drraythe's version from 2016-04-29 15:11

Tests of things

Question Answer
Mandatory additional toxicological testing--> what are two additional things we have to test for?(All substances that is used intentionally in animals and in the food production chain must undergo this type of testing) Reproductive toxicology and teratogenicity must be tested for. We need to test for Effects of toxic substances of reproductive organs, Effects of toxic substances on the hormonal balance (endocrine disrupters), Effects of chemicals on the embryo/fetus (teratogenicity)
why do we use hamsters when we test example of reproductive toxicology?THEY HAVE NO BLOOD- TESTES BARRIER! This makes them a vulnerable species-- so if it's safe in THEM, then it's prolly def safe in us.
why is there a lag time between when testes are exposed to a toxin and when you see toxic effects of the testes?because of the Germ cell maturation (mitosis and meiosis)-- it takes time for spermatogenesis to occur and you wont be able to test the adult sperm's morphology and function (motility and integrity) until it has been exposed when developing and then fully matures to assess this
which gonad has EXPOSURE RESULTING IN DELAYED EFFECTS?testes!!! because spermatogenesis has a lag time. (diff from female where oocytes are already there)
how can you easily test the effect of a substance on oocytes?if want to look at specific repro tox in cattle, so to slaughterhouse, culture oocytes easily, then put compound on, and within 24--46hrs have your answer.
What animal do we use to test MALE repro tox on?HAMSTER
which animal do we use to test FEMALE repro tox on-- and why?RABBIT-- because they breed rapidly, so you can see the effect on litters without waiting for forever
what are the two things you can analyze female repro tox with?(1) Analysis of cumulus expansion (incomplete expansion leads to loss in viability) (2) Analysis of oocyte maturation (44hrs) (<--can get oocytes from a slaughter house and culture them)
if oocyte maturation is affected, what implications might this have?can have an effected oocyte function, and this will affect the second generation.---> see Small litters in multiparous species
Endocrine disruption and transgenerational effects--> explain how this might happen with a substance, and how it can have transgenerational effects tooOften substances will work as Agonists/antagonists of ER (estrogen/hormone) receptors regulating transcription. They can also act as Substrate for enzymes regulating the synthesis & inactivation of endogenous hormones (disrupting the endocrine system greatly). (Interferes w/ cytochromes producing steroidal hormones causing a hormonal imbalance in the individual.)
***explain age-dependant sensitivityYou might not see effects until there is a certain age reached. And example of this is like when a fetus is exposed intra-uterine, and then you don't see some effects of that until they hit puberty. Fetii are the most vulnurable age group because fetus has no defense mechanisms for it. *Ex. Zearalenone has transgenerational toxicity: mom ingest mycotoxin, show clinical effects in newborn piglets, and then when reach puberty, see all kinda of repro effects- often more in females than in males (In pigs ZEA exposure reduced the quantity of healthy follicles in F1 females. Daughters of the sow that was affected are genetically modified and carry mutations. Sons are not affected) So exposure of mom carries trans-generationally and you wont see full effects until piglet reaches puberty. (Ex. Human DES increases risk of cervical cancer at young ages in the daughters of women who took the drug. Mothers saw no effects, had beneficial effect from drug.)
*a drug must be ___ to get through the blood brain/placenta barrierlipophilic
what are examples of MINOR malformations of the fetus caused by teratogens? what makes them MINOR?generally mild= ***reversible. Might be Reduced birth weight, or physical retardation (evidenced by waved ribs)
what are Teratogenicity(or transgenerational tox) MAJOR irreversible malformations?(major= not reversible). Usually have noticeable physical malformations like cleft lips. (picture of messed up kittens from mom eating mycotoxins-- carnivores dont have the tools to break down plant stuff)
REPRO tox--> REGULATORY toxicology: what is this, why do we do it?This is a Important parameter. Might result in the recommendation: not to be used in pregnant females (humans and animals). High level of uncertainty due to species differences (in vitro - in vivo: toxicokinetics)
**why do we use RABBITS in repro tox testing?**same placental structure as humans, and a high repro capacity to make studies faster. Then you can pick a second species along with rabbit, but always need rabbit
REPRO tox--> CLINICAL toxicology: what is this? keep in mind with this?(see it clinically) High level of uncertainty due to species differences. Poor predictability (DISCLAIMER on VMPs). Even slight effects [are] of significant economic relevance in farm animals
Transgenerational toxicity of zearalenone--> explain thisusually interferes with steroid hormone synthesis and estrogen receptors, and with anatomy and production of occytes. So oavries smaller, dont produce similar quality transgenerational effects. Dams show some clincial signs-- smaller litters than usual. potentially leads to infertility. effects much more dramatic on offspring on dams though. always daughters affected with zearalenone-- sons have no problems.
Carcinogenicity, mutagenicity and genotoxicity testing--> In vitro screening: What kinda assays do we use? examples? What should you know about if we want to do in vivo?doing Mutagenicity assays. Examples include: AMES: Salmonella/ microsome assay. Micronucleus assay. COMET and SCE. DNA adduct ( 32 P post labelling). DNA methylation assays... what you need to know is that ****VMP & pesticides will not be licensed if they are positive on more than one assay. Many natural toxins are mutagenic and potential carcinogens --> so if positive in vitro, not even going to bother trying in vivo (will not try product) however, many natural toxins are positive in many many of these assays- so keep that in mind.
Carcinogenicity, mutagenicity and genotoxicity testing--> If in vitro assays are passed, what do you do to test the substance in vivo? Rodent assays!!!! 18 months exposure study in mice, >24 month exposure study in rats.
VMP & pesticides will not be licensed if they are positive on ...more than one in vitro assay. If post on in vitro, will never make it to be in vivo
what is a Golf-stick dose response? What are the implications of this type of graph?relatively 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.
what is a non-specific problem toxins can cause DNA? give example(discuss this more later) oxidative damage. Milk thistle: silymarin and other compounds in it can stop oxidative damage to the DNA. (antioxidants).
Biotransformation-dependent mutagenicity--> explain what this is, and how it affects what tests we do.sometimes its an activation of compound which makes the compound toxic-- if use parent compound on cells you dont see any tox, but if we use microsomes (which contain CYP450), in the cell assay, then the parent compound is metabolized which produces toxin and hence producing cell death. so have set of cells we genetically engineer to express CYP450 or use hepatocytes too, so to see toxic effects of metabolites. (So take NIH/3T3 transfected cells expressing human CYP450s--> expose to toxins--> Rescuing of the reporter gene: mutation analysis).
how might we be able to measure toxicity caused by free radicals? (ex of when free radicals are a prob)eukaryotic cells will allow for the measurement of oxygen free radicals, which have been found to be involved in the genotoxicity (DNAtox) of for example heavy metals
exposure to a genotoxicant results in DNA damage, which can be seen with what 3 diff methods?(1) Direct reporters (example: AMES) (2) Indirect reporters (ex: umu, chromotest). (3) direct visualization (ex: electrophoresis has comet tail)
DNA damage--> explain an example of a DIRECT REPORTER of genotoxicitya direct reporter assay is the Ames test. ((wiki says: The Bacterial Reverse Mutation Assay, also known as the Ames Assay, is used in laboratories to test for gene mutation. The technique uses many different bacterial strains in order to compare the different changes in the genetic material. The result of the test detects the majority of genotoxic carcinogens and genetic changes; the types of mutations detected are frame shifts and base substitutions. In the example given, they have bact that cant make his (are his-) and then they are exposed to genotoxicant-- they mutate, and then when you culture them on a his neg plate (meaning if they couldnt make it themselves they wouldnt grow) you suddenly see all these his+ colonies. ( growing bacteria on agar plates and comparing natural mutation rates to mutation rates of bacteria exposed to potentially mutagenic compounds or samples)
DNA damage--> explain an example of an INDIRECT REPORTER of genotoxicitythis is a test like umu/SOS. ((wiki says: The SOS chromotest is a biological assay to assess the genotoxic potential of chemical compounds. The test is a colorimetric assay which measures the expression of genes induced by genotoxic agents in Escherichia coli, by means of a fusion with the structural gene for β-galactosidase. The test is performed over a few hours in columns of a 96-well microplate with increasing concentrations of test samples. This test was developed as a practical complement or alternative to the traditional Ames test assay for genotoxicity. The SOS response plays a central role in the response of E. coli to genotoxic compounds because it responds to a wide array of chemical agents. Triggering of this system can and has been used as an early sign of DNA damage.
DNA damage--> explain an example(s) of DIRECT VISUALIZATION of DNA damage from genotoxic substancesex: comet, chromosomal aberration
how does biotransformation affect a compound?can ACTIVATE OR INACTIVATE compound
Analysis of liver biopsies--> what do target microarrays do?(he skipped this) identifying toxic pathways


Question Answer
Interpretation of LD50 datathis is ACUTE tox. Dose to kill 50% of them. Done is EXPERIMENTAL (not clincial) tox and we can get indirect info from comparing LD50 info across diff gender, species, etc. (LD50s related to Quantal dose-response relationships)
Which cells types are used for acute toxicity tests and what is the endpoint?In vitro we use cell types not animals to test acute tox. The end point is death of the cell (cytotoxicity). we look at Fibroblast type cells, HepG2 cells (liver derived), Neuro 2A cells (neuroblastoma), Caco cells (colon carcinoma)
Describe the toxicity tests for acute, subacute and chronic toxicityACUTE: LD50-- use animals, see how much till they die. ---or---- TC50- this is in vivo, we look at cells and see what CONC. of substance causes CYTOTOXICITY. SUBACUTE This is repetitive-dose. Think 90 day rodent assay. CHRONIC i think is things like Carcinogenicity, mutagenicity and gentoxicity testing (long rodent assays)
Give the definition of NOAEL and ADI and how is the ADI calculated?NOAEL is the tested dose without any effect (no-observable (adverse) effect level) (determined from 90day rodent assays aka subacute testing). Once you know the NOAEL, you use that along with the avg body weight of a human, and the uncertainty factor (going between species and variation in humans) to get the ADI (acceptable daily intake)
Reproductive toxicity is primarily measured in which species?male gonads: hamsters. Female gonads: rabbits. repro tox in general: rabbits bc placenta like ours and also fast repro turnover
L50 represents which kind of tox?acute
Repetitive dose studies--> some of the variation in this is?diff exposure routes and their diff tox effects
what are the "additional" toxic tests?Reproductive toxicology and teratogenicity, Endocrine disruption and transgenerational effects, Reproductive toxicity, Carcinogenicity, mutagenicity and genotoxicity testing