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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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« Our Buddies at the FDA | Main | The Ames Test and the Real World »

July 29, 2002

The Ames Test

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Posted by Derek

One hears a lot about the Ames test (as a measure of carcinogenicity and other Bad Things.) It's sometimes held up by animal-rights types as a model of the sort of testing that could be done if, presumably, we weren't all so much into torturing the lesser species. I thought a look at the test would be worthwhile. This is another post where many of my readers in the industry will know the material, but since almost no one outside of it does. . .

The original Ames test dates to the mid-1960s, and the idea behind it is even older. First, you need some mutated bacteria, in this case mutant Salmonella bacteria that have lost the ability to produce their own histidine (an essential amino acid.) These guys can be grown on a medium that contains histidine, but if you try to grow them on something else, they'll die. Unless, of course, they mutate back to being able to make it on their own, and that's the basis of the test in a nutshell.

The original protocol was worked out after a long search for Salmonella mutants - some naturally occurring (although not for long!) and others induced by radiation or exposure to toxins. The modern form uses some engineered bacteria as well. There's a panel of standard strains, with different types of defects in the gene that's responsible for histidine production. Several also have hand-introduced glitches in their best DNA-repair enzymes. Losing those force the bacteria to use more low-tech cellular machinery to do the job, increasing the chance of random mutations. Finally, all the Ames test strains have defective polysaccharide outer coats, to make them more permeable to the test chemicals. These are not exactly the most robust Salmonella you'll ever see (but robust Salmonella would laugh at most of our test chemicals, which wouldn't be too informative.)

There are a number of different ways to run the test, but they all come down to this: you expose your bacteria to the test substance, and try to grow them without their required histidine. That really puts the selection pressure on - stumble back to making your own histidine, or die! So anything that survives is a mutation, and the more of those you get, the worse your test substance was. That's because the mutation occurred while the DNA was being repaired or replicated. Your test compound either caused the damage that had to be repaired, or it caused the replication process to fumble.

An added wrinkle involve taking your compound and exposing it to liver enzymes first, then taking that mix and running the Ames on it. That can pick up toxic metabolites that might form in vivo(not all of them, but it's a start.) Drug companies routinely do it both ways, just to be sure.

So that's an Ames test. What does it mean? Here's where the arguing starts, but there are some agreed-on facts: A dose-responsive positive in means that your compound damages DNA. There are a lot of ways for that to happen, and the behavior of the different Salmonella strains can point toward what kind it probably was. As you pile up the DNA damage, odds increase that you'll finally bollix some gene that shouldn't have been messed with, one that can start a cell on to road to cancer.

And that's what the test has traditionally been used for, as a proxy for carcinogenic potential. It's not a good surrogate for general toxicity, because there are plenty of toxic compounds that don't work by damaging DNA. Nerve gas, for example, would probably pass an Ames test, although I'm pretty sure that no one has been insane enough to actually take a look. Long-term carcinogenic potential is not a big issue for the typical nerve gas user.

Here come the harder questions, though: are all carcinogens positive in the Ames test? Do all the things that light up an Ames test cause cancer? And at what level of positive activity should you start to get worried? Bruce Ames himself started to wonder about these questions as the years went on. The answers were a bit surprising. . .(to be continued.)

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