The AMES TEST aims to test whether avoiding a certain chemical’s best 🧐 Is it a MUTAGEN? What does that mean? What can (& CAN’T) you learn from this REVERTANT SCREEN? 🤷‍♀️ Yesterday we saw how scientists used chemical mutagens to show that the genetic code isn’t overlapping. But what is a mutagen? And how’s it different from a carcinogen?

Before we start, remember 👉 EVERYTHING is made up of chemicals 👉 even water is a chemical 💦 So don’t be scared off by that term (& don’t let companies encourage that fear to make a profit 😡). Ok. Now let’s 👀 at why *some* chemicals may be harmful & how we can check 👇

Cancer can occur when your cells’ regulatory mechanisms get disrupted and they start growing & dividing out of control.

The instructions for those regulatory mechanisms are written in the DNA it’s controlling 👉 talk about self-policing!👮‍♀️ So mutations in the DNA instructions for the regulators, if unfixed, can lead to cancer.

Things that can cause cancer are called CARCINOGENS. Things that can cause DNA mutations are called MUTAGENS. Since some of those mutations can cause cancer, mutagens are often also carcinogens & mutagenicity is easier to test for. The AMES TEST is a way to test if a chemical is a *mutagen* – the test is carried out in bacteria, so it doesn’t test if something is a carcinogen because bacteria can’t get cancer – cancer is the uncontrolled growth of abnormal cells within an organism and bacteria are single-celled so there aren’t cells “within them” to grow out of control.

How do we know whether bacteria are acquiring mutations? Well, bacteria are *always* acquiring mutations – randomly – and so is everything including you. Mutations are how evolution proceeds. Mutations happen randomly and, if they provide a growth advantage (or at least don’t provide a disadvantage) they’ll stick around and get passed on (in multi-cellular organisms this is only true if the mutations occur in the germ line).

Mutagens increase the rate at which mutations occur and we can check for an increase in this rate by comparing the mutation rates of bacteria treated with a compound or to those not treated. But how to easily measure these rates?

We test for the ability of bacteria to acquire a new mutation that “cancels out” or REVERTS an original mutation that made them unable to synthesize HISTIDINE. Histidine (His) is an amino acid – one of those protein building blocks. And it’s ESSENTIAL for His- bacteria meaning that it’s essential that their food has it because the bacteria can’t make it themselves. (non-essential amino acids are still important, but an organism can make their own if the food doesn’t have it).

So, with regards to His, the so-called his- mutants are AUXOTROPHS (require auxiliary supplements) as opposed to the normal (wild-type) AUTOTROPHS which are his+ and can make their own.

We take 4 Petri dishes containing bacteria food with BARELY ANY histidine. Just enough for it to live long enough to mutate. And we add some His- bacteria. Since they need His, and they can’t make it, they can’t grow UNLESS they acquire a mutation that allows them to make it or somehow get by without it. If they acquire such a “back mutation”, they will be able to grow, and they’ll show up as “spots” of cells on the surface of the plates we call colonies.

We treat 2 of the plates with a chemical we want to see if is mutagenic. We treat 1 of those (and one of the no-chemical-added ones) with rat liver enzymes – what? why do we still need rats? one of the ways our bodies protect us from toxins is the liver. The liver metabolizes chemicals, often neutralizing them, but it’s not equipped to handle everything so sometimes the liver “gets it wrong” and the products of the metabolism (metabolites) are more harmful than the original substance. When this occurs we call the original substance PRO-MUTAGENIC. It’s not mutagenic by itself, but it can become mutagenic when processed in our bodies.

Bacteria don’t have livers, so, by themselves, they don’t allow us to test for this pro-mutagenicity. This is where the rats (at least their livers) come in. We add enzymes from their liver (similar to the enzymes in our liver) to the bacteria. This allows us to test whether our liver is likely to make the chemical safer or more mutagenic (or have no effect)

So, 4 plates:

1️⃣ just the bacteria 👉 this allows us to determine the background rate of mutation – remember, mutations happen naturally too! so we subtract the # of colonies we get on these plates from the other plates when we compare

2️⃣ bacteria + chemical 👉 this allows us to see whether the chemical is mutagenic (you’d see MORE colonies) or maybe even protective (you’d see fewer colonies)

3️⃣ bacteria + rat liver enzymes 👉 another control plate, this one for plate 2 that lets us check whether the liver enzymes themselves are affecting the background mutation rate in plate 4👇

4️⃣ bacteria + chemical + rat liver enzymes 👉 allows us to see whether a chemical is pro-mutagenic (you’d see MORE colonies than plate 2) or if the liver neutralizes the mutagenicity (you’d see FEWER colonies than plate 2)

Even if you identify something as mutagenic or pro-mutagenic, that does not necessarily mean it’s carcinogenic 👉 that requires more testing & evidence showing how it acts in a body. Also, not all carcinogens give a positive Ames test. But it’s an important, useful test that’s been around since 1973, when its namesake ,UC Berkeley scientist Bruce N. Ames, introduced it.

Since then there have been modifications 👉 you can use bacterial strains with different types of mutations that need to get reverted in case the mutagens cause different types of mutations (like swapping a letter back versus adding in one that got removed). And you usually use bacteria who’ve been made vulnerable to the chemicals due to a defect in their cell wall and mutations in their DNA repair machinery so they can’t fix the “mistakes” that we’re looking for.

Speaking of what we’re “looking for” – lots more changes are actually occurring that we’re not detecting. In fact, there are probably lots of mutations occurring that are just killing off the cells they occur in. But we’re not looking for them, so we don’t see them. But that’s ok because the sites at which mutagens mutate DNA are usually random so the chance of a mutation occurring in the thing we can measure is likely tied to the overall rate of mutations.

Some genetics experiments involve using mutagens to randomly mutate DNA – they look to see what changes occurred and then trace those changes back to what was damaged (this is called forward genetics). In contrast, in the Ames test we look for specific changes and don’t really care what caused them just the output. And, in biochemistry, we often use site-directed mutagenesis. This is a lot different than this random mutagenesis – we introduce precise mutations at precise locations to test how they affect different things. And I’m working on a post to tell you more about it but I had a really busy day in the lab using some of those mutants I made!

Going back to the whole mutagen/carcinogen thing again 👉 Growth control is a complicated process that involves lots of different components whose instructions are spread out throughout your DNA. This offers a lot of potential sites for things to go wrong if you add things that start randomly mutating DNA.

So our cells also have lots of mechanisms to detect and fix mutations, so, even though random mutations are actually really common, they usually get fixed. And if one does slip by and say, causes a protein to stop working, there’s usually a similar protein that can compensate and pick up the slack (though maybe not quite as well). When you get into real trouble is if a mutation occurs in one of those regulators or the things that detect and/or fix the mutations. These allow lots of mutations to accumulate. And often in cancer, some of those mutations occur in the systems that sense sense that the cell is getting out of whack and initiate its killing. Instead, these cancer cells keep growing and replicating and causing problems.

more on that mutagenesis to show non-overlappingness:  http://bit.ly/2old6h6

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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