Lecture 5f - Ames test

Hi. I'm Dr. Lesley Blankenship-Williams, and in this microbiology lecture we are going to cover

the principles of the Ames test. The Ames test is so named after the person who developed it,

Bruce Ames, in the late 1960s and the early 1970s, and his test "aimed,"- haha, that's a pun -aimed

to test this question: "If I have some sort of chemical or physical agent, is that chemical or

physical agent a mutagen?" And I've kind of reformatted that question to just look at a

chemical. So if I have some sort of chemical, is this chemical mutagenic? Now why would I be

interested in that question? Well it turns out that that is not exactly the question I really

want to know. From a human health perspective, the question I really want to know is this one:

is chemical X a carcinogen? But it turns out those questions have a lot of overlap in their answer.

So what do I mean by that? Most mutagens are also carcinogenic, and almost all carcinogenics

are mutagens. So let's quickly define each term. A mutagen is a substance that increases the rate of

mutations. It causes induced mutations, often by fooling DNA polymerase when it goes to replicate

the DNA. A carcinogen is a substance that increases the likelihood of cancer developing,

usually a specific type of cancer. Now how are they related? Cancer develops when a cell stops

listening to the body and starts replicating out of control. Normally the body kind of shuts down

the cells and says "Don't, stop replicating, I don't want you to replicate." And it says

"I'm not listening, I don't see that, I'm not following your directions, I'm just gonna go rogue

and start replicating beyond the body's control." And that creates a mass known as a tumor,

and then if the tumor dissipates throughout the body that would be an example of stage four

cancer, which is very very difficult to manage. So prevention is the best cure. To prevent cancer,

we should minimize our human exposure to carcinogens, substances that would increase

the likelihood of cancer. Now how are carcinogens and mutagens related? Well we know that mutagens

increase the risk of mutations in DNA. A cell also incurs mutations through replication,

and if those mutations land in a gene that codes for the cell listening to the body's instructions,

and the outcome is a non-functional receiver protein that says "I'm not listening anymore,"

human cells make certain proteins that are listening in to the body's signals and if

they become mutated into something that doesn't work, then they no longer hear the body's signals

and they might continue to replicate. So in other words, cancerous cells are ones that have incurred

quite a few mutations, and so it would make sense that something that is mutagenic and, therefore,

increases the mutation rate would also increase your risk of cancer. Now as I mentioned they're

not a perfect overlap, but there's a pretty strong overlap. So just as an example of some, kind of,

fringe chemicals here, caffeine is an example of a mutagen that doesn't tend to be carcinogenic.

And there are chemicals out there that are carcinogenic that don't seem to be mutagenic,

and benzene is one of them. Why there are some discrepancies there is really beyond the scope

of this lecture, although it's- it's really fascinating, but, again, for our purposes,

most mutagens are also carcinogens, but not all of them. So where are we going with this? This

is a microbiology class and I'm sitting here talking about human cancer. Well it turns out

that in order to answer the question about whether or not something is carcinogenic,

we should probably first look to see if something is mutagenic, because almost all carcinogens

happen to be mutagenic. And I'll give you an example of a substance that is both mutagenic

and carcinogenic, and it was the substance that you saw in the first slide, benzo(a) pyrene.

Benzo(a) pyrene is a pretty large fibrin structure. You can see benzenes are in there,

everywhere. And when it gets inside of our body, we have enzymes that change it or metabolize it.

And we change it into a substance that has some additional hydroxyl groups on it,

and an ester group, and then when that modified benzo(a) pyrene, which we've modified- so we've

actually modified -it gets access to our DNA in our nucleus, it tends to bind to guanine.

And when that happens, it actually creates a covalent bond with guanine. So this is guanine,

here, and this is this new modified version of benzo(a) pyrene that's now covalently bound to it.

And what that does is that actually blocks guanine, so it can no longer pair with cytosine.

So when DNA polymerase gets ready to replicate this area, it just sees this big mess right there

and has no idea what to do with it. And so- hence mutations arise. The more mutations show up, the

more likely cancer is going to show up. So let's get back to 'How is this related to microbiology?"

Because so many carcinogens are also mutagens, let's take a look at how easy it is to answer

each question. It turns out that testing for mutagenic properties can be done with bacteria,

which are cheap and expendable. Testing for carcinogenic properties requires animal testing,

which both has ethical concerns and, of course, is financially expensive.

So every time you get a new product out onto the market, you don't necessarily want to go right

into the animal testing. Let's first determine if it's mutagenic. That's cheap and easy.

And then if it is mutagenic, you better do your animal testing to see if it's also carcinogenic

before exposing humans to this chemical. So now that I've covered that, let's go ahead and talk

about the question of "Is substance X a mutagen?" Which is what the Ames test is designed to do.

So the basic overview of how the Ames test works is a little bit like pin the tail on the donkey,

but instead of pinning the tail on the donkey, we're gonna pin the mutation on the chromosome

somewhere. In other words, we're going to expose bacteria, with their chromosome,

to something that we think might be mutagenic. The more mutagenic it is, the more likely mutations

are going to show up somewhere in this chromosome. You don't know where. It could be there,

it could be there, it could be there. We just don't know. It's random. So instead of pin the

tail somewhere on the donkey, you're pinning the mutation somewhere on the chromosome,

but I do know this: the more mutagenic a substance is,

the more mutations are going to show up. So that's the basic principle. And then

we have a way to test for the frequency of those mutations. So let's take a look at how this works.

To perform the Ames test, we are going to need a culture of salmonella bacteria.

This particular culture has been genetically engineered in a laboratory,

and is designated HIS negative. In addition, we'll need some special media

and, of course, our mutagen. So what exactly does HIS negative mean? Well HIS stands for histidine,

and histidine is one of the 20 amino acids. So all 20 amino acids are required

for bacteria to live. If they don't get all of their amino acids, they won't be able to make the

proteins that they need to make and they will die. So for bacteria, histidine is required for life.

So where do bacteria get this histidine that they require to live? Well there's essentially

two options. One option is that they get it from their food. If we grow them in media, that means

histidine is required to be present in the media in order for them to acquire it in their food.

Alternatively we might provide them with a precursor to histidine and, if they have a special

enzyme that allows them to convert that precursor into histidine, then, technically, they can make

their own histidine from the precursor. But it's important to understand that for option two,

they must have an enzyme that allows them to synthesize this histidine. Without a functional

copy of this particular enzyme, these bacteria would be unable to synthesize their own histidine.

HIS salmonella come equipped with a gene on their chromosome that codes for that particular enzyme

that allows them to make their own histidine. So the very fact that they have a gene encoded HIS

means that these bacteria should be able to synthesize their own histidine,

and therefore do not require it in their food,

but the caveat is that the histidine gene must produce a functional enzyme.

Assume that this is the original HIS gene. This is referred to as the wild type.

If it is transcribed and translated, it should produce a functional enzyme that is capable of

converting the precursor into histidine. In other words, bacteria that possess this HIS

gene do not require histidine in their food. Rather, they can make their own as long as the

precursor is provided. We refer to these bacteria as HIS positive. So we put a superscript positive

up there to denote the fact that this gene works and produces a functional enzyme

that allows these bacteria to make their own histidine. In other words, HIS positive salmonella

should be able to make their own histidine, and would not require it in the media.

But what happens if this gene incurs a nonsense mutation. Remember that a nonsense mutation

is actually a point mutation, and I've drawn that here. I've taken one of the letters that

was a T and I've changed it into a G. And so, in this case, the G, let's assume, it's a nonsense

mutation. Such that, when it is transcribed and translated, it does not produce a functional

enzyme, and therefore these bacteria that have this mutated version of the HIS gene

cannot make their own histidine. Since they cannot make their own histidine, they would die

if it was not provided in the food. We call these bacteria HIS negative. So HIS negative salmonella

refers to salmonella bacteria that, once upon a time, had a functional working histidine gene,

but it has incurred a mutation such that the gene no longer works. It's still there,

it's just been mutated so that the product doesn't work. Now that we understand a little

bit more about what HIS negative refers to, let's return to our experimental design.

Imagine that I have this culture of salmonella that is HIS negative,

and I have media that contains histidine. So there is histidine embedded in the media.

If I plate this bacteria on this media, they should grow. In other words, the bacteria have no

reason to not live since the histidine that they so desperately need, but can't create, is provided

for them in the media. So if I plated this culture, I would expect to see a lot of colonies.

But what would happen if I used a different type of media? Let's say that I use synthetic media,

and my synthetic media was built in a way that no histidine was added to the media.

So this media has no histidine. Now I'm going to plate my bacteria. What's going to happen?

Well since these bacteria don't have histidine in their media, and they can't make their

own histidine, they're going to die. So 48 hours later, I would expect zero colonies.

But wait, I might get one colony or maybe even two colonies that show up. Are they contaminants?

Well probably not, if I used aseptic technique. Instead, these single colonies, maybe one or two,

are the result of what is known as a back mutation. What's a back mutation? It's a

mutation back to the original form, and these do happen. In this case, we might have had billions

of cells in our test tube, and one lucky cell hit the jackpot and experienced a spontaneous mutation

back to its original HIS positive form. Because we have one cell that is HIS positive, it is able to

synthesize its own histidine and therefore survive on this plate. And that represents the one or two

colonies that we can sometimes see. Again, these are spontaneous back mutations.

Let's take another look in our case. A back mutation would mean going from the mutated G base

back to the original T base, and the outcome of that back mutation means that the mutated,

or back mutated, salmonella cell has become HIS positive again. In other words, it is

able to now synthesize its own histidine, and therefore grow on a plate that lacks histidine.

So it turns out this is what we're actually going to test for. Well these back mutations can happen

spontaneously. Under the presence of a mutagen, we expect them to happen a lot more frequently.

We now have all the information we need to understand the experiment. So in the Ames test,

we always run an experiment and a control in the experimental procedure. We add our suspected

mutagen to a culture of HIS negative salmonella species. In our control, we add water. Water is

not a mutagen, so we let the salmonella cultures mix with the suspected mutagen X or the control,

and then we add them to a plate that lacks histidine. So there is no histidine in the media.

So what are the expected results? Why don't you try to predict the expected results,

based on what we've learned so far. Pause the video and see if you can guess.

All right, let's take a look. The control should have zero, or maybe one or two colonies on it.

Remember, the control just had water, so any colonies that do show up, again

assuming good technique without contamination, are going to be the result of spontaneous mutations.

So these would still be back mutations. We would still go from HIS negative to HIS positive,

but they are the result of spontaneous mutations. The reason why we run a control is to try to get

an assessment of how many spontaneous mutations are likely in a given culture.

Now let's look at our experimental plates where the suspected mutagen

X was added to the salmonella cultures. There are really two possible outcomes.

In the bottom outcome, which is highlighted in green, there are only two colonies. This

is a very similar result to our control. It is so similar that we basically assume

that those two colonies are probably also the result of spontaneous mutations. So that means

that substance X is not classified as a mutagen, where substance X was not mutagenic in this case.

But what if we see a lot more colonies on our experimental plate, as shown with the

purple highlighted option? If that's the case, then substance X is probably mutagenic. Here

we see that there are many more colonies, which means many more back mutations from HIS negative

back to HIS positive, compared to the control. And the only way that's possible is if there

were a lot more mutations. Well what would cause a lot more mutations?

A mutagen. So therefore substance X must have induced more mutations,

and therefore substance X is a mutagen. So we can compare the results of our experimental plate

where substance X was added to our control to determine if substance X is a mutagen or not.

Here is a brief look at the same type of description

from an image in your textbook. Here the top row is showing the experimental sample.

The bottom row shows the control. In both cases we have cultures of salmonella that are HIS negative.

The book refers to them as histidine dependent, which is effectively saying they require histidine

in their food in order to survive. Notice that the top tube has the suspected mutagen added

and the bottom one does not. You might also notice that rat liver seems to be added.

What's that all about? Well that kind of is beyond the scope of what we want to get into,

but it is added because the liver has enzymes that might modify the mutagen, like a human's body

might modify the mutagen. It's effectively an addition to make this test more applicable to

something that might be carcinogenic. For our purposes, you do not need to know the purpose of

rat liver extract or even that rat liver extract is used, so you can ignore that. So once you add

your mutagen to your salmonella, and then you've got your control, then you're going to go ahead

and plate them. Notice that when you add them to the plates, the plates do not have histidine.

So unless they can make their own histidine, those bacteria are going to die.

And you see in the results that the control still has two colonies. That means there were

two successful back mutations from HIS negative to HIS positive, And those are the result of

spontaneous mutations. Whereas the experimental plate shows many colonies. The fact that there

are many colonies tells us that additional back mutations occurred in the experimental sample,

and the only way that could have happened is if there were additional mutations,

induced mutations. So that tells us that, in this case, the suspected mutagen is in fact a mutagen.

Now one final comment before I leave you,

just because something is a mutagen does not also mean it is a carcinogen. It's likely,

but not a hundred percent for sure. So once you determine that a substance is a mutagen,

you will need to do additional tests to determine if it is also a carcinogen.

And that concludes this lecture on the Ames test. Thank you so much.