p-benzoquinoneimine (NAPQI).
Usually, a low concentration of this compound is not harmful to the body. This is because NAPQI is conjugated with glutathione. However, if the concentration of NAPQI increases significantly it fails to conjugate with glutathione. As a result, N-acetyl-p-benzoquinone accumulates and interacts with different enzymes in the liver. Such mechanisms lead to hepatic dysfunctions including acute hepatic failure and death. N-acetylcysteine (either in its intravenous or its oral form) is recommended for mitigating the harmful effects of paracetamol poisoning. N-acetylcysteine maintains the titer of glutathione in the liver. As a result, N-acetylcysteine mitigates the harmful effects of N-acetyl-p-benzoquinone on the liver. Regular prolonged consumption (say 10 – 15 tablets daily-assuming each tablet has 500mg of PAR) would effectively deplete liver cells of GSH and lead to hepatoxicity.