Week 2 – Brine Shrimp Viability Studies

Welcome to BME
209, 219, week two of the nanoparticle
toxicity lab. Last week you made gold
and silver nanoparticles. You also made gold
and silver solutions to try to get a sense of whether
gold or silver are toxic, either in their ion form or in
their nano-particulate form. This week you’ll actually
be testing the toxicity of gold, silver nanoparticles
and gold and silver solutions in a whole animal
assay of toxicity. So let me give you a quick
introduction to toxicity. Broadly defined, toxicity is
the degree to which a certain substance– this could be a
material, a small molecule, a drug, pretty much anything– can harm humans or animals. We generally break toxicity
down into two parts. The first is acute
toxicity, and that involves harmful
effects in an organism through short term exposure. So if you are exposed
to a substance for a short period
of time, do you start showing signs of toxicity? The other form of toxicity
that we can talk about is chronic toxicity. And that is when you are exposed
to a substance for a really long or an extended
period of time, often either through
repeated bouts of exposure or through continuous
exposure, say in the air. Sometimes the exposure
lasts for your entire life. And the question becomes,
was this continuous exposure causing some sort of
toxicity that we might not see if we’re only looking
at acute toxicity? Determining possible
toxicity of substance is absolutely crucial for
any biomedical application. But not just
biomedical applications such as implants and drugs. Determining toxicity is
really important for having clean water and for figuring out
what clean air actually means, and how to sustain clean air. It’s important if you’re
making cooking vessels or any vessels that touch food
or water that go into you. It’s also important in
industrial processes that might create waste products
that could get into the water or might get into the air. But measuring toxicity
is not trivial at all. There are many different
ways to measure toxicity, and all of them have their
positives and negatives. So at the very,
very simplest we can start with what’s called
in vitro cell studies. And that’s when we grow
mammalian cells in a flask. We can add substances
to these flasks or to wells that
have cells in them. And then we can look at
toxicity for the cells. And the toxicity for the cell
may mean that the cell dies. So cell death could be
a measure of toxicity. But we could also see changes
in the metabolism of that cell. We might see changes in
the shape of that cell, or even changes in
the salt content or the iron content of the cell. There are many ways to
evaluate toxicity in a cell. There are a lot of pros
to this method, which is why it’s usually the
first one that gets tried. It is easy and it is
cheap to do these studies. And you get direct information
about how these substances affect mammalian cells. There’s also virtually
no ethical issues with doing these
kinds of studies. However, single cells
are not an organ. And they’re not
representative of what happens when an entire organism
takes up a certain substance. While these substances
are inside a person, they may change
form, change shape. There might be
chemical reactions that take place that make these
substances much more toxic. Or whole organisms
might effectively clear a certain substance,
making a substance that looks toxic not toxic. So we can look at whole animals. We can take animals– whether these are mice,
rats, Guinea pigs, or pigs, or cows, or dogs, or
cats, or any other animal– give them substances,
usually the same way that a human would take it in. And then look for
signs of toxicity. One sign of toxicity
might be weight loss. Are they losing weight
compared to a control group? Are they losing their hair? Do they look fatigued
or less energetic? Are they looking dehydrated? Or we can actually do
necropsies on these animals and look at whether their
organs have regenerated or whether their organs look
different under a microscope. These studies are
relatively inexpensive and relatively fast. Now, the more
relevant an organism like a pig, the more expensive
and slower the slide it gets. But the major pro of this system
is you get direct information about how a substance
affects an entire organism. But there are some
major cons to doing these kinds of animal studies. First of all, there are
ethical considerations to consider when you’re
testing on animals. Different people have
different opinions on what is acceptable and not
acceptable to test on animals. Moreover, animals
are not humans. And there are numerous
examples of substances, like chocolate, that are toxic
to other animals but not toxic to humans. And substances like poison
ivy that are toxic to humans but not toxic to other animals. Finally, oftentimes it’s hard
to understand how a toxic effect will scale with size. Mice are not just
smaller than us. They also have very
different metabolisms. So the much faster
metabolism of a mouse might metabolize a substance
much faster than us. How would that influence
the toxicity of a substance? It’s really hard
to predict, which is why in most cases, once we
show that a substance is not toxic to animals we actually
do a controlled clinical study of these substances in humans. These trials are
called phase one. And we actually give
a substance to a human and look for signs of toxicity. These signs of toxicity might
be something like weight loss, or with a human,
you can actually ask them directly are
you feeling different? Are you experiencing
any side effects? The pros is these are
really good predictors of acute toxicity. And these humans are
monitored very closely for any sign of
toxicity in them. But there are some cons. These trials are
incredibly expensive and there are some major
ethical considerations. By and large, the people
that do these trials do them because they’re getting paid. So you’re essentially taking
advantage of the impoverished in order to learn whether a
substance is toxic or not. Finally, most of
these studies are done to evaluate acute toxicity. There’s virtually no
controlled human trials for chronic toxicity. They simply take too long and
are simply far too expensive. The last way of
measuring toxicity is to do environmental studies. You can actually look at
a human population that’s exposed to a substance
and try to compare them to a similar population that’s
an unexposed control group. So maybe one town has
substance X in their water, whereas the next town
over doesn’t have substance X in their water. And you can look at
whether the first town has a higher incidence of a
certain disease like cancer, or other unexplained
deaths or illnesses. The major benefits to this
are that they’re directly relevant to human health. And they can expose
chronic toxicity. Many of the pivotal
studies that showed the link between
smoking and lung cancer were these kinds of studies. Unfortunately, there
are also some cons. It’s often hard to define
a control population. You don’t know whether the
control population is exactly the same except
for that substance or has other differences. A major example is
if you wanted to test whether wearing sunscreen
reduced the chance of skin cancer, if you just look at
people who don’t wear sunscreen they might not get skin
cancer because those are the people that don’t
go outside and just stay in their basements. And you might get
a false result. So there’s lots of environmental
variables to consider. And these studies
get very complicated to identify those kinds
of extraneous variables. So every toxicity study is
accompanied by limitations. That’s the bottom line. The limitations might be
in the scope of the study. It might be in the time period
over which the study looks. It might be in the
cost of the study. It might be in its
relevance to human health. The best we can do is weighs
the pros and cons of each study and make rational conclusions
from the entire body of work. Oftentimes it takes numerous,
numerous studies on many levels to convince policymakers that
a substance is actually toxic. So in this lab we’ll be
looking at brine shrimp. And let me tell you why. Brine shrimp are a great
way to quickly test the toxicity of substances
on a whole organism. They’re small,
relatively inexpensive, the toxins can be incubated
with the shrimp for hours or even weeks if you want to
try to look at chronic toxicity. And the shrimp move
in response to light, so you have an easy read out. Moving shrimp are
presumed to be alive, shrimp that are not moving are
assumed to be sick or dead. Let me show you an example
of what your data should look like in the end. This is a single well
of a 24 well plate. In it I have some
live shrimp but I also have some shrimp
eggs, which here are shown with a red arrow. But they are circular in shape. We can play this video. And what you can see
is that the shrimp are moving around this well. The eggs and any dead
shrimp that might be in here are not moving. You will be taking
a video like this of every single
one of your wells on day one of your
lab experiment. And then your TAs will
be adding to this a video of the same wells on day three. So two days after you
first set this up. I have a couple of
pieces of advice for you. First, make sure
that your camera is focused on the shrimp. Sometimes there might be a speck
or something in the background. And your phone is going to
auto focus on that speck. So take a look at
it and make sure that it’s actually
focusing on the shrimp so you can count and watch
the shrimp move around. Second, when you add
shrimp to the plate try to avoid shrimp eggs. As you can see here, it is
possible to distinguish shrimp from shrimp eggs. But they’re just an
extraneous factor there that might make it
more difficult to count the shrimp later on. 20 to 30 shrimp per well
is a pretty good number. If you get below 10,
you start to have issues with sample sizing. If you get above 40,
you start to have issues with counting all the
shrimp because they’re just moving around and
there’s too many of them. So shoot for about 20
to 30 shrimp per well. As you take these videos,
try different lighting or background conditions. Make sure you can really see
the shrimp move around so that later on you can
go frame by frame and count the shrimp
in your video. I recommend taking the
video for at least five to 10 seconds on each
well before moving on to the next well. This way you have a nice
continuous period of time over which you can actually
analyze how many living shrimp you have in your sample. Finally, when you
move to the next well, I recommend you keep
the phone where it is and move the plate under it. This will allow you to keep
the positioning, the background lighting the same as you move
from one sample to the next so that you can more easily
take good videos of your shrimp. I wish you luck in
figuring out the toxicity of these gold
nanoparticles on shrimp, and silver particles as well.

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