Rhonda: Right. You also mentioned that the most of the changes are happening during development. And this is also really interesting, mostly...Steve, when Dr. Steve Horvath was on the podcast last time, and this kind of gets into the next section, which is, like, underlying mechanisms causing these changes in the epigenetic patterns, but he had mentioned that, like, he had developed at least, you know, and maybe there's something new now, but back then, a few years ago, there was a few clocks that he had used that could really beautifully measure gestational age.

Which was interesting because measuring the aging process during gestation where you've got this really coordinated program that has very little background noise, right, inflammatory processes aren't going off and all this damage and, you know, this stuff, I mean, it's just a very clean place to, like, you know, measure aging.

So, and then he's also...I think there's a preprint I saw pretty recently where he had developed a universal aging clock.

Dr. Levine: Pan-mammalian.

Rhonda: Yeah, there was like I don't know how many different mammalian samples that were, you know, used to generate the clock, and I don't understand everything that goes into generating it. But I just looked, you know, skimmed it and it was really talking about...this universal clock was also really coordinated with development. And so, you're mentioning development, it poses the question, like, do you think that the epigenetic aging could be sort of like a program, like a program that's regulating aging? Like, is that a possibility?

Dr. Levine: Yeah, I don't think it was a program designed...you know, some people would argue that it is, you know, a program designed to drive aging. Because I think, you know, for species selection, you need things to age and die so that the species [inaudible 00:28:16]. I think it's a developmental program that just doesn't really get turned off and maybe goes a little bit awry as all these other changes start to accumulate in our bodies. But yes, the epigenetic clocks are really tracking something very central to development because most of these changes we can see during development and a lot of the genes that seem to be involved are these developmental genes. We're still, again, not sure what this means or what this program actually is but it definitely is tied to development. But people would also argue that aging is very tied to development. So, there are beautiful experiments in flies where, if you can extend kind of the developmental period, it extends the lifespan of these animals.

And so, development and aging are kind of two dichotomous things that we usually think of, right, that you're going through development, you hit age 20 and...okay, maybe age 20 or 30 is when your aging starts. But there's a lot of great work, even one of my colleagues at Harvard, Vadim Gladyshev, showing kind of when he thinks this ground zero when aging starts, which is, according to him, day eight of gestation. So, there's...

Rhonda: In humans?

Dr. Levine: In humans, yes. So...

Rhonda: When you said, "In flies and fruit flies," Drosophila probably...

Dr. Levine: Yeah, yeah.

Rhonda: "When they extend the development," can you explain that, what do you mean by that?

Dr. Levine: Yeah, so, I think, in this case, actually, the study I'm thinking of, they extended kind of the reproductive kind of age of the fly. So, they can push flies to, what we would consider, like a late fecundity, so, they can develop a little bit longer and don't reproduce until slightly later.

Rhonda: And how do they do that?

Dr. Levine: It was more like selection. So, they're selecting for flies over generations that are going to be these later-fecundity flies, and they show that they also live longer in the end.

Rhonda: Like, is it a genetic...genes that are controlling it?

Dr. Levine: Yeah.

Rhonda: Okay. So, a couple of things here, then back to this...it's very interesting, the development thing. And another thing came to mind from the conversation I had with Steve was he had mentioned, like, if you take a cell that has not been immortalized in tissue culture and then you immortalize it with a component of telomerase, TERT, and you, essentially, overcome cellular senescence, which is one of the hallmarks of aging. Right? When a cell undergoes senescence, I mean, it's pretty much not...I mean, it's still metabolically active but, you know, it's considered sort of the end, right?

Dr. Levine: Yeah.

Rhonda: And these cells, if you continue culturing them and tissue culture, their epigenetic age just keeps going and going and going.

Dr. Levine: Yeah, we're actually doing this exact thing in my lab right now where we use hTERT to immortalize cells and we are just seeing how long...like, there has to be some...like, eventually, can it just continue to change forever? I think two people have looked at things like HeLa cells which have just been changing, you know, they've been evolving for decades. And like at what point does the epigenetic age kind of reach a saturation point? And again, I don't think we know but, yeah, definitely with these immortalized cells, every time you passage them, their epigenetic age keeps...at least it seems to continue to increase over time.

Rhonda: It's interesting. And sort of, on the flip side of that, would be, like you mentioned, like, are the epigenetic aging clocks biomarking something, like, something else that's causing aging? And a study that you were a co-author on, as I was preparing for this podcast, I was thinking about it and I was, you know, in my mind, I was like, "Well, how could you, like, cause something like that would be massive damage to accelerate aging?" And so, I googled "cancer chemotherapy epigenetic clock" and, like, the paper you were a co-author came up on it. I was like, "Oh, this is Morgan's."

Okay, so, I was reading the paper and these patients that had head and neck cancer and they were getting treated for it, radiotherapy, chemotherapy, you know, which causes massive damage, inflammation, these patients, their epigenetic age was measured before the treatment, after the treatment, and then six months later and a year later. And it was so interesting to me because they had aged...like, their epigenetic age had accelerated by 4.9 years right after the treatment. But then, six months later and a year later, like, their epigenetic age had, like, normalized back to baseline. And sub-analysis then showed actually not only did the epigenetic age acceleration of almost five years correlate with inflammatory biomarkers but people that had extremely high inflammatory biomarkers one year later did still experience the age acceleration. So, I'm curious as to what your thoughts are on what that means? Like, to me, I look at that and I go, "Wow, inflammation is causing epigenetic age acceleration," because you see this like graph, right, I mean... So...

Dr. Levine: Yeah. I think, definitely, when we measure aging in blood, we need to think, you know, what is, you know, probably driving these signals that we see. And I would guess that epigenetic age acceleration in blood is mostly reflective of inflammation. Unless, again, you're developing a clock that's specifically tuned to some other thing. Although inflammation seems so, you know, vast and systemic it affects so many different things.

But I don't think everything that epigenetic clocks are capturing is inflammation. Because again, when you look at immortalized cells, it's not because they're becoming more inflammatory every time you're passaging them per se but definitely, I think, epigenetic aging measured in blood is very much tied to inflammation. Which again, is probably why it's highly predictive of a number of diseases which we know inflammation can be a major driver of.

Rhonda: Is that where the extrinsic and intrinsic aging clock...or, I don't know exactly, one of them considers the external factors in blood and one doesn't or something, is inflammation calculated in that or not really? Is it sort of...

Dr. Levine: Yeah. So, these are two of the first-generation clocks, so, I think, you know, Steve kind of called them intrinsic-extrinsic aging. I think he called the original Horvath pan-issue clock was the intrinsic aging, it wasn't that tuned to differences in kind of cell turnover or inflammation. Whereas a clock, that was developed by [inaudible 00:35:08], he kind of added these different kind of cell composition measures that actually ended up picking up inflammation a little bit better. But this was before these second-generation clocks came into being. And then I think, once they came into being, they're probably picking up inflammation a lot more than even the first-generation clocks.

And again, we can make these kind of systems clocks and one of our systems is inflammation, and we can show that it's highly predictive of outcomes, it's definitely capturing things related to inflammation.