Biochemical Pathology FULL

5 reasons for Jaundice to Occur?

1. Blocked Bile Duct (cancer, gall stone, scar tissue)

2. Increased Heme breakdown

3. Liver infection e.g. Hep A

4. Liver toxicity e.g. paracetamol

5. Cirrhosis from Alcohol

Normal vs Jaundiced range for serum bilirubin?

Normal 0.2 - 1.2 mg/dL

Jaundiced >2.5 mg/dL

Paracetamol poisoning overview

Symptoms: Jaundice, high serum bilirubin, Abdominal pain

Outcome: slow decline over about a week → death!

Treatment: Liver Transplant

Paracetamol overdose pathway

Excess paracetamol overwhelms excreting routes e.g. Sulfonation and Glucuronidation.

Thus more goes through oxidation pathway which forms NAPQI (highly liver toxic).

Normally mopped up by GSH; there is not enough GSH to deal with it all.

This causes liver necrosis.

DDT and the membrane

DDT is apolar and so accumulates in the fatty layer of phospholipid membrane.

Every single cell has at least 1 molecule of DDT which is sus.. but we don’t know what its doing in cells :(

DDT is insecticide.

Monofluroacetate (1080) overview

Toxic to anything w/ Krebs Cycle. Mammals also can’t bypass it!

1 molecule stops 1 Krebs cycle.

Normally acetate goes thru to isocitrate BUT fluoroacetate gets to fluoroaconitate and gets stuck i.e. it can’t form fluoroisocitrate.

Kochumen Family Case study

Wild boar eaten; mum and dad hospitalised while grandma died.

Initially claimed that poisoning was from Botulism. BUT symptoms incongruous with this. Suspected 1080 poisoning.

5-Flurouracil for Bowel Cancer

Mimics thymine, stops DNA replication specifically targeting fast replicating cells.

Side effects: loss of hair cells, gut cells, skin cells.

Antibiotics which target translation

Chloramphenicol, Erythromycin, Tetracyclines, Streptomycin

Pharmacokinetic parameters

C max: is the maximal concentration reached by the drug; must be greater than the limit for pharmacological effects.

V_D: total dose (mg) / ([plasma Drug] (mg/L)) = How much blood u need to account for the amount of drug given.

Routes of administration from fastes to slowest

IV

Intra peritoneal

IM

Per Os (oral)

First pass metabolism

Stuff from stomach and upper intestine diffuses to portal vein and into liver; immediate detox for the stuff you ate. Then it can go to rest of body after being detoxed.

Polar poisons

Confusion as to how these would cross the membrane and exert toxicity…

• LEAD (Pb2+) goes thru carrier channels for calcium. Evolution worked out Ca2+ channels before lead was a significant environmental toxin.

• 4-Chlorobenzoate: mixture of hphilic and hphobic regions allows transmembrane permeation

• Tetrodoxin: binds to Na+ channel on the OUTSIDE of membrane

2 step oxidation

Start w/ oxidising aliphatic chain of toluene then can do aromatic ox blah blah

Predicting metabolism outcomes: Phase 1

Going to be oxidising stuff! Aliphatic or aromatic…

Predicting metabolism outcomes Phase 2

Glucuronyl transferase can conjugate the metabolite as either the ester or the ether Glucuronide.

Sulfonation can also occur.

Further oxidisation

Could liberate organic compounds from the main structure e.g. formaldehyde could leave.

Sulfo vs Glucurono

Glucurono: higher MW than sulfo; this determines the excretory pathway.

High mass leaves in bile (800 Da cutoff minimum) while lower mass leaves in urine.

Glycine conjugation

Conjugation onto a carbonyl group is a big excretory pathway in humans.

Glutathione overview

Is at 5mM in all cells, during paracetamol poisoning it gets used up

GSH conjugates to our metabolite we want out, via the sulfonyl group.

THEN we chop it up so as not to excrete the whole thing and piss out Mercapturic acids.

Example breakdown pathway

Aspirin broken by ESTERASES to form Salicylic acid.

This can then be OXIDISED then GLUCURONIDATED
OR Glycine conjugation

OR direct GLUCURONIDATION

Alternative phase 2 approach : “"Mopping up the nasties”

Methyl Bromide is an example chemical that can get mopped up with GSH. Then it can get pee’d out. MeBr is super toxic and fucks w/ DNA and proteins.

BUT it can get depleted. :(

PHASE 3: EXCRETION

Urine and feces are considered the normal excretion pathways…

BUT sweat semen saliva are also other ways.

• The semen here is of specific interest since it means that sperm is being exposed to bad chemicals!

• Allylthiocyonate is the smelly thing in garlic and its why u smell bad after eating it. SWEAT AND SALIVA

Enterohepatic REcirculation

Bacteria in the gut can use Beta glucuronidase to remove the conjugated glucuronidate from the metabolite; then we end up with this chemical going round and round (entry into hepatic portal vein and over )

Some drugs utilise this pathway since this increases the lifespan of drug in system.

The big 3 pathogens in food tox.

Campy. jejuni (poultry feces)

Noroviruus: MOST COMMON foodborne pathogen in world. Vomiting and poopy

Listeria monocytogenes: super motile!!!

Campy deep-dive

Difficult to cultivate and highly temp sensitive. Chickens are main source

Chickens poop when being slaughtered and therefore the juice covered in campy.

Need to therefore cook chicken!

Incidence of campy infection in NZ increasing because GP visit expensive and ppl only going when they get super ill and have to go to hospital.

Shiga toxin producing E. coli (STEC)

Causes dysentery. 2 subunits to the toxin: a does toxic effect and b allows for cell entry.

Shigella did HGT w ecoli.

Botulism

C. botulinum is soil bacterium; bad canning can lead to infection! potent paralytic.

Mode of action: Prevents SNARE cmplx formation thru protease activity and thus is an acetylcholine inhibitor.

Saxitoxin

Paralytic shellfish poisoning: Is heat stable and h2o soluble made by Dinoflagellate blooms. SHellfish eat the d.flags and acquire toxin. MOA: binds Na+ tsporter and inhibits!!!

Cucurbitacin

Zucchini gets infected by fungus and then as a defence mech it makes this toxin/. VERY BITTER

Psoralen

Parsnips produce this when damaged, potent carcinogen that is highly stable. Potential to cause skin cancer due to subcutaneous accumulation.

Aflatoxin.

Made by aspergillus flavis. INTENSELY POTENT CARCINOGEN.

Grows on peanuts, shitty china peanuts are bad and infected. THEN we make peanut butter :(

Organnophosphate based pesticides

Are acetylcholinesterase antagonists. Leading to TETANY (spasm!) due to ACh accumulation.

Example is PHORATE: used on carrots… but it could get stuck in the carrot divot on the top of the carrot and lead to HUGE PHORATE CONC. in the root!

So now they use spray on…

TERMS AND DEFINITIONS TO KNOW FOR AGROCHEM

MRL: max residue limit; the residues in food that occur when pesticides are used within the limits of “good practice”

ADI: acceptable daily intake; max intake of chemical for lifetime with no toxic effect!. ADI = NOAEL / 1000

NOAEL: no observed adverse effect lvl; highest dose in animals that has no observed effect.

The cocktail effect

Since pesticide fx are additive, perhaps we should consider MRL as a combo…

Some places use cocktail sprays which don’t exceed MRL for a single agrochem BUT together exert a massive effect.

concern about neurotoxicity in developing fetuses and babies.

LINDANE CASE STUDY

UK 1995, pesticide used for first cotoledon growth of sugar peas which are cow feed.

MASSIVE SPIKE to the ADL in sept, and that months feed was sourced from the Ukraine. Solution was to stop using feed from Ukraine and also just make all the milk into cheese in the meantime.

Food colouring agents, preservatives and flavours

Erythrosine

• Used in cherry cola and maraccino cherries

• KNOWN CARCINOGEN stimulates division. Is nongenotoxic.

• BUT its ADI is 0.05 mg/kg and this is higher than how much it is in food. ALSO the food its in isnt consumed frequently.

Aspartame

Phenylalanine and aspartic acid; is metabolised to these individually. Reasonably likely to cause hepatocellular carcinoma BUT DOSAGE MATTERS

Big deal for phenylketonuriaks: These guys have deficiency in Phe Hydroxylase and thus no melanin and just Phenylpyruvic acid (neurotoxic!)

Folate case study

Need for fetal development; deficiency causes spinal bifida. Fortified flour with folate.

Estrogen Mimics (Xenoestrogens)

Likely cause of breast cancer increase in the world; 70% of BrCa cells have ERs which stimulate cell division when activated by stimulating dimerisation leading to DNA binding and upregulation.

ER has hphobic binding pocket and a phenolic bind site.

3 amino acids that help bind the estrogen: His524, Arg394, Glu353

ER isoforms

alpha present in breast, uterus, liver, lung, kidney, spleen

beta present in intestine, monocytes, macrophage, uterus, prostate

4 example estrogen mimics

Genistein (soy), Bisphenol A (bpa), DDT (synthetic insecticide), 4-nonylphenol (Mr muscle)

Evidence of estrogenic effects

Male fish producing egg precursor protein

Alligator penis shrinkage

Girl puberty starting younger

Oysters turning female

Sperm count decrease

BrCa Drugs: Tamoxifen & Arimidex

Tamoxifen : Metabolised by CytP450 (i.e. is a prodrug) that gets hydroxylated to fit into ER. This makes it an antagonist - if it were more hydroxylated, would be an agonist!

Arimidex : inhibits aromatase activity so no 17B estradiol

Experiment done on placenta to see if it can protect baby from estrogenic compounds

BPA disappeared at the same rate as fetal flowthrough (No stopping)

Genistein had slower appearance: Glucuronidation??

Cancer has 2 key stages

Initiation:

• Mutation in DNA

• OR Inflammatory response

Promotion:

• Cell proliferation e.g. receptor activation

• OR inflammatory response

Main cancer types and their overviews

• BrCa: 80% of these express ER.. rest of info already discussed

• CoCa: 3rd most common worldwide, increase by 14% each decade. These cells express ERBeta

• PrCa: 2nd most diagnosed in world. Metastasis very common! More info later

Prostate Cancer meds (5)

Docetaxel: blocks Androgen receptor and reduces Test production.

Goserelin: Stops Testosterone production, by exploiting Hypothalmic-pituitary-testicular axis. When [Test] in blood increases the HPT axis STOPs test production. BUT TEST also made in adrenals and thus does not stop it completely.

Bicalutamide: Distorts AR structure (antagonist) BUT prolonged use leads to mutation which makes it into an agonist

Hydrocortisone: is a SAID that looks like test but is not agonist.

Chemical Castration w/ Abiraterone Acetate that inhibits test synth.

Prostate Cancer diagnosis

Prostate Specific Antigen (PSA). Is a serine protease that can be measured.

Lung Cancer treatment drug and complications

Cyclophosphamide treatment, and so is Isofamide. These are metabolised to kill (prodrug) and kill cells that reproduce fast.

Cyclophosphamide can spontaneously ring open and form Aldophosphamide, which leads to fatal haemorragic cystitis in bladder via breakdown to Acrolein and Chloracetaldehyde.

Solution is MESNA, which acts like GSH to stop the toxicity. So MESNA gets put in chemo bags. It gets pee’d out benignly.

Things to consider when assessing risk (6)

1. Structure/activity relationship

2. Chronic (lifetime) toxicity

3. Mutagenicity (AMES) Assay

4. Metabolism study

5. Acute toxicity

6. Reproductive toxicity

2 measures for toxicity

LD50: unethical

NOAEL.

What is an ADME study?

Absorption distribution metabolism excretion

Weird mechanism its like a big cage thingy that an animal is strapped into where we collect all its stuff and measure it like slobber, pee, poo, vomit, breath sweat etc. Then we kill the animal and slice it up and measure these slices.

Amyloid fibril formation

Amyloid precursor protein (APP) is acted upon by B and Y secretases releasing monomer AB40/42 → after enough form oligomers they can polymerise.

Early vs late onset alzheimer’s

Early is aka familial AD. accounts for 1-5% of all AD cases. Stipulates that AD develops before 65 years of age.

Late is the most common variant.

Hypotheses

Amyloid cascade/Oligomer hypothesis

Tau hypothesis

Inflammation hypothesis

Amyloid cascade

Aggregation of Amybloid beta drives AD pathology.

For: Down’s syndrome fun fact, mouse models showing oligomer concentration etc.

AGAINST: plaque concentration does not correlate with progression.

Tau hypothesis overview

Tau is MAP, and an IDP. When phosphorylated it forms aggregates.

Tau is GOF and LOF: losing microtubule integrity while forming aggregates.

FOR: cognitive impairment lines up with tau tangles

AGAINST: Tau formn is not earliest changes in disease

Brain cell composition

50% neurons, 50% other cells (Microglia [immune cells], Oligodendrocytes [perform myelination], Astrocytes [star like cells that do a bunch of stuff], Ependymal cells)

Inflammation hypothesis

Microglia are normally involved in Phagocytosis, Amyloid monomer uptake, synaptic pruning…

WHEN THEY FUCK UP this cause destroy viable cells, synapses etc.

So chronic inflammation of brain matter can lead to microglial dysfunction.

Oxidative stress hypothesis

SOD1 reduces ROS; when SOD1 was knocked-out an increase in [ABeta] was observed.

Possible treatment brainstorm ideas PERTAINING TO ABeta

• Adult neurogenesis

• Secretase inhibitors

• Aggregation inhibitors

• Immunotherapy

Possible treatment brainstorm ideas PERTAINING TO Tau

• Aggregation inhibitors

• Kinase inhibitors (cuz phosphorylation..)

• Immunotherapy

Possible treatment brainstorm ideas PERTAINING TO Inflam

• Anti inflam drug

• Activation OR BLOCKING of immune cells

• Cytokines?

Possible treatment brainstorm ideas PERTAINING TO Oxidation

Antioxidants lmao

Diagnostic tools for AD. Hint: scans… Biomarkers?

FDG-PET scans to see brain glucose use. ALSO Can use this to track disease progression.

Fluid biomarker in CSF. e.g. Abeta40/42; Tau/pTau ratios.

11C Pittsburg Compound B which binds ABeta plaques.

USING RATIOS FOR BIOMARKERS

Amyloid ratios are better than on their own… but not great :(. The distribution is tighter tho

pTau biomarker ratio to normal Tau is better than CSF there’s a visible epidemiological difference. Still not good enough for diagnostic use.

Methods for biomarker measuring

Proteins can be imaged with PET, Dye methods, Immunohistochem like ELISA, Mass SPEC>…

Genes but this is only predictive..

Small molecules like metabolomic techniques…?

Therapies for AD (3)

Aducanumab:

• ABeta directed Mab specific for fibrils and oligomers, can cross BBB.

• Engages w/ microglia to stimulate phagocytosis

• Caused reduced amyloid plaque concentration but MASSIVE CONTROVERSY SURROUNDING

Lecanemab:

• ABeta directed Mab; reduces the instance of protofibrils

• Proven safe and sort of effective… Lowers brain amyloid conc.

Donanemab:

• Specific for ABeta isoform p3-42, a form of ABeta which is aggregated in plaques.

• Shown to clear plaques in mice!

• Proven to reduce cognitive decline.

Cholesterol biosynthesis

AcCoa to HMG Coa to Mevalonate; to Squalene; to Cholesterol.