Toxicity and Ames L20 Flashcards
what do different chemicals do
affect microbes in different ways and a critical threshold is required to achieve killing
what happens when microbes are exposed to sub-lethal concentrations
microbes can survive
cells can repair damage (NOT VIRUSES)
what does repeated exposure to sub-lethal concentrations of chemicals do
chemicals, including antibiotics can lead to resistance due to mutations
what can occur in large populations to do with mutations
even rare mutations will occur
what is toxicity
relative phenomenon
what does toxicity depend on
1) innate toxicity of a substance
2) dose administered (dose being exposed to, drugs or can think about environmental exposure)
3) the biology of the test organism
4) the chemical properties of a substance (if hydrophilic likely to diffuse through environment and get to the target)
what is toxicity like
non-linear dose-response relationship
what is maximal response in dose-response curves
may not be 100%, may never see 100% response, but will reach a threshold – no matter how much more you add
what is the dose-response curve
% response could be death, treatment etc
As response goes up is fairly linear till it tails off and then reaches a maximal response
what is NOAEL
No Observable Adverse Effect
what causes NOAEL
threshold
what is the NOAEL curve
no effect at start, then it starts to increase, then is linear then tails off again
what does the NOAEL tell us
how much is it safe to be exposed to before our body’s cannot tolerate it anymore, there will be a response
what is NOAEL
everything up to the point where there is a response
what response do we want biocides have
maximal response
when is a maximal level reached
when no further Adverse Effect is seen
what is needed to determine 50% effect
end point reached, graph can be used to determine point at which 50% effect is seen
what is LD50
50% of population is killed (Lethal Dose) (all dead)
LD50 the maximal response will be 100%, 50% of the population will be killed
what is TD50
50% of population shown unwanted (Toxic) effect (may have some surviours)
what is ED50
50% of population show desired effect (Effective)
what is maximal response set at
maximal response is set at 100%, but may not be all the population, is just where it plateaus
what is important to think about when giving dosages
Need to think about how much to give regards to the volume (body mass)
what are the phases in the toxic compound pathway
absorption
distribution
metabolism
excretion
what does absorption involve
passage across cell membranes whatever the route of entry into the organism
what are lipid membranes like
selectively permeable
what does passage of the chemical through the membrane depend on
size
lipid solubility
similarity to natural molecules (analogues)
polarity/charge
what are the ways foreign substances pass through a membrane
filtration through pores (size) (porins in bacteria)
passive diffusion through (lipid solubility)
active transport
facilitated diffusion
what uses facilitated diffusion
movement of toxin depends on charge/polarity – exchange transport where getting change in charge or polarity looking at facilitated diffusion
what process happens in multicellular organisms
phago/pinocytosis - engulfment of membrane vesicles and things taken inside cells, does usually happen in microbial populations, but is important in higher organisms
what is phago/pincyotosis independent of
independent of the chemistry e.g. size and charge is just whether or not you are taken up
what processes require receptors and are the basis of some biological variation
active transport
facilitated diffusion
what may NOAEL be affected by
efficiency of:
- efflux pumps
- detoxification pathways
- excretion pathways
number of receptor sites (binding saturation)
varies depending on different types of cells
which cells will absorb more molecules
cells with more receptor binding sites will take up more – difference in accumulation rates, difference in how much you tolerate before see an effect due to the variation of receptors
what happens when the antibiotic e.g. streptomycin introduced into cell
if strep in cell quickly get resistance in population sue to single point mutation in a protein that makes up small subunit of ribosome - target site for strep to bind to
mutated strep can’t affect cell
what is a downside of streptomycin in the cell
ribosome change due to preventing strep binding to target cell is that it is slightly worse at making protein
what happens to the antibiotic resistant mutants when no antibiotic is present
mutants that are antibiotic resistant in the cell without antibiotic present will likely be outcompeted
what happens to the antibiotic resistant mutants when the antibiotic is present
they survive and grow, population become antibiotic resistant
what happens to microbial cells when microban/triclosan are added to plastic
leach out and contact with microbial cells
bacteria use an efflux pump it already had, up regulates it which leads to faster pumping of triclosan so cells become resistant
why may microbe be resistant to molecules
molecule can go in at a standard rate, but have ability to pump it out, if have more pumps, can pump out faster
what does the cell need if accumulate molecule
enzymes to detoxify it
what effects the metabolising effect
how many enzymes you have
which bacteria become resistant in metabolism
bacteria can gain enzyme that detoxifies an antibiotic
selected in population, cells with enzyme become resistant population
how can excretion occur
urinary system
lungs
biliary system
what is ADI
acceptable daily intake
what is ADI based on
NOAEL
need to understand what the safe threshold is
what is ADI used to determine
safe intake of food additives and contaminants
what is the safety factor
built in depending on the severity of consequences of overexposure
how is ADI calculated
NOAEL mgkg^-1day^-1 / 100*
* = safety factor
what is dose
total amount administered
what is dosage
includes reference to biological variation
e.g. body weight
what is MEL
maximum exposure limit
how are non-ingested compounds regulated
maximum exposure limit over 8h working day (to do with skin or inhaling)
what is used as test organisms
animals, commonly mice, rats, rabbits aquatic organisms invertebrates bacteria cell culture systems
what test is used to determine if a chemical is a mutagen
Ames Test
what is the Ames test based on
assumption any substance that is mutagenic may also turn out to be a carcinogen
what is a mutagen
agent capable of introducing mutations into DNA
what is a carcinogen
agent resulting in the formation of cancer cells
are all mutagens carcinogens
not all mutagens are carcinogens
what bacterium is used in the Ames test
a strain of Salmonella enterica
what is a phototroph
organism is able to grow on a medium lacking amino acid histidine (His)
what is an auxotroph
strains carrying point mutations in a single gene in (His) biosynthesis pathway isolated
strains require His in medium for growth as can’t make it
what are the point mutations in the different Ames strains
deletion
insertion
transition
transversion
what is a back mutation
mutation reversed and regains its function
returns to be ableto grow
what is prototrophy
be able to grow
what happens in the Ames test
bacteria on petri dish test chemical disk in middle
only things that should grow is mutation
mutant can make own histidine and grow into colonies
what is the filter paper soaked in
with known carcinogen (2-aminofluorine)
what causes back mutations
mutagenic effect of the chemical
what is the little clearing on the petri dish around the chemical disk in Ames test
DNA mutation that has occurred reversed mutation that we started with, but hasn’t caused lethal damage anywhere else in the cell
likely everything died as knocked out something essential as well
what do colonies present in Ames test mean
first indication this is causing DNA damage
what is usual to see in an Ames test petri dish
clear ring round chemical disk then a ring of mutations – more mutagenic more there will be
colonies outside ring area likely to be spontaneous mutations
when can non mutagenic chemicals be converted to mutagens
converted into mutagens (and carcinogens) as they are metabolized
what is the Ames test used for
first screen for possible carcinogenicity
why is the Ames test good
simple
low cost
what has been adapted since the Ames test
rapid in vitro tests have been adapted for some eukaryotic cells such as:
Strains of Yeast
Mouse or Human cells grown in culture
what is a negative Ames test
only a few spontaneous revertants
