wilsbach's version from 2016-02-24 14:03

RECAP from mutagenecity lecture, extra stuff from kirk review

Question Answer
which sp do we use for tests of effects of OTA and why?PIGS-- bc most similar GI anatomy (omnivores like humans) and also most affect species that isnt human
how does OTA affect pigs?Pigs develop severe renal damage --> Porcine “Moldy” Nephropathy. Animals show ONLY PU/PD (have enlarged kidneys) (humans liver longer so you see a dramatic loss of kidney fxn and the kidneys SHRINK bc chronic damage.--> Balkan Endemic Nephropathy [BEN]. )
(kirk review) what are the two urinary tract tumors OTA causes(1) Renal Cell Carcinomas (A NO RETURN Dz – patiend WILL DIE) (2) Transitional cell carcinoma (bladder- can try to remove, better prog than the carcinoma)
(kirk) ALWAYS start with that test to determine mutagenecity?Always start w/ Salmonell Typhirium Ames Test
Basic elements of toxicity testing include: what 6 things?Acute toxicity (single dose: LD50), Repetitive dose studies (often different exposure routes: oral, dermal, ocular), 90-days rodent assay determines NOAEL (basis for ADI), ADI provides the basis for acceptable residue levels in food, MRL (VMP) in edible tissues of animals, ML: maximum permissible level: contaminants - all foods
what are the Additional (mandatory) tests?Reproduction toxicity and teratogenicity, Immunotoxicity and myelotoxicity (to be discussed later), Mutagenicity - genotoxicity and carcinogenicity (understanding the basic assays and the golf-stick dose-response curve versus a normal log-linear dose response curve)
the basic assays for mutagenicity are?AMES, micronucleus assay, COMET assay, DNA adduct assays ( 32 P post labelling) (he didnt talk about DNA methylation assays)
understand the golf-stick dose-response curve and how it's diff from the linear curverelatively safe portion at dose doesnt usually lead to dramatic inc in adverse effect, until reach certain conc and then HUGE effects. With these it is hard to predict whats gonna happen in the next 1 additional milligram (BMD= benchmark dose. BMDL= benchmark dose level. BMR= benchmark response) (so takes a while to see bench mark response, but once we see it, it goes up very steeply. BMDL is the corresponding lower limit of the 95% confidence interval. So if look at a group of animals, with 95% accuracy we can say that here probably (the BMDL line) potential response can be observed. Compounds with a golfstick dose response curve are very difficult to evaluate.
Understanding the difference between genotoxic and epigenetic mechanisms in carcinogenicitygenotoxic compounds directly damage the DNA (directly alkylate DNA), epigenetic has to do with the compound causing oxidative damage, and the body reacts in such a way that tumors form from cumulative effects (Epigenetic agents operate largely as promoters of cancer and usually require high and sustained exposures. For example, dietary fat can act as an enhancer of cancer induction at about 40% of calories. The effects of epigenetic agents, unlike genotoxins, are reversible. Accordingly, distinction between these two types of carcinogens is critical for informed risk assessment.)
***(kirk) so we use the AMES test to test the mutagenicity of OTA/etc. to simulate effect in the liver, you need at add WHAT which does WHAT?need S9 mix** Microsomal Fraction which is COMPETENT to carry out biotransformation reaction (phase 1 bioactivation) ..Derived from livers of rats stimulated w/an INDUCER of CYP450 system. Used as a metabolic system, resembling Microsomal Fraction of NORMAL Cell that carries CYP450. (~MICROSOMES ONLY DO PHASE 1 METABOLISM! ( phase 1= Oxidation, Reduction, Hydrolysis)
*(kirk) What is the difference between a microsome and an intact (liver) cell? what conclusions can you draw from this?Microsomes can only do Phase 1 metabolism (Oxidation, Reduction, Hydrolysis), Hepatocytes and Renal microsomes can do phase 1 and phase 2 metabolism, but renal has more phase 2 and prostaglandin H synthase. Therefore, there must be more than just CYP450 for a substance to become Toxic! (needs more than Phase 1!!!) Therefore, it can only ID w/ high certainty, compounds that have a Biotransformation-Dependent Toxicity that is EXCLUSIVELY predominantly mediated by CYP 450!
(Kirk) not explained/really gone over in werner's notes, but... what is the reporter gene assay "LacZ"?
(kirk) what is an affordable way to help with DNA oxidative damage from ROS/etc?Antioxidants. Vit E/ MILK THISTLE has SILYMARIN which can be given to cats and dogs as a therapeutic agent
***(kirk) explain golf stick response curveHappens with these genotoxic substances.. Shows low doses w/ no response then a sudden increase. There is no strict linear dose response curve for carcinogenic substances! Benchmark dose (where the bend occurs)= a dose that produces a predetermined change in response rate of an adverse effect (BMR) compared to background. Take Home Message: For a Carcinogenic compound, there is NO LINEAR RESPONSE CURVE!
OTA's primary target in kidneys is?prox tubule. (Karyomegaly, desquamation, focal degeneration, focal peri-tubular fibrosis)
who has the highest estimated dietary exposure to OTA?CHILDREN (might exceed PTWI)
2 types of tox that OTA causes?GENOTOXIC and EPIGENETIC (relating to or arising from nongenetic influences on gene expression) Toxicity