An Overview of Pharmaceutical Innovation

So our mid-semester for this unit is on Wednesday, and some of my friends kindly pointed out to me that this special lecture from the first week is likely to be examinable. The lecturer confirmed it as well. Guess I'll have to look over it then :P

This lecture is relatively short, but it doesn't come with a nice little lecture outline slide, so I'll have to actually make some decisions and decide what to include. Le sigh.

Drug Development

Drug development can be classified into three broad stages:

1. Discovery Phase- The phase in which the drug is "discovered." This starts by figuring out what you want to target and figuring out if the target is "druggable" (i.e. able to be targeted by a drug). Next up is the selection of a "lead compound" (that's the "lead" that rhymes with "bead", not the periodic table element) that might be able to bind to the drug, and then some optimisation to make it bind better or otherwise give it better pharmacokinetic properties. This gives you your "candidate drug."
2. Development Phase- The phase in which the drug is trialled and tested. This starts off with some preclinical development, in which some of the technicalities (like how to administer the drug) are sorted, and then the actual clinical trials and so forth.
3. Commercialisation Phase- The phase in which the drug is approved and commercialised, ready to make the drug companies some $$$.

Of course, in real life it isn't always that simple. In this post I'll cover three drugs that were discovered in different ways: omeprazole, paclitaxel and flecainide.

Omeprazole

Omeprazole treats gastro-oesophageal reflux disease (GORD), also known as GERD in North America (since they leave out the O in "oesophageal"). I've blogged about GORD/GERD here. As mentioned in that previous post, there are drugs to treat GORD. These include histamine receptor blockers (as histamine leads to acid production, as noted here) and proton pump inhibitors (PPIs). Omeprazole, produced by the UK company Astra, is one of the latter.

There were several obstacles in omeprazole development. The first test compounds that they used were not very efficacious. Also, another company managed to make some histamine blockers, filling the GORD treatment niche and putting pressure on Astra to come up with something different. Later on, however, Astra found another compound that was promising, but it was patented in Hungary, further halting development until the patent was found to have expired. This promising compound still wasn't what the company was looking for, as it had serious thyroid and vascular toxicity issues in the animal models that they tested. Eventually, however, promising human trials of omeprazole helped its development to come to fruition. In the early 1990s, omeprazole reached "blockbuster" status (i.e. it was a popular drug).

Paclitaxel

Paclitaxel is a drug that treats ovarian cancer. This lecture had a lot of random facts about ovarian cancer, and I doubt we have to remember them in that much detail, but here they are anyway. The categories of ovarian tumours include serous, mucinous, endometrioid, clear-cell, transitional and squamous cell, with serous being the most common. Ovarian cancer can also be subdivided into benign, intermediate and malignant.

Paclitaxel works by binding to β-tubulin. β-tubulin is an important component of microtubules, which form the spindles during cell division. Now, the slides don't say, and I didn't exactly take great notes from this lecture, but I'm guessing that that's how paclitaxel works: by inhibiting the spindles which would normally help a cell to divide. (This random abstract I found on PubMed seems to agree.)

Paclitaxel is derived from the Pacific Yew. Just like omeprazole, paclitaxel's development was also fraught with challenges. In the beginning, this compound wasn't seen to have much activity in tumour cell lines, so nobody really cared about it until President Nixon declared a "War on Cancer." It was then found that paclitaxel actually had an effect, but there was another problem: it was poorly soluble in water, and solubilising agents could be toxic. Eventually they figured out that they could solve this problem by using slow infusions. Yet another problem that paclitaxel development faced was a shortage of the Pacific Yew: it grows slowly and was endangered. Later on, however, chemists figured out how to produce it by using plant cells in vats, solving that issue.

Flecainide

Flecainide treats cardiac arrhythmias, which I've written about here. At first, dysrhythmic drugs had serious side effects. An example of this was procainamide, which had a very narrow therapeutic window. Flecainide is actually a fluorinated analogue of procainamide.

Once again, there were several challenges in flecainide development. For starters, they weren't really sure how it worked or what it targeted: they were mainly just testing it to see what would happen. Also, flecainide was slow to produce, which made the whole process slower. Eventually, though, flecainide was released as the first intentionally produced antidysrhythmic drug.

Smooth Muscle Response to Stretch

Last post on smooth muscle!

Explain how myogenic tone develops in vascular smooth muscle

As mentioned here, some smooth muscles, including those of the vascular system, contract in response to stretch. This process requires calcium to enter the cell from outside, and is thus blocked by nifedipine (a blocker of L-type calcium channels). But how exactly does this process work? Read on...

Describe the possible pathways for myogenic tone including the role of TRPC3,6, TRPM6, L-type channels, GPCR and autocrines.

Stretch-Gated Channels (SGCs)

There are many non-selective, stretch-activated channels in vascular smooth muscle. These are called stretch gated channels, or SGCs. The main ones include the epithelial sodium channel (ENaC), TRPC3, TRPC6 and TRPM4. Now, that's a lot of letters, so lets break it down. The TRP refers to the Transient Receptor Potential (TRP) family, the C or M refers to the family, and the number refers to the specific member of the family.

As there are a lot of different channels that might be involved, they can compensate for one another. Mice that lack the genes for TRPC6 appear to have upregulated TRPC3. This is probably why these mice still have the myogenic response.

G-Protein Coupled Receptors (GPCRs)

Now it's time to expand even more on the signalling pathways covered here and here! Yup, there's even more stuff that you're going to need to know. If you're not familiar with the signalling pathways that have already been covered, I suggest that you look back on these before proceeding or you're going to be lost in the alphabet soup to come.

Stretch can activate some G-protein coupled receptors, such as the AT₁ (angiotensin) receptor. Furthermore, some of the signalling molecules in the G-protein coupled receptor signalling pathway can increase the opening probabilities of the SGCs mentioned above. DAG (diacylglycerol) can bind to and increase the opening probability of TRPC3 and 6, whereas IP₃ does the same for TRPM4.

Autocrine and Paracrine Signalling Molecules

Smooth muscle can also release some mediators that can act in an autocrine (on the same cell) or paracrine (on neighbouring cells) fashion.

Phospholipase A (PLA₂) is a membrane-bound enzyme that is activated by stretch. When activated, it catalyses the formation of arachidonic acid, which can be further broken down to form 20-HETE and thromboxane A2 (TxA₂)- yup, the same TxA₂ mentioned in PHAR3303. These can diffuse out of the cell and bind to receptors (such as the G-protein receptors mentioned above) on the same cell or on neighbouring cells. Other signalling molecules that may act in this fashion include sphingosine-1-phosphate (S1P), UTP and ATP.

Describe DI induced bronchodilation and its relevance to asthma and COPD

DI = deep inhalation. When we take in a deep breath, it reduces bronchoconstriction. That's why it's so much easier to exhale after we take a deep breath in first. In patients with asthma or COPD, this mechanism may not work- a DI may cause no change, or maybe even bronchoconstriction.

Explain how length-adaptation and stretch induced dilation might be due to rearrangement of actin and myosin filament
Describe the labile nature of actin and myosin in smooth muscle

Smooth muscle is very adaptable. If you pin it out at a longer length, it will adapt so that that longer length becomes its new optimum length for contraction. If you pin it out at a shorter length, it will likewise adapt so that that shorter length becomes its new optimum length. Interestingly enough, no changes in calcium or myosin light chain phosphorylation are associated with this phenomenon, so something else must be at play.

That something else, believe it or not, is the ability of smooth muscle to add or remove contractile units (a property that is unique to smooth muscle). When smooth muscle contracts, it assembles its thick filaments (i.e. myosin filaments). This may seem counterintuitive, but when myosin is phosphorylated (as happens during contraction), it is more stable, which may help with polymerisation. Stretch triggers disassembly of these filaments so that they can reassemble at a new length. We're still not sure on all of the details, but the end result is that a muscle that is adapted to a shorter length will have fewer myosin filaments, whereas a muscle that is adapted to a longer length will have more myosin filaments.

Actin filaments are also important. Contraction triggers polymerisation of actin as well as myosin. Inhibiting the polymerisation of actin will also reduce the amount of force that can be produced.

Of course, it's not that simple in vivo. As loads on muscle are often oscillating (such as in airway muscle where you are breathing in and out all the time), the muscle may never be able to finish adapting to any length. This may be why airway smooth muscle often relaxes with oscillatory loads (such as the loads placed on it during regular tidal breathing).

There's also another phenomenon to take note of called shortening inhibition. Shortening inhibition basically means that the longer you leave a muscle contracted, the less able it is to contract.

Define Latch

Latch is a state in which the cross-bridges are attached, but are not cycling. We're still really not sure about how this happens, though: maybe the cross-bridges are dephosphorylated or something like that, preventing them from releasing, or maybe this is due to the polymerisation of actin filaments after contraction. In any case, the latch state is probably the reason why smooth muscle can maintain tone without using up a lot of ATP, despite smooth muscle contraction being less efficient than in other muscles (smooth muscle is consistently polymerising and depolymerising its filaments, and that shit takes energy).

The TCA Cycle

In my last post for BIOC3004, I wrote about glycolysis: the process in which glucose is broken down to pyruvate. That post did not cover the stuff that happens before and after this process: the breakdown of glycogen into glucose, and the entrance of pyruvate into the citric acid cycle.

Describe the reactions of the TCA cycle, where they occur within the cell and the regulation of the pathways.

From Glycolysis to Acetyl CoA formation

After glycolysis, which occurs in the cytosol, pyruvate enters the mitochondria where it is irreversibly converted to acetyl CoA with the help of pyruvate dehydrogenase, producing a little NADH along the way. Pyruvate dehydrogenase is actually a large complex made up of three subunits, each of which catalyses a single step in this reaction. Pyruvate dehydrogenase can exist in a phosphorylated (active) and dephosphorylated (less active) form. It is held in place by negative feedback mechanisms: when acetyl CoA, NADH and ATP levels increase, they can inhibit the phosphorylation of pyruvate dehydrogenase.

If pyruvate dehydrogenase is deficient, this can cause a condition called lactic acidosis, which may result in neurological defects and death. This is because pyruvate cannot become acetyl CoA, so it gets converted into lactate instead. Sometimes, this condition can be managed by eating fewer carbohydrates so that less pyruvate will build up.

The TCA Cycle

Enter long chain of reactions! All of these happen in the mitochondria.

1. Acetyl CoA and oxaloacetate (a four-carbon molecule) combine to form citrate. Requires citrate synthase.
2. Citrate forms isocitrate with the help of aconitase.
3. Isocitrate forms α-ketoglutarate with the help of isocitrate dehydrogenase. Forms NADH and CO₂.
4. α-ketoglutarate forms succinyl CoA with the help of α-ketoglutarate dehydrogenase. Also forms NADH and CO₂.
5. Succinyl-CoA forms succinate with the help of succinyl-CoA synthetase. Forms GTP.
6. Succinate forms fumarate with the help of succinate dehydrogenase. Forms FADH₂.
7. Fumarate forms malate with the help of fumarase.
8. Malate forms oxaloacetate with the help of malate dehydrogenase. Forms NADH.

Aaaand we're back to the beginning of the cycle!

It should be noted that this cycle is not strictly a cycle: some things can enter at different spots along the way, and some of the substances in the cycle can leave and do their own thing.

It should also be noted that NADH, FADH₂ and GTP don't come out of nowhere. NAD⁺, FAD and GDP are also required for the cycle, providing another mechanism through which the TCA cycle can be regulated.

Electrons carried by NADH and FADH₂ can then enter the electron transport chain in the inner mitochondrial membrane, but that's a subject for a later post.

Draw Pyruvate & Acetyl group of Acetyl CoA.

We have to draw these? I DID NOT SIGN UP FOR THIS SHIT >_>

Drew these on ChemDoodle, with a bit of wrestling with my computer. Enjoy.

Understand the energetics of aerobic metabolism.

Aerobic metabolism produces a net ATP yield of 38 molecules. You see, each molecule of NADH yields 3 ATP after going through the electron transport chain (which I'll talk about in a later post), whereas each molecule of FADH₂ forms 2 molecules of ATP. Hence you get the following:

• Glycolysis: 2 ATP + 2 NADH (x3 = 6ATP) = 8 ATP
• Pyruvate to acetyl CoA: 1 NADH (x3 = 6 ATP) = 3 ATP x 2 (because you get 2 molecules of pyruvate for every molecule of glucose) = 6 ATP
• TCA cycle: 3 NADH (x 3 = 9 ATP) + 1 FADH2 (x 2 = 2 ATP) + 1GTP (=1 ATP) = 12 ATP x 2 = 24 ATP
• 8 + 6 + 24 = total yield of 38 ATP

Know the pathway of glycogen breakdown.

Glycogen can be broken down to glucose in the process of glycogenolysis. I've covered this in more detail in an earlier post: Carbohydrates- Metabolism of Glycogen.

Toxicology of Tobacco II

In my last post, I wrote about the carcinogenic compounds in cigarettes. In this post, I'll be talking about free radicals.

Demonstrate basic understanding of the main chemical species formed during oxidative stress

Firstly, I should give a quick definition of oxidative stress. Oxidative stress occurs when there are more free radicals produced than removed. (Free radicals can be removed by antioxidants.)

The main species formed from oxidative stress can be classified into two main categories: reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS include superoxide, hydrogen peroxide and hydroxyl radicals. RNS includes NO radicals and ONO₂CO₂^-, which I know virtually nothing abut other than that it can be very damaging.

Show an understanding of how redox-cycling quinones are a source of superoxide radicals in tobacco smoke-exposed lungs.

Redox-cycling quinones in tar can produce free radicals, such as superoxide. Some quinone-containing compounds, such as the hydroquinone-quinine complex, are formed during the incomplete combustion of tobacco leaves.

Identify major products of oxidative damage to proteins and unsaturated fatty acids, and their use as biomarkers.

8-isoprostane is a common product of oxidative damage. It is formed by free radical attack on arachidonic acid. Carbonyl-adducted proteins (i.e. proteins with carbonyl attached) are also common products of oxidative damage. These products can also be used as biomarkers, i.e. their concentrations can be measured to determine the extent of oxidative stress,

Show how smoke-derived ROS may cause 2 pathological features of COPD: elastase activation and proinflammatory gene expression secondary to chromatin remodelling.

Elastase Activation

Human neutrophil elastase (HNE) mediates degradation of elastin fibres in alveolar walls, leading to emphysema. Usually, HNE is inhibited by α₁-antitrypsin, but when α₁-antitrypsin is oxidised due to ROS, it can no longer bind to and inhibit elastase.

Gene Expression

ROS may regulate kinases that affect the acetylation, methylation and so on of histones (proteins that the DNA are wound around). This can affect cell signalling and gene expression. For example, ROS can deplete SIRT1, which is a deacetylase that normally represses chronic inflammation, sensecence (cell aging) and apoptosis. When SIRT1 is depleted, it can no longer repress these processes, so cell aging, death and so forth become more prevalent.

Glycolysis and the Pentose Phosphate Pathway

Now we're getting into the nitty-gritty details! (At least it's pretty easy to see the relevance of this lecture though :P)

Describe the reactions of glycolysis and the mechanism of energy production in the cytosol.  

Glycolysis, as you should know, is a pathway in which glucose is broken down to form energy. The main end product of glycolysis is pyruvate, which has three different fates: in the presence of oxygen, it can be converted to acetyl-CoA and enter the citric acid cycle; in anaerobic conditions, it can form lactate; and in some organisms it can be broken down to ethanol (such as in the fermentation process in yeast).

Glycolysis has two major phases: the preparatory phase and the payoff phase. (Not sure if those are official names, but they're on the slides.) In the first phase, 6-carbon glucose is converted into two 3-carbon sugars called glyceraldehyde 3-phosphate. In the second phase, glyceraldehyde 3-phosphate is converted into pyruvate, generating ATP and NADH. It should be noted that glucose is not the only fuel that can enter this pathway: it's the main fuel, but other sugars can enter the pathway at different stages.

Phase 1: Preparatory Phase

The preparatory phase consists of five steps:

1. Glucose --> Glucose 6-phosphate. Requires ATP and the enzyme hexokinase.
2. Glucose 6-phosphate <--> Fructose 6-phosphate. Requires the enzyme phosphohexose isomerase.
3. Fructose 6-phosphate --> Fructose 1,6-bisphosphate. Requires ATP and the enzyme phosphofructokinase-1.
4. Fructose 1,6-bisphosphate <--> Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Requires the enzyme aldolase.
5. Conversion of dihydroxyacetone phosphate into glyceraldehyde 3-phosphate (so you end up with 2 molecules of glyceraldehyde 3-phosphate for every molecule of glucose). Requires the enzyme triose phosphate isomerase.

Note that 2 molecules of ATP are required (steps 1 and 3). Don't worry- we'll more than make up for that later.

Phase 2: Payoff Phase 

The payoff phase also consists of five steps:

1. Glyceraldehyde 3-phosphate <--> 1,3-bisphosphoglycerate. Requires the enzyme glyceraldehyde 3-phosphate dehydrogenase. During this step, NAD⁺ is also reduced to NADH, with a little help from HPO₄^2- which supplies the hydrogen.
2. 1,3-bisphosphoglycerate <--> 3-phosphoglycerate. Requires the enzyme phosphoglycerate kinase. Produces ATP. (1 molecule of ATP is produced for each molecule of 1,3-bisphosphoglycerate, but since one molecule of glucose produces two molecules of 1,3-bisphosphoglycerate, you actually get 2 molecules of ATP for every 1 molecule of glucose.
3. 3-phosphoglycerate <--> 2-phosphoglycerate. Requires the enzyme phosphoglycerate mutase.
4. 2-phosphoglycerate <--> Phosphoenolpyruvate. Requires the enzyme enolase. Produces some water.
5. Phosphoenolpyruvate --> Pyruvate. Requires pyruvate kinase. Produces ATP (the ATP yield of this step is the same as the yield for step 2 of this pathway).

As you can see, this pathway has two ATP-producing steps (2 and 5). These steps combined produce 4 molecules of ATP for every molecule of glucose. As 2 molecules of ATP were consumed in the first half, this results in a net yield of 2 ATP molecules per molecule of glucose.

Explain the difference between reversible and irreversible reactions.

Reversible reactions are reversible while irreversible reactions are irreversible...? Also, the reversible reactions tend to have a small free energy change, whereas irreversible reactions have a much larger one (I think).

The three irreversible reactions in the glycolysis pathway are steps 1 and 3 of the first phase as well as the final step of the second phase.

Have an understanding of the purpose of the pentose phosphate pathway.

The pentose phosphate pathway produces pentose sugars, such as ribose 5-phosphate, that are required for the production of nucleotides (and, by extension, DNA and RNA). Along the way, NADPH is formed, which is required for biosynthesis of fatty acids and production of glutathione (GSH).

The first step of this pathway is catalysed by glucose-6-phosphate dehydrogenase (G6PDH). If this is deficient, the red blood cell membrane might be lost, leading to haemolysis and haemolytic anaemia.

Respiratory Reflexes

This lecture had a fair bit of new content. It's not as difficult conceptually as that last cardiovascular lecture, but it is still way too much for a Monday afternoon IMO :P (Okay, it wasn't that bad, but I feel like there were a few little bits and pieces that just slid past me.)

Describe the inputs to the central rhythm generator.

As mentioned in my previous post, respiratory rhythm arises largely from the medulla. The centres in the medulla involved in control of respiration can be influenced by a variety of factors, such as feedback from peripheral receptors, emotion and temperature. (Temperature doesn't really have a massive effect on ventilation in humans, but in some animals, such as dogs, it can cause panting.) Voluntary control of ventilation is separate to the rhythm control in the medulla.

List the three types of lung receptors

The three types of lung/lower airway receptors include slowly adapting receptors (SARs), rapidly adapting receptors (RARs) and C-fibres. I'll describe them in more detail soon.

Describe the Hering-Breuer reflex.

As mentioned here, the Hering-Breuer reflex prevents overinflation of the lungs. It does this by suppressing inhalation when the lungs are already inflated. The vagus nerve is vital to this reflex- if you cut it, the reflex disappears. The Hering-Breuer reflex is not particularly strong in conscious humans, but it is important in animals (and may also be important in anaesthetised humans).

Explain the role of SAR and their stimulation and effects.

SAR, or Slowly Adapting Receptors, help detect the volume of the lungs. They are activated when the lungs are stretched, and as they do not adapt to the stretch (or at least they adapt very slowly, hence their name), they keep firing while the lungs are inflated. These may contribute to the Hering-Breuer reflex, which I just described.

SARs are myelinated fibres which have a conduction velocity of 15-30m/s (which is pretty standard for myelinated fibres). As mentioned above, they are stimulated by stretch. They do not respond to deflation, unlike RARs as I'll discuss shortly. The effects of SAR activation include reduced tidal volume, shorter respiratory time and bronchodilation.

Describe the reflex effects of RAR and the stimuli that activate them

RAR, or Rapidly Adapting Receptors, help detect changes in volumes of the lungs. Located within or near the epithelium, they are activated when stretching occurs, but their response is rapidly "switched off." They respond in response to both inflation and deflation, and the frequency of their impulses depends on the rate of change of volume of the lung (which is also related to inspiratory flow rate).

RARs, like SARs, are myelinated fibres with a conduction velocity of 15-30m/s. As well as being stimulated by stretch, they are also stimulated by irritants such as acid, smoke and dust. They produce reflexes such as coughing, tachypnea (abnormally rapid breathing), hyperventilation and bronchoconstriction.

Describe the activation and reflexes produced by C-fibres.

C-fibres, unlike RARs and SARs, are unmyelinated and thus their conduction velocity is much slower (1m/s). They come in two flavours: bronchial and pulmonary. They are present in the airway epithelium, as well as in other places around the lung.

Stimulants of C-fibres include capsaicin (as I'll explain shortly), acid and/or hypertonic saline (for bronchial C-fibres) and oedema and/or large amounts of inflation (for pulmonary C-fibres). It should be noted, however, that they are generally not very responsive to inflation. Activation of C-fibres produces bronchoconstriction, bradycardia, and sometimes cough. Initially, C-fibre activation can produce apnoea, but later on hyperpnea can be produced instead. (See here if you don't remember what these terms mean.)

One thing that is kind of unique about C-fibres is that they contain both afferent and efferent fibres, allowing the signal to branch out more easily (I think... there were quite a few tidbits in this lecture that I didn't quite get). C-fibres also contain neurotransmitters, including peptide neurotransmitters such as tachykinins. These can cause vascular leak (contraction of endothelial cells, increasing the amount of space between them), mucous production and bronchoconstriction. (I think. As I said, there were quite a few bits in this lecture that slid over me. I blame the time in the afternoon.)

Recall the receptors activated by capsaicin.

C-fibres can be activated by capsaicin, the component of chilli that makes it hot. Capsaicin activates vallinoid receptors (VR1), which are TRP (transient receptor potential) channels. Activation of VR1s produces the sensation of heat. Over long periods of time, capsaicin can actually kill off C-fibres, making you more tolerant to hot foods.

Explain the detection of cough.

This was hardly covered in the lecture, but the summary slide says that RARs and maybe C-fibres can detect cough. Receptors in the upper airways might help as well.

Describe the effects and activation of upper airway cool receptors.

Receptors in our upper airways can respond to changes in temperature, such as what happens when we exhale air from outside. Stimulation of these receptors makes us feel like we are breathing, so receptor stimulation suppresses further ventilation. On the other hand, when these receptors are blocked, we can feel like we can't breathe even though our blood gas concentrations might be normal.

Aside from cool receptors, there are quite a few other receptors in the upper airway (nose and larynx). These can cause laryngospasm (closing off of the larynx). These receptors can respond to flow, temperature, pressure, upper airway muscle contraction, snoring and obstruction.

Describe the effects of menthol.

Menthol stimulates cool receptors, making it feel like you are getting more airflow than you actually are.

Explain the muscle proprioception reflexes including the spindle and tendon organ

Muscles have a couple of different proprioception reflexes. We didn't go into too much detail on them in the lecture though. Apparently this was covered to some extent in PHYL2002, which I didn't do, so...

Muscle spindles can measure muscle length, and sudden change in spindle length (as might happen when you tap someone on the knee) can cause a reflex contraction. The Golgi apparatus can also play a role in measuring force on the tendon. Such receptors are present in a lot of skeletal muscles, but the diaphragm has few of them. Hence, a lot of the proprioception reflexes involved in breathing actually come from the abdominal muscles and intercostal muscles.

Define dyspnea.

Dyspnea is the feeling that you can't breathe, even though blood gases might be normal. There are several different mechanisms that might trigger this, such as mucus covering the cool receptors (as happens during a cold).

Toxicology of Tobacco I

Now we're moving onto the respiratory system in PHAR3303! (And what good timing too, considering that we're learning about the respiratory system in PHYL3002 as well :P)

Demonstrate understanding of the main steps in the manufacturing of cigarettes together with an appreciation of the toxicological implications of some processing steps.

It can take months to prepare tobacco. In the first step, the leaves are dried through a process known as "curing." This preserves the leaf by decreasing its water content. Curing also destroys the chlorophyll, hence turning tobacco yellow. Other processes then take place, such as partial rehydration and removal of the stems. Additives can also be added to the tobacco in order to improve its flavour or aroma later on. Once the leaves are ready, they are chopped up, blended and then packaged into cigarettes.

Crop production may introduce some contaminants into the cigarette. Aside from pesticides, herbicides and so on used when growing the plant, tobacco can also become contaminated by microbes.

Now let's talk about the cigarette itself! The cigarette paper may have been treated with whitening agents, inks, adhesives and so on, which can add even more contaminants to the smoke. There are also other features of the cigarette that can affect how much stuff actually gets to the smoker's lungs. The rolling paper can affect the air flow and yield, the filter can prevent some (but not all!) of contaminants from getting to the smoker's lungs, and vents can help to dilute the smoke. Of course, this is all far from perfect, as there are still many tobacco-related deaths every year.

Show an understanding of the two common ways of thinking about the different types of smoke released from a burning cigarette.

There are two main ways of thinking about the different types of smoke.

In the first classification system, smoke can be classified as mainstream, sidestream or environmental. Mainstream smoke is the stuff that gets inhaled, sidestream smoke is the smoke that comes off the end of the cigarette, and environmental smoke is a mixture of sidestream smoke and the mainstream smoke exhaled when the smoker breathes out.

In the second classification system, smoke can be classified as first-hand, second-hand or third-hand. First-hand smoke is analogous to mainstream smoke, whereas second-hand smoke is analogous to environmental smoke. Third-hand smoke refers to the smoke residue that gets deposited on surfaces.

Identify 3 major carcinogen classes in tobacco smoke, showing awareness of any role of metabolism in converting such species to genotoxic or DNA adduct-forming metabolites.

This lecture covered three main toxic compounds: nitrosamines, 1,3-butadiene (BD) and acetaldehyde.

Nitrosamines

Nitrosamines are bioactivation-dependent: that is, they need to be metabolised before they can start causing damage. Nicotine can undergo a process called N-nitrosation which can form NNN and NNK. NNN and NNK are potent carcinogens as they can attack DNA and form methylated bases. (These altered bases are also known as "DNA adducts.") These methylated bases can then cause incorrect base pairs to form. For example, guanine can be methylated to form O⁶-Me-G (the "Me" stands for "methyl"), which pairs with thymine rather than cytosine.

1,3-butadiene (BD)

1,3-butadiene is quite important, partially due to its high prevalence in tobacco smoke. BD is also present in other things such as in the synthesis of polymers or the exhaust gases from cars. In rodents, it can cause cancer in multiple different organs.

1,3-butadiene, like nicotine, is also bioactivation-dependent. CYP2E1, an isoform of CYP450, catalyses its breakdown into 1,2-epoxy-3-butene, or EB. EB can attack guanine, forming a deoxyinosine adduct which can cause incorrect base pairing and mutations.

Acetaldehyde

Acetaldehyde, which is also prevalent in tobacco smoke, does not require bioactivation to become carcinogenic. It's not 100% confirmed how it causes cancer, but it's possible that DNA adducts play a role. Deoxyguanosine, for example, can form a weakly mutagenic adduct when combined with acetaldehyde, but this adduct can also block DNA replication.

Understand the major biological outcomes of DNA adduct formation, namely DNA repair, apoptosis and mutagenesis.

DNA adducts have three main outcomes. Firstly, DNA repair mechanisms might kick in and fix the damage. Secondly, damage to the DNA may trigger the cell to undergo apoptosis (cell death). Thirdly, DNA adducts may cause incorrect base pairs to form, essentially causing mutations. Depending on the genes that are altered, cancer may result.

Also, it's important to note that not all of the bad stuff in cigarettes cause cancer. Some toxic chemicals are related to other negative outcomes, such as COPD. For example, acrolein is a chemical that might play a role in COPD development due to its reactivity with proteins and DNA.