pbhati17's version from 2018-02-24 00:08

(he is going to talk through this section with an example, ochratoxin A)

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there are a variety of tests we can do to look at Carcinogenicity, genotoxicity and mutagenicity. One such test looking at MUTAGENECITY is a micronucleus assay. Explain thisexposing erythroblasts (early RBCs) to a particular compound. If that compound leads to mutagenicity, then we start seeing micronuclei. Based on the dose of that compound given, we start seeing smaller than normal nuclei in the erythroblasts. (can be done with other cells though, also-- after mitosis there is just two smaller nuclei instead of a normal one) as soon as you see a number of micronuclei, you know that compound is mutagenic.
explain the AMES test (what is it looking at?)another in vitro-mutagenicity assay. Use salmonella-- does not grow without histidine. So put salmonella with test compound on a plate, and you can see what happens-- if nothing happens, it's not a mutagenic compound. if it IS mutagenic, changes DNA of salmonella resulting in a salmonella which DOES grow without histidine and you will see growth on histidine negative agar.
explain the comet assayin vitro mutagenicity assay which looks at DNA damage. If DNA is damaged, it gets fragmented, which if you do electrophoresis you will see this "tail" instead of a nice round sample which are all about the same weight-- the fragments are might smaller and lighter and distribute along the electrophoresis gradient in this tale shape.
if you wanna do an in vivo mutagenicity assay with mice and rats, what do you need to do in regards to how long you are testing?needs to be relatively long (for their lifespan) so that we know it doesn't lead to chronic toxicity/carcinogenicity/mutagenicity. So do test for 18mo in mice and 24mo in rats.
when is the only time you will do in-vivo Carcinogenicity, genotoxicity and mutagenicity tests?only if they were not positive in vitro!!
what is ochratoxin A? how about B and C?a mycotoxin found worldwide. Ochratoxin A is the most tox one, Ochratoxin B has a 10-20% reduction in tox, and Ochratoxin C has virtually no toxicity. (he said we don't need to know the molecule structure stuff but i dont trust him) A has a isocoumarin group on R side, coumarin is a precursor of coumadin, so if it gets metabolized it can have coumadin effects (vit K antagonist--> bleeding tendencies). but the amino acid part of the molecule is actually the most toxic part. (also important in protein binding of OTA)
what are some fungi which can produce OTA?Aspergillus and Penicillium species (Aspergillus ochraceus , Aspergillus carbonarius <--natural contaminant of many foods, Penicillium verrucosum)
**OTA is a regular contaminant of what kinda food products?Cereals (rye, wheat, barley, oats) rice, maize, Hazelnuts, Brazil nuts, palm products, Wine, grape juice, dried vine fruits, resins and currants, Figs and licorice, Coffee and cacao, meat and sausages
(pathophys) when OTA first started affecting people in europe (Balkan area) they noticed what kinda things were happening to ppl? (2)Balkan Endemic Nephropathy (BEN) and Urinary Tract Tumors (UTT)---- most affected were pigs and humans. In a short amount there is dramatic loss of kidney function. In short amount of time there is CHRONIC kidney failure where it is difficult to do anything about it.
what is karyomegaly? how does it relate to OTA?this is cells with big nuclei. (large cells with large nuclei).
prolonged exposure of OTA leads to what prob?urinary tract tumors
explain the Balkan Endemic Nephropathy (BEN) and Urinary Tract Tumors (UTT)-- what are the main effects on the body and why(1) karyomegaly= large cells with large NUCLEI. Also see End-stage renal failure (Clinical signs: glycosuria, proteinuria, increased urinary GGT concentrations). There is Progressive tubulo-interstitial nephropathy, tubular atrophy, peri-globular fibrosis and cortical cysts. there are also the UTT - urinary tract tumors (chronic exposure) which are Poorly characterized renal and urothelial tumors
toxicokinetics of OTA-- what is the oral bioavailability like (since it is usually consumed this is the thing we care about) and what is the plasma protein binding like?has decent oral bioavail-- 66%. It also binds to plasma proteins a lot- 99%.
how does half life of a substance relate to tox?bigger the half life, more chance for tox (bc its sitting in the body longer)
what is the marker of exposure we measure for OTA?Blood serum concentration is a marker of exposure (highly plasma protein bound)


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what can you get out of this chart showing the half life of OTA? longer the half life, the more toxic- and so humans and pigs are affected the most (And this is seen clinically)
OTA is highly plasma protein bound (99%) so what does this mean for tissue distribution?you would think it was minimal tissue distribution bc it is so plasma protein bound. And since it is plasma protein bound, it is now a big complex, and wouldn't cross membranes. But then why do we see all these clinical effects? There are ABC transporter proteins! They transport stuff almost anywhere in the body. 2 examples of ABC transporters in the kidney are MRP2 and BCRP (see pic). It delivers well in the kidneys, and mammary glands (so actively transports compound into milk)
why do we see toxic effects of OTA in the milk, and why are the kidney's and urinary system affected so severely?Thats because thats where the ABC transporters are that transport the OTA. (pic= 2 kinds of kidney xporters)
implications of ABC transporters in mammary tissue..?OTA transported into milk, which means mothers who consume products with OTA are then breastfeeding their babies and intoxicating them with OTA right after birth. (and coffee is highly infected with OTA-- and mothers who drank MORE coffee had HIGHER levels of OTA in their milk= direct correlation)
explain how you would test the mutagenecity of OTA with a AMES salmonella test.inoculate a plate with a salmonella strain that does not grow without histidine (slide listed some of the strains they use- TA100, TA98, TA 1537, TA1538, TA1535) . And there is no histidine in the medium. we put a test chemical in the middle. If the test chemical is mutagenic, it's gonna change the DNA of the salmonella so that now it does grow without histidine. It is a genetic mutation. If negative, you just see a FEW colonies form (bc there are always spontaneous mutations). Also if it is positive, we will see more colonies growing around the test compound (bc conc is highest there= more mutations). (if see it only on the prolly messed sthing up). The specific mutation is a point mutation leading to a frame shift which creates the growing-without-histidine mutation.
so why do we like the AMES test over, say, a mouse test?takes a long time for these urinary tract tumors to form in a mouse- bact mutations are fast (rats took 2 years) Straight forward and easy to do also
what if you want to see if the metabolized products of the toxin are also toxic? Or if compound is only toxic when metabolize? what can you do to test this?add microsomal fraction to the AMES culture. You can combine diff microsomal fractions (such as CYP450 for a human, some cat and dog microsomes, etc) Then inoculate the plate as you normally would and wait and see. If it is more toxic WITH the microsomes added, then we know the metabolite is toxic. If both parent and metabolite are toxic, then there are even more mutated colonies than in just the parent test.


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MICROSOMES are only CPY450. If you want to look at metabolism pathways that aren't cyp450, what can you do?hepatocytes do all kinds of metabolism. So if you add hepatocytes, you might diff a different response than if you just used microsomes. (bc dont just see cyp450 but also other oxidative reactions and such). You also should know that there is a diff between HEPATIC and RENAL microsomes. the Renal ones will have some additional phase II metabolism. So you can do this with all different kinds of plates (microsomes, hepatocytes, combos, etc)
explain what a post-labeling technique is, what it does ( (32)P post-labeling technique)you are looking at DNA adducts ( a piece of DNA covalently bonded to a (cancer-causing) chemical.)-- because we added the compound to cells, leading to a DNA adduct. Each type of adduct results in a different kind of blot on an electrophoresis. Then you have to ID the individual adducts (difficult). The fact that there are adducts means that there is DNA damage-- just need to figure out what's what now. If DNA damage= potential for compound to be mutagenic/carcinogenic (remember you still need to figure out what the adducts all mean though) You can do this in vitro or in vivo
explain how you do post-labeling techniques in vitro versus in vivoVitro is where you add compound to cells and then electrophorese the results to look for DNA adducts (fragments/damage attached to the compound). in VIVO you are giving the compound to mice/rats, and then extract blood and see if there are adducts.
explain DNA damage and the COMET assayIt shows in A SINGLE CELL whether or not there is damage (yes or no). Normally we want to see (if there was no harmful chemical in there) a nice ellipse shape of the electrophoresis. But if we see lots of DNA material outside of the normal size/weight range (the ellipse) scattered behind it (which is why we call it a comet assay)- all these fragments in the "tail" imply there is DNA damage. dont forget this is in a SINGLE CELL (also if you test cell with cyp450 you can see if the compound is more toxic when metabolized)
OTA and carcinogenicity: epigenetic mechanisms (how does mutagenicity work?) (explain this crap) This is what happens to a lot of toxic compounds- they lead to oxidative stress (reactive oxygen species, reactive nitrogen species, etc) and they alter a variety of things in the cell, and because they do that, it ultimately leads to mutation, apoptosis, cytotoxicity, DNA damage, mutagenectiy, carcinogenecity-- leading to cell tranfromation/prolif--> tumor. (vast majority of tumors come from oxidative stress).
how do OTA and ROS (reactive oxygen species) relate?inc doses of OTA lead to inc doses of ROS. ROS causes DNA damage. If we add an antioxidant (Vit E aka alpha-tocopherol), we see less ROS formation. ROS are one of the ways OTA leads to mutagenicity. (kirks notes said; See GSH (intracellular antioxidant glutathione) depletion in different cell lines)
list of problems OTA causesProximal tubule damage (the primary target), Karyomegaly, Desquamation (skin peeling), focal degeneration, Focal peri-tubular fibrosis
Risk assessment: so we know OTA causes renal damage and tumors etc. so how do you determine how bad these things are in a quantifiable way?need to look at NOEL and LOAEL. Then figure out a uncertainty factor. So if sthing comes up positive on the AMES test or some of the other tests, then we DRASTICALLY inc the uncertainty effect (remember how anything above a 100 was iffy? well for OTA they made it 450). so with a bunch of other calculations they came up with a provisional tolerable weekly intake (meaning we should prolly be ok with an intake of whatever that PTWI was determined to be)
Conclusions of OTA risk assessment..what are the conclusions they came to after all the testing they did?(1) OTA is a frequently occurring food and feed contaminant (2) Intoxications in farm animals are reported in pigs and poultry (3) Toxicological testing indicated a specific renal toxicity, driven by the accumulation of the compound in proximal tubular cells followed by tubular injury (4) A typical pathological marker is karyomegaly in proximal tubular cells (5) Genotoxicity and carcinogenicity testing revealed genotoxic and epigenetic toxic mechanisms (6) A recent rodent bioassay confirmed the induction of renal cells carcinomas (7) The ADI/TDI/TWI derived from previous rodent studies amounts to 100 ng/kg bw (8) Exposure assessment indicates that children may exceed this PTWI (provisional tolerable weekly intake) - the uncertainty is increased because OTA is also excreted with breast milk
what is a typical pathological marker of OTA tox?karyomegaly in proximal tubular cells