#609: Unprocessed Red Meat & Cancer Risk

[Music] Hello and welcome to another episode of Sigma Nutrition Radio. My name is Danny Lennon and with me is Dr. Alan Flanagan. Alan, how are you? I'm very well, thanks. I'm surprised we're back after episode 600, but. There is enough of a fervor for us to return that we gave into pre-opressure from the public and we've decided to return. Like all good at retirements from sports, you always see them come back. It's the classic. It's the last dance. Yeah, it's the boxer that's going to return and we'll stay returning until they get knocked out. We're punch drunk. Yeah, we just couldn't have had to give up. Or the classic one where they say, "I should have retired and should have stayed retired." When would you pass our peak? Yes, I think certainly the deadpan nature of our last discussion, maybe I caught a few out because I had to answer a number of messages and emails, laying people's fears that the podcast was indeed gone. So maybe we need to signpost our deadpan delivery of those comments in the future. But indeed, we are back and we are taking on a topic that continues to generate debate and also interest. In the past, we have talked about the topic of unprocessed red meat on chronic disease risk, broadly mainly focusing on the epidemiology in the area. So we're not going to rehash all of that in today's episode, although it's important that we do touch on it. Recently on the podcast, I was talking with Dr. Gil Carvalio and Dr. Mario Kratz, specifically on the outcomes of type 2 diabetes, insulin resistance, and then some of the stuff around cardiovascular disease, specifically deciding not to touch on the outcome that we'll look at today, which is cancer. And I suppose the outcome of cancer is particularly important because it's perhaps the one where we have a lot of the research that gets debated and is perhaps one of the major concerns around consumption of unprocessed red meat. And again, with the same caveats that were applied to that episode, today we are going to keep our focus to unprocessed red meat, so not including processed red meat. And so focusing on everything that comes under that umbrella of red meat in an unprocessed sense and looking specifically at the outcome of cancer. And as we'll discuss that is typically relegated to one particular cancer type or a subtype with it. There's a few within them. And we're going to walk our way through this. So maybe a good place to start here, Alan, is when most people hear about this topic around red meat and cancer, one of the things that gets referred to is that it is being classed as a carcinogen. And there's some differences here between processed and unprocessed. But even in relation to unprocessed meat, we have these designations by, for example, the international agency for research and cancer as red meat having being probably carcinogenic to humans. The WCRF has a very similar label or being a probable cause of colorectal cancer. Can you maybe just touch on these designations of what these classifications mean, what they are built on and anything else that people might have heard that we might need to add some nuance to? I think both are relatively similar in the conclusion that they arrive at from a causal standpoint, which is probabilistic language of unprocessed red meat as probably carcinogenic to humans. They slightly differ in the emphasis on evidence and the types of evidence that are emphasized. So with the IARC definition, that's something that prioritizes long-term perspective cohort studies coupled with mechanistic research. And the test that they apply is that there are consistent associations in different populations that then mean that other factors like confounding or chance can be ruled out. And the WCRF, which also arrived at this determination of probable cause for red meat and colorectal cancer, is based largely again similarly on identified mechanisms and those specifically in conjunction obviously with the long-term epidemiology. And those specifically relate to the formation of what are known as heterocyclic amines and other compounds known as polycyclic aromatic hydrocarbons and the potential for carcinogenesis to be induced by a group of compounds known as n-itrosocompans or n-itrosamines. And those compounds, the production of those compounds, can be stimulated by hemyron, which is obviously highly concentrated in red meat. The main distinction and where the controversy lay with the IARC and WCRF designations was in relation to the distinction between processed meats and unprocessed meats. And processed meats were much more weighted towards a kind of causal determination in terms of the strength of the evidence and the consistency and the clear dose response relationship evident in prospective cohort studies, which would all satisfy a kind of probabilistic causal framework from epidemiological data. Processed meats, sorry, unprocessed meat or what we might otherwise term fresh red meat, was the more controversial finding because some of the dose response analyses were not significant for example for unprocessed red meat per 100 gram a day increment increase. And so the conclusions and the use of probabilistic language was essentially in spite of a lack of significant association in some of the cohort studies with unprocessed meat intake specifically and more of an emphasis on some of these mechanisms. And that was where most of the, shall we say kind of issue was taken with certainly the WCRF classification. But I think as we will hopefully highlight today, it wasn't a misguided evidence assessment. And while we do have issues that arise in the epidemiology of unprocessed meat in relation to dose thresholds at which risk may be observed, there is when people tend to look at this evidence of tendency to say, well this is all based on epidemiology so we'll never get further to reconciling that. That's actually not the case and there is a body of evidence that we'll discuss today that really goes back to the mid 1990s looking at in humans, contrary controlled elegant feeding studies looking at some of these intermediate mechanisms that may explain a long term relationship with coloractyl cancer risk from unprocessed meat. And that will become important because it's often maybe left out of some of the conversations or debates that are commonly had and they end up devolving into whether someone wants to actually discuss the epidemiology and appraise that properly or they just want to dismiss it outright. But of course, that still serves as an important part of this evidence triangulation. We've talked about that previously on an episode more in depth. So if people really want the details of some of the best epidemiology in this area to answer some of these questions, that is worth revisiting. We also have an article on the website that might discuss a bit more and indeed some of the work, for example, Catherine Bradbury was on the podcast discussing their specific work in this area answering these questions around red meat coloractyl cancer risk and their epidemiology there based on the UK bio bank data. So I'll put those in the show notes for people want real details on that. But for now, given that we want to get into some of those real controlled studies looking at these controlled trials and humans, first let's touch on that epidemiology because it can't be left out and it gives us a really important basis for seeing why we're even investigating this here. And there's probably a few of the most important pieces of evidence in this area that might do a good job of summarizing generally some of not only the direction of those effects, but some of the nuances within interpreting it, which is why there's maybe not one necessarily black or white answer when we're looking at the epi in this area a lot depends on those cohorts, those exposures we're looking at, what specifically we're answering with these questions. So if we're to talk about some of the epidemiology in this area and from this overview level, so we without us getting all into the details of every single study, what are some of the most important points that we can touch on related to the epidemiology that you think lead us to be able to say some things with some degree of confidence that we can come away with the epi with some clear signal relation to this question. Yeah, for me, the most striking feature of this and this relates to most of the epidemiology or in fact, all of it of unprocessed red meat and any disease outcome, including in this specific instance of our focus today, coloractal cancer is regional variation in the presence of positive associations, i.e. higher risk associated with higher intake. And where we see a regional variation like that in any sort of exposure outcome relationship, that's a signal to look a little closer at the characteristics of the exposure in those different populations. And what we tend to see is a kind of general overview of this area is that in, for example, European or North American cohorts, we more consistently see higher risk associated with higher intake. And of course, as we've discussed on numerous podcasts that relate to nutritional epidemiology. The problem is this abstract use of the term "high" quote unquote. And so we really need to start to then define what we mean by high and what we mean by low and what a range of actual intake in terms of grams per day, for example, in absolute terms is in that population. Because a relative comparison of high and low without further describing that tells us very little about the characteristics between populations. And when we do that due diligence, we typically find that you will have certain populations North America in particular, where the absolute actual intake is almost two to threefold higher than it would be, for example, if you took a cohort from the Asia-Pacific region. And that gives us something to then start to work with. And generally speaking, a relatively consistent picture emerges where dose thresholds of at least around 100 gram a day or less of unprocessed meat intake don't really produce any sort of signal in the noise for any of these outcomes. And yet if you go over then in terms of some of the North American and indeed within Europe, some of the European cohorts that tend to have higher intakes of 160, 170 over grams a day. And that's factoring in the kind of well-established underestimation that tends to result from dietary assessment methods in nutritional epidemiology. We're likely looking at higher again actual intakes on an average terms. And so the regional variation is important because that regional variation speaks to differences in absolute intake. And that appears to particularly for unprocessed meat be a really crucial factor because the absolute intake, the intake in grams per day, is what is going to be important in trying to explain how unprocessed red meat could in fact drive some of these intermediate carcinogenic processes such as chemiremediated and itrosocompound formation or the production of polycyclic aromatic hydrocarbons or hydrocyclic amines as well. And that for me, I think, is probably the top line most important consideration that we can have in this literature. And for colorectal cancer, I think a good example with the Chan and colleagues, well, it's now quite dated, but it's still a good meta-analysis of that distinguished red and processed meat and colorectal cancer risk in a meta-analysis. And so you had 24 prospective cohort studies that spanned North America, Europe, or which was 10 countries from the epic cohort, Asia-Pacific region, including Japan, China, Singapore, and then you had Australia and Canada. And in that analysis, for example, red meat alone on processed red meat per 100 gram a day increase was associated with a 17% higher risk of colorectal cancer. The same, obviously, for processed meat, that's typically a stronger association. But again, in terms of stratifying by geographic region, that was strongest in Europe and North America, whereas in Asia-Pacific cohorts, there was no significant association. And that's because you can't model a per 100 gram a day increase necessarily in a population that are consuming 70 grams or 60 or 50 grams a day on average. You can model it, but again, you're not getting an association or a signal in the noise simply because the habitual intakes in those populations is relatively low and within this range which we don't see risk. And I think that gives us a kind of general overview of the epi in a way that allows us to then take some of those questions to stress test in some of the controlled feeding studies that we have. I think that's a particularly important point that we can turn to these control trials because they allow us to maybe fill in gaps that are very difficult to do otherwise. So for example, one thing people may be thinking now is that as you mentioned, when we have these lower doses that we're looking at and maybe a comparison where everything is under 100 grams and let's say we're comparing 70 as the quote, quote, high in a certain position. And how we interpret that might be worth talking about because maybe there's a temptation to say, well, below these certain thresholds you've just mentioned, there is no risk as opposed to saying, below these certain thresholds, we are unable to detect a risk from this particular epidemiology. And those are two different things. And you see, depending on how someone interprets that, they might say, well, that is true. We're not detecting effect, but based on all this direction, we would still assume there is risk even at these lower intakes. Someone else might say, well, we can't say that. So at these levels of intakes, it seems that there's no increase in risk and so therefore they're fine. And so those might be different conclusions to saying we're not detecting a risk. Can you maybe talk a bit about the interpretation of the epidemiology given this kind of gray area that we're referring to? Well, I think it obviously depends on the outcome that we're looking at, but you've outlined very well, essentially the two main interpretations that were offered by an association like that. So what I think is always really important, then, is to really look at our risk estimate. And the point estimate of that hazard ratio or the relative risk and the corresponding confidence intervals to see if there's any kind of direction that we can glean. Now often, let's take, for example, that Chan and colleagues cohort, the relative risk by geographic area for the Asia-Pacific analysis, specifically was a relative risk of 0.94. So a 6% lower risk, but the confidence intervals went from 0.69 to 1.27. So that is spread entirely across. There's no direction of effect there evidence in terms of that range. And so that's something that I think you would generally interpret more as a no association rather than perhaps interpreting it as not. And it could, the potential there is that is something because of the confidence intervals that could, if obviously, no association and that analysis, but there is room open for it to be, could not detect an association. Whereas with some of the cardiovascular studies, for example, when cardiovascular disease is an outcome in some of these East Asian cohorts, the entire point estimate and the confidence intervals range are actually in a direction of under 1.0. And in some cases, statistically significant under 1.0. And that's often sex based, those statistically significant differences in Asia-Pacific or East Asian cohorts, specifically lower risk observed. That's statistically significant in women, but not men. And again, the paradox of hem iron in women and reproductive age, it may be because it's making a contribution to hem iron, who knows. They're all the speculative components. But I think in terms of how do we then say, is this just no association, but with the door open for potentially the particular analysis, of that particular dose comparison range, not being sufficient to detect an association. Or whether we can say we're actually looking at more of a kind of a lack of risk, it depends really on what the effect, or what the risk estimate actually is, and what we can clean from the overall and the confidence intervals of that risk estimate in particular. So that turns to why it might be important for us to look at these other areas that could fill in that gaps to suggest, well, these areas where either were unable to detect an effect, in those situations, that could be down to, first of all, not they're not being an effect or negative effect, let's say, at those levels of intake. Or it could be, as we've said, it could be that there is some degree of effect, but just not that we're able to detect it here. And so to be able to try and answer which one of those is, we can try and look to some of this other work. And as you've alluded to, these are really nicely done studies, but maybe are oftentimes let out left out this conversation we spend all the time looking at this epiate and debating what we can take from it or not. And this might be useful to walk through. You've already discussed those primary mechanisms that have been typically hypothesized as to why red meat could be particularly, could be playing this role in the development of colorectal cancer specifically. Some of those relate to one another, for example, the hemion potentially setting the stage for some of these other compounds that are having a problem. So if we start walking through some of these and referencing some of the studies that you've highlighted as particularly useful and particularly interesting to touch on these topics, that might be a way to give people an idea of what we actually know in this area in these controlled trials in humans. One of the first and one of the ones you've highlighted that talks to some of this issue around these in nitroso compounds that we've referenced was from Bingham and colleagues in 1996. Can you walk us through why this particular study stands up to you, why this is a useful one for us to take into account? Yes, so there's, like I said, a body of work in very nicely controlled trials that we can draw on. And this, I think, is quite a seminal paper in this area published in 1996. And it was a metabolic ward study where eight participants, male participants, age 24 to 32, so young, lived in a controlled metabolic facility and consumed diets that were fully controlled, prepared under metabolic kind of kitchen conditions. And they had two different protocols that the participants were exposed to with different setups for their diet. In the first, they had a low-red meat diet with 60 grams a day of red meat. They also had a high-red meat diet with 600 grams a day of red meat. And then they had a high-red meat diet that was supplemented with additional 20 grams of wheat bran to add a fiber-based supplement to the high meat diet. And then they also had a second protocol with a low-red meat same, sorry, low white meat, 60 grams a day, high white meat, and then they had a low-red meat diet that was the same as the first two diets. And then they also had a low-red meat diet with a low-red meat, sorry, low white meat, 60 grams a day, high white meat, 600 grams a day, turkey chicken and fish primarily. And then the high-red meat again, 600 grams a day diet. And they were primarily interested as their primary outcome in fecal excretion levels of total Nitrosocompans. So these compounds are these alkylating agents that can promote potentially musogenic and carcinogenic processes in the colon. And they then also looked at fecal nitrite, nitrate, iron levels and other outcomes. And for the primary outcome of the fecal Nitrosocompan formation, those compounds, the generation of those increased significantly on the high-red meat diet, as we might expect with a dose maybe of 600 grams a day given kind of hypothesis in this area. And then interestingly, on the high-red meat with the addition of the brand, the Nitrosocompan formation, was still significantly higher than the low-red meat 60 grams a day condition, but not significantly different from the high-red meat diet. So effectively, the increased red meat consumption led to this significant and quite rapid increase in endogenous fecal. And the level of Nitrosocompans that was produced from the 600 gram a day red meat diet was comparable to the carcinogenic exposure that you might get from tobacco smoke. The whitefish condition, that second protocol, the white meat or the whitefish condition, didn't increase fecal in nitrosocompan formation or nitrite, which again would be consistent with the epidemiological evidence that we have, which you see no real association for white meat, you do and you see benefits in substitution analysis that model replacing for example 100 grams of red meat with 100 grams of white meat. And the brand supplement, this is important, did not reduce the Nitrosocompan formation. And so at that high dose of 600 grams a day, the addition of that brand supplement made no difference to the Nitrosocompan formation. So now, with the same effect we've seen with an intermediate dose of something in the region of 250 grams or 300 grams, that's the limitation of this study. It was deliberately in its set up, creating a very wide exposure contrast, but it would also in terms of ecological validity, a dose of 600 grams a day, probably not reflect, unless we're talking full carnivore, the way that regular meat eaters might actually consume. But from a mechanistic standpoint, it's given us something to go with. One, is there some plausibility to the lack of association we see in East Asian cohorts at doses of under 60 grams a day? Or 100, well in some way yes, because this study used a dose of 60 grams a day, didn't see that significant increase. And then we do see this increase with the high red meat intake that's unprocessed driving an Nitrosocompan production and duchinously to very high levels. But there's the artificiality of the dose that was used in this specific study. There's some really nice components that they've thought of here. Obviously we, as you mentioned, there are the limitations being this wide exposure, which was done because we're trying to teach out what's going on here. But that aside, we have this really nice comparison of not only the low level of red meat to the high, but then taking to account this additional fiber, which is another classic line we hear that once you have enough fiber in your diet, then you don't need to worry about any red meat consumption. Here, even in those situations, we're seeing this, it almost looks like a threefold increase in some of these formation of these compounds. And then when we look at then the white meat or fish, you mentioned that there was no increase there in the so straight away, we're seeing that we can isolate this increase to red meat driving that and it's not offset by at least a supplementation of a weak brand in this case. Now, you mentioned there that obviously this is comparing that 60 grams per day to a 600 grams per day as opposed to some intermediate numbers that might give us a bit more information. There was another trial that came after that. This was the Hughes and colleague study that you mentioned that did indeed look at different ranges from 060 to 40 and 420. Can we maybe walk us through the important parts of this trial and maybe what this adds on top of what we've just discussed? Yes, so this was also a metabolic ward, tightly controlled study where participants completed in a kind of crossover randomized order design for different 10-day dietary periods. In one they consumed 0 grams a day of meat of red meat in the other in the second condition 60 grams a day. So that same dose from the previous study and then 240 grams a day and then 420 grams a day. So we're into slightly more ecologically valid absolute thresholds of intake and we still have a very high dose with the 420 gram a day dose. This was provided primarily as roast beef and other meat preparations and because it was a fully controlled study again in a metabolic facility protein intake total fat and fiber levels in the diets were all matched between these diets differing only in their dose of red meat intake. It was designed specifically to look at dose response relationships then across that range from 0 to 420 primary outcome was still L and nitroso compounds in feces and then they had similar secondary outcomes to the previous study. So for this the generation of fecal and nitroso compounds from 0 to 60 grams a day there was no significant increase. So at 60 grams a day the difference from 0 there was no difference. It was 53 micrograms per day in the 0 grams per day meat group and in the 60 grams per day meat group it was 52 micrograms per day so there was no difference whatsoever when you then went to the 240 gram a day meat dose that jumped up to 159 micrograms a day and when you went to the 420 gram a day meat group that fecal and nitroso compound formation jumped up to 198 grams per day. So there was evidence of significant increased levels of N nitroso compounds once they went from the 6,240 to 60 to 240 gram a day dose and then again an increase from 240 up to 420. And that was obviously then evidence of a kind of dose dependent increase in nitroso compound formation with those increases evident out of more moderate dose of 240 grams a day which again in terms of when categorical analysis and epidemiology model particularly in North American cohorts and some of the European cohorts model there are very high group. That is again often in this region of over certainly 180 grams a day possibly into this kind of range that's reflected more by that 240 gram dose. So at that moderate level you were still seeing quite a significant increase in an nitroso compound formation there was a lot of inter individual variation which is potentially obviously a factor always in why some see people see outcomes related to these mechanisms and potentially some don't. And for an interesting aspect of this study they maintained a subgroup of four of the participants voluntarily obviously on the 420 gram a day read meat diet for 40 days and in that fecal and nitroso compound formation showed no source of adaptation effect. The level is just remained high over time and did not decline at all so there was no kind of adaptation there was no and this is outside of the kind of control of the metabolic word where they're just sustaining the meat intake and other aspects of diet of kind of loss their control in this. So again prolonged intake of a high red meat diet is not going to we hear this a lot right and push back and sometimes in some of the kind of ancestral myths around diet is humans are adapted to that word adaptation is often. and invoked as humans are designed to eat this way. And this clearly showed that there was no source of adaptation against the production of these carcinogenic compounds. So I think this particular study is possibly more persuasive than the previous study in linking back to the epidemiology. What we have is an even more now plausible mechanistic support for stating that when we don't see risk at these doses of under 100 grams a day in and around this average dose of 60 grams a day, that could very well be genuinely because there is no risk. We didn't see any difference in an Ichoceo compound formation at that dose of 60 compared to zero compared to a no-meat diet, a no-red meat diet. So I think that lends to more of the plausibility for making that statement in relation to what we see in the epidemiology and we now have a degree of biological plausibility to associations that are observed with high-red meat intake in populations at more moderate red meat doses that could be reflective of more consistent intake knowing that the production of these compounds is effectively immediate and knowing that production is essentially sustained as long as the meat intake is consistent in that diet. There's a couple of things you've just said that I think are really important and worth restating and emphasizing for people. One is this connection to the epidemiology and helping us explain maybe what some of those observations are. Now of course there's a number of candidate mechanisms that really read me to this color right to a cancer risk. So we can't say with certainty just based on what we've said, but indeed if we are to accept this formation of in Ichoceo compounds as important or maybe one of those driving factors or if that is indeed the mechanism, then we can see based on this work that we've just mentioned that up to that 60 grams per day level, we're not seeing any change in the production of those compounds. We're not seeing an increase which then would then fit in and at least make plausible sense as to why maybe we aren't detecting those effects at that level of intake in the epidemiology. So it marries up nicely there. Once we start pushing it beyond that to these ranges we used in this study to 40 or 420 grams per day. We are seeing this much elevated level of these compounds and again that might map on to why we would be seeing more risk being detected in the epidemiology and I think that's a really crucial point that helps us connect some of these dots. The other thing that was nice about the study that you outlined was this maintenance of that intake of the high intake over that 40 day period after the controlled feeding study. And again this gives us again room to at least for the moment say we can call into question some of those claims people have of oh this is just a transient response or seeing over a couple of days after that there's going to be an adaptation there's nothing to worry about. Whereas we see here that's not the case if you continue to eat these higher levels of red meat that was described here you would continue to see that high level of Nitroso compound formation. So really nice useful work there. Next on what we've discussed so far we're starting to see that with red meat we've been able to specifically say it's red meat as opposed to let's say white meat or fish we can also say it's specifically red meat even in the context of this added brand that we mentioned earlier here we're now starting to see that maybe there's a dose threshold to pass. The next step is then to say well if we are detecting this effect what component of that red meat actually is it what is the thing that's driving some of the formation of these Nitroso compounds what aspect of red meat leads to that and this is maybe where we can get into the cross study because this is really useful and perhaps one of the most important mechanistic trials people should be aware of in this area and helps us try and answer some of that question of what are the components of concern that might be driving these effects. Can you maybe speak to the setup of this cross study and again what this adds to our understanding. Like you say I think this is probably one of the most important of the studies that that kind of came out from this group in this period from kind of 96 to the early and this was a 2003 paper published in cancer research and there were two protocols again randomized crossover dietary interventions in healthy male volunteers that were living in a metabolic ward consistent with the kind of rigor and control that we've described for the previous studies. Each diet period in this study lasted 15 days with no what or sorry with a wash out period basically for the first 10 days leading into each study arm each protocol. The first protocol had three diets over those three 15 day periods where you had a low red meat again the 60 gram a day dose high red meat this 420 gram a day dose and a vegetarian diet matched for protein content to the high meat diet and then the seconds and this is the real kind of genius to speak of this study is the three period crossover again with the 60 gram a day red meat diet alone or 60 gram a day diet supplemented with heme iron at around 8 milligrams per day or the same diet supplemented with inorganic iron which was 35 milligrams a day of a ferrous gluconate tablet again and I chose a compound formation was the primary driver or primary outcome but what they were specifically interested in particularly with this protocol too was heme iron and the type of heme iron a potential driver of that nitrosocompan formation and what was brilliant about the design with the full control and the rigor that they had over the study was that between the vegetarian diet matched for protein content and the red meat high and low diets they actually controlled for nitrate intake so that actual exogenous nitrate intake through the diet was matched between those diets. So for the outcomes for the first protocol if you remember compare the 60 grams I'm 420 grams a day of red meat with the vegetarian diet with the fecal nitrosocompan formation the low red meat diet the high red meat diet was just under 1300 is in 1300 micrograms the way they expressed it was micrograms per kilogram of body weight what that translated to was about 190 micrograms per day in an individual in contrast the low red meat diet was 42 micrograms per day that's consistent with what we saw in their earlier work where the nitrosocompan formation at their 60 gram a day diet in these otherwise healthy participants was around 52 grams and micrograms per day and the vegetarian diet then in terms of micrograms per day was around 63 so there was no significant difference between the low red meat 60 gram a day diet and the vegetarian diet despite the fact that the vegetarian diet was protein matched to the high red meat diet but the high red meat diet was a kind of for full increase in an nitrosocompan formation relative to both the low red meat diet and the vegetarian diet and then when we then look to the second protocol for the addition of heme iron you've got the low red meat 60 gram a day diet which was similar in this range if this time it was 77 micrograms per day of nitrosocompan formation on the low red meat diet but when they added the 8 milligram a day heme iron supplement to that that jumped up to 156 micrograms a day when they added the inorganic ferrous iron supplement there was no significant difference between the low red meat diet that had no iron added whatsoever so the increase in nitrosocompan formation was driven by the addition of heme iron even in the context of a low total red meat diet whereas inorganic iron had no significant effect and so this I think is where we build on what we've seen in some of those previous studies in that because sometimes you can see associations or suggestions that protein content is a kind of relevant factor in red meat and in unprocessed red meat in some of these associations but it wasn't the protein content that was driving the nitrosocompan formation it was heme iron that was responsible for that increase associated with red meat consumption and so what this indicated was that heme iron itself catalyzes the formation of potentially carcinogenic nitrosocompanes in the colon and that provides if we think back to the epidemiology and we can look to specific cohorts in epidemiology that have modeled for example or measured heme iron or nitrates or and what you will see in several analyses including analysis of for example the national institutes of health retired person cohorts in the US or the UK biobine cohort is in mediation analyses in epidemiology heme iron explains substantial proportion of the association between unprocessed red meat and protein. processed meat and colorectal cancer risk. So we now have a plausible biological mechanism evidenced in a controlled intervention with a really elegant design that specifically isolated independent effects of the addition of hem iron as a supplement. That clearly showed that the increase in an Ichozo compound formation observed when you give people 420 grams a day of red meat was achieved when you give people 60 grams a day, but add 8 milligrams of hem iron to that dose. And I think this is an incredibly powerful kind of line of research building on some of their previous work with the dose response relationships that ties together. Yes, you've got these end points of N-Ichozo compounds that are potentially mutagenic and carcinogenic, but now you actually have an explanation for why unprocessed red meat, which is low in preformed nitrates as opposed to processed meat, which people typically are happy with the kind of to conclude that there is that association with processed meat. And I think this study really gives us the causal chain that would link back to hem iron rather than the protein content or other component of an unprocessed red meat. It's a really beautifully done study and well thought out study some of these components that help us isolate those. As you say, we have this initial comparison of the low red meat at 60 grams per day to the 420 to the vegetarian diet match for protein, which gives us not only that we see this increased N-Ichozo compound formation with the high red meat compared to low, but even then if we match the vegetarian diet for protein, we don't see an increase thus allowing us to conclude well, it's not the protein that's driving it, which was maybe one of those hypotheses. We can then look at even in the low red meat intake that 60 grams per day, if we give the addition of a hem iron supplement, we see these compounds jump to the same types of levels we're seeing with the high red meat intake. Again, just really nice work that allows us to isolate down that what we're seeing is not a function of the protein load. It's also not a function of iron per se because we're comparing that hem iron to the inorganic ferrous iron that you mentioned. We can specifically say it's this hem iron that seems to be driving it and with this nice clear causal pathway. There's some really nice work. With that then we turn into another area and there's a collection of studies here. There's two or three at least that you've highlighted that. Speak to this topic of genotoxicity. Essentially we're thinking about the colonic environment and where maybe that can become genotoxic. Can you maybe give us an overview of some of these studies, what they might be referring to, and again, what this collection of studies might add to our understanding? Yes. There's a couple of studies that I think we'll go through now for listeners. Before we do, I think it will be just helpful to describe the outcome that they use in these studies. The outcome used in a couple of the papers that we'll discuss now is called fecal water genotoxicity. What this is, it's a kind of ex vivo measure of DNA damage in a colon cancer cell line. This is a model that has been used for years in colorectal cancer research. What it does is you feed your participants your dietary intervention or exposure. You then take fecal samples and you incubate the participants fecal samples with a specific colon cancer cell line. The fecal water looks then when it's incubated with these particular cell lines, you're then looking at the level of DNA damage that the fecal water from your sample causes when it interacts with these colon cancer cell lines. It's a marker, and yes, it's not sticking something into your participants colon to directly measure this as it's happening in vivo, but it's quite a good substitute methodologically to try and assess DNA damage and genotoxicity on human colon cancer cell lines using this method. When we're talking about fecal water genotoxicity, which sends a bit strange, that's essentially what it is. You're looking at the potential DNA damage in a colon cancer cell line caused by taking a sample of fecal water from a participant and incubating it with that particular cell line. A early paper to do this that I think is quite helpful to look at was a 1999 paper published by regorant colleagues in the journal Carson a Genesis. It compared two diets that were consumed sequentially for 12 days. It was a one week wash out period in between where they just consumed their normal diet. The first diet was 50% of total energy from fat, was high in meat, and it was also high in sugar, but low in vegetables and dietary fiber and free of whole meal products, roughly getting at a crappy western diet type of approach. The second diet was high in vegetables and whole meal products, but lower in total fat, which was now just 19% of total energy and low in meat. The primary outcome was as I just described this fecal water genotoxicity. When they looked at DNA damage as this outcome after incubation with fecal water after the high meat and fat diet, that was 28% and whereas the second diet that was high in fiber and vegetables was 17%. The DNA damage from the fecal water incubated with these cancer cell lines following diet one was basically twice that of the second diet. There was in terms of some of the other kind of outcomes, they looked at oxidative DNA damage as well, significant increase in oxidative DNA damage compared to control. This was an analysis then that basically you could include that the diet, high in meat and fat, but low in fiber and vegetables increases the genotoxic potential of fecal water that's reflected in increased DNA damage evident and oxidative DNA damage in these human colon cancer cells. Again, that could be something that contributes to an elevated risk of colon cancer with these kinds of diets. Now, the caveat of this study as you probably already inferred is this wasn't really conducted with the isolated control of some of the previous research. We were really looking at dietary pattern type of interventions where there's multiple component parts and that are not matched between those diets. We're not really left with, as I said, describing the et cetera, diet one was high in meat and fat, but it was also high in sugar. We're left with a lack of ability to more specifically isolate the independent effects of red meat in this particular analysis. We have this support for the broader idea that diet itself and certain types of dietary changes can alter this genotoxic potential in the colon, but given the limitation of this study, we can't isolate this effect to red meat because we have all these various different things changing within the diet from fiber to fat content to the amount of vegetables we're consuming. But it does show us this potential and one of the things that maybe other work can allude to that we might see these changes within the colon relative to these types of differences between diet. Yeah. So there's, I think there's two papers that we could look at to look at some of the fecal water genotoxicity outcomes. There is a Dino and colleagues paper, which was actually more recent, was published in December of 2025. And there is a Hebbels and colleagues paper, which was published a decade earlier, which was 2012, which was over a decade. The Hebbels and colleagues paper basically had individuals who had a baseline average red meat intake of 88 grams on average per day. So again, not dissimilar to some of the kind of low intake, low wear intakes that we've seen in some of the amount of jane cross and Bingham and colleagues papers. They then went on to a seven day high red meat diet with around 300 grams a day, consisting only of beef products that were pan fried in terms of their the cooking method. And their specific outcome of interest was this fecal water genotoxicity and also anitrosocompan formation. But the fecal water genotoxicity that increased significantly with the addition, obviously, of this 300 gram a day meat diet compared to the kind of baseline lower red meat intake. It didn't show an increase interestingly in fecal anitrosocompan formation following the 300 gram a day red meat intake. And so this particular study is the first that you don't see that increase in fecal anitrosocompan formation at a dose of 300 grams a day, which is actually somewhat intermediate relative to some of the doses used in the previous research. But the previous research I think was better controlled. So I would still run with the conclusions we've seen with some of that kind of metabolic ward studies. But this basically added to that previous study by looking at this kind of fecal water genotoxicity outcome and seeing a significant increase in that in terms of a seven-day sustained diet that was high in the, that was high in red meat. Now, I think the problem with this study potentially is that you also had participants with moderate grade inflammatory bowel disease. So I think that somewhat limits in terms of obviously making inferences based on intestinal health, six of them had diagnosed inflammatory bowel disease, and another six also had irritable bowel syndrome. And while that is something that we wouldn't expect in underlying pathology in terms of gastrointestinal function, I still think that kind of presence of half the subjects with IBD probably limits some of the kind of inferences that we can make particularly in relation to the M.I.T.R.S.O.C.A. compound formation, whatever may be going on in that regard or overlaps with inflammation. And in the previous studies, we were looking at otherwise healthy individuals. So with that, I think the more recent paper, the denu and colleagues paper is actually probably more instructive, at least in my opinion, because it was a really nice design that thought through some of the potential moderating or modifying factors that you can see in the literature. So they had three exposures, diets, intervention diets. It was a cross over design, sorry, a parallel group design where they were either consuming a 900 gram per week red meat intake, high meat intake. And that was a mix of unprocessed red meat, processed meat, and they also had 150 grams serving of poultry. So I think important to bear in mind that we're looking at weekly doses of intake now in this study rather than high daily doses. Then they had the same diet, so three, sorry, what they were asked for 150 grams servings of unprocessed red meat, 350 grams servings of processed meat, and 150 grams serving of poultry. That is same high red meat diet, but supplemented with 100 milligrams a day of alpha to cough for all. And then there was a pescovegetarian diet where participants excluded all meat and poultry and included 350 grams servings of fish per week. They were prescribed a similar macronutrient intake, and this was also looking at this outcome of fecal water genotoxicity as its primary outcome. And for fecal water genotoxicity, DNA damage increased on the meat based on the high meat based diet by 12% compared to the pescovegetarian diet, and interestingly by a similar amount compared to the meat based diet plus alpha to cough for all. There was no significant difference in the generation of DNA damage measured through this fecal water genotoxicity assay between the meat based diet with added alpha to cough for all and the pescovegetarian diet. And when you looked at the within group difference, it was the high meat diet that basically showed this significant within group increase from their before diet by 16% increase in DNA damage compared to baseline, whereas there was no significant difference in either the meat based diet, same diet plus alpha to cough for all or the pescovegetarian diet. And there were some other interesting changes in terms of markers of lipid peroxidation, although some of the more robust measures of lipid peroxidation that you can look at, there were no significant differences between groups. And this trial for me is a really good example of the potential for moderating factors. In the previous study we discussed, there was the addition of this kind of high fiber wheat brand to a high meat condition, and that didn't abolish the generation of anitrosocompend formation at a dose of 600 grams a day of red meat intake. This is a weekly dose and I think we have to bear that in mind, distributed across the week. But it's still very interesting that the addition of the primary fat soluble antioxidant, which is alpha to cough for all, to the meat based diet effectively abolished the increase in fecal water genotoxicity observed on the high meat based diet. So that I think speaks to some of the complexity that we can sometimes see in some of the epidemiology and the potential for moderating factors to influence associations. And some of these are obviously not necessarily always captured in prospective cohort studies. And it does suggest at least some degree of moderation by fat soluble alpha to cough for all in at least this specific outcome, what it would say for other potential mediating effects. And what it might mean if the study focused exclusively on unprocessed red meat, where is this actually included white meat as well, although much less of a dose of that. All of those are open questions. The study doesn't particularly answer, but it is consistent with suggesting overall that the diets obviously lower in unprocessed red meat have a lower coloractyl cancer risk profile. But there are other factors within a diet that may moderate the relationship, at least with some of these kind of intermediate outcomes. And so when we're looking at some of these, this mechanistic work to try and piece together, is there this causal effect? One of the other studies maybe were touching on before we start pulling all of this together was 2021 paper, Gerjow and colleagues. And this was interesting because this specifically looked at human coloractyl cancer tumors and found this association with the higher red meat intake to these, this kind of mutational signature on these tumors. So essentially these signatures that they're able to trace back to see what might have been involved here in a causal way and connecting it to some of what we're seeing with these components of red meat. Is there anything within this particular study that's maybe worth highlighting that might be of interest that could add something else? I think for this, so this was an analysis of the US, the kind of two longstanding US cohorts, the nurses health study and the health professionals follow-up study. And it was specifically looking at, like you said, this alkylating mutational signature in coloractyl tumors. And this analysis, I think, is probably so they looked at, they identified seven different mutational signatures that could be identified in the coloractyl cancer tumors, one of which was this particular alkylating signature that they highlighted was undescribed previously in coloractyl cancer. It was enriched in tumors that promoted methylation as a kind of process by which cancer cell proliferation can occur. And this was associated with a high intake, not just of processed red meat, but also unprocessed red meat and overall red meat. And no association was found for either white meat and chicken or fish. So yeah, I think this is an interesting additional analysis from these cohorts that gives us again some degree of linkage between some of what we saw in the kind of short term, mechanistic studies, and some of what we might see in the longer term prospect of studies. These are studies of hundreds of thousands of men and women in the US. It is very limited demographically and does not represent the wider US population. So I think that has to be said as well. But in terms of these signature kind of these kind of metabolomics essentially based alkylating signatures associated with unprocessed meat, but not with chicken or fish, I think that's relatively consistent with what we've seen with the outcomes of an Ichoce of compound information generated by hemeyer in the controlled interventions. So it provides I think another link in the evidential picture of there are mechanistic carcinogenic processes in the colon that are influenced by and these are cohorts that do have high intakes of unprocessed red meat. And I think that's important. And we're thinking about the dose response I mean typically in the NHS or the HPFS. The high quintile is often in this range of over 180 grams a day. So yeah, I think it's a nice analysis that kind of adds to the picture of causal pathways that could explain a relationship between this exposure of unprocessed red meat and color rectal cancer as an outcome. Yeah, it's another one that kind of fits in and aligns with some of that and again still with open questions. And I think here we do have within that red meat consumption, it was unprocessed and processed here, but we still see that kind of ten, the 10% of highest red meat intake had the highest amount of that alkaline signature and that intake ends up corresponding to those types of figures we mentioned, I think about 150 grams or above. So so fits in there, but just maybe adds another little piece. So I suppose to start pulling this together and do it to what people really want to hear from you on a topic that as we've noted is got some lots of interesting questions that maybe still we can say certain things with confidence, but maybe when we come to clear conclusions that's maybe another thing. So if we were to pull this together from every thing we've said, Alan, as opposed to what people want to get to is first from purely talking about the evidence we have in this area. What are some things you think we can conclude with some degree of confidence that are fair conclusions to say about the current state of evidence related specifically to unprocessed red meat and this outcome of color actual cancer risk. What would you have, yeah, a fair degree of confidence in saying this is what we can state based on the current evidence base? Yeah, I think we've got based on evidence from the controlled feeding studies that we were discussing. If we're trying to understand is the relationship between unprocessed red meat as an exposure and color actual cancer as an outcome that we observe in epidemiology is that a true effect or is that something that people can just dismiss as well as just correlations and aquasization and all that kind of jazz? I think we have for unprocessed meat red meat, we have clear evidence of an increase in carcinogenic compound production. We've clear evidence that is primarily mediated by heme iron and we have clear evidence of a dose response relationship that this effect is primarily observed at doses of at least based on the evidence we can say over 240 grams a day. Now, is there likely to be similar effects at something like 200 grams a day? arguably. When we do look at the total body of these controlled feeding studies, those increases in carcinogenic compound production are relatively consistent at these higher doses of unprocessed red meat intake. That is generally consistent with the type of threshold effects that we tend to see in the epidemiology. So I do think that it is fair to say that there is a degree of biological plausibility to the effect of a high on processed meat diet sustained over time in terms of long term coloractal cancer risk. But I also think that there's an equal plausibility based on these same studies for the lack of association that we see with intakes of less than 100 grams a day. And I think that is not simply an absence of being able to detect an effect which we could potentially be left with sometimes wondering in the epidemiology because of the fact that so many of these trials had controlled or comparative exposures of 60 grams a day where we didn't really see any sort of difference in the production of these same carcinogenic compounds that we see at higher doses. And I think when you have a dietary pattern with red meat in that average range, that also typically would be uncommittedly higher in either other white meats or fish or indeed higher in fiber-rich whole grain foods, legumes and vegetables and fruits, typical of dietary patterns that are lower in red meat. I find it very difficult to conclude on that basis that unprocessed red meat is still "cozel" in that kind of context. And so I think that what is offered by the epidemiology that there is some sort of dose threshold in this relationship between unprocessed meat, I believe is supported by the evidence that we have from these controlled feeding studies. And again, there's a lot to discuss maybe for another time. I know we have a plan to revisit this topic of substitutions, comparisons. We have a previous episode that I'd encourage people to go back and listen to. I was a 300 and something called "Compared to what?" We'll be revisiting that topic in an upcoming episode, but there's also that is quite pertinent to some of the things that you've just discussed, given that as people have noted, not only through the studies we've discussed today, but also the epidemiology we see in the trials, maybe different comparators that have been compared to red meat in each of these situations. And then with epidemiology, depending on what type of substitution analysis we're doing, we can have it being compared to not only different things, but typically the background diet that is filling in the gaps. And there's some nuance for us to discuss, but again, that's maybe for another point. So given that those conclusions that you've just put in place to maybe put you on the spot, even further as we finish here, there's people that maybe are thinking, "Okay, going beyond that as an individual that they might want to make some decisions based on this evidence and thinking about the question of to what level of intake should I feel safe consuming, let's say, unprocessed red meat, given that there is this zone where maybe we're not seeing an effect, polls will be that we're not there is no effect at these certain levels that there's some evidence that as you said could support that, but given that we're seeing that for depending on what we're comparing it to and what to maybe replacing that red meat or not in the diet, there could be differences here. What would be an intake that someone should say, "Okay, if I am going to reduce my red meat intake because currently it's now high, where should that go?" And would I foreseeably continue to see a benefit if I continue to reduce that down to, let's say, ultimately zero, depending on what then replaces that. How do they work through that at an individual level given the current gaps we might have in the evidence? Yeah, I think given those gaps, the only thing that you could really conclude is that under 100 grams a day is a desirable target for someone that currently consumes a high red meat diet. I think it's pretty clear that the substitution that would be desirable for that reduction would be primarily if the diet, if they are eating high red meat diet and it's low concurrently in other protein sources, likely gooms and stuff, that that would be the primary substitution that we want to make. And secondarily, if they also eat an omnivorous diet, then in terms of getting that red meat consumption down to those intake levels, then obviously then white meat or fish probably would be preferable even to white meat, I think, for various reasons. And that would probably be the kind of order of hierarchy by which they would prioritize changing their diet. But I don't think that we can, based on the evidence that and the gaps that do exist fill in the gaps with anything more than the kind of dose threshold that we've identified. And I think that really the only way that we could say that, and obviously this then depends on individual risk. But I think the only reason really threshold level or range that we could say with any degree of confidence that this is somewhere where if you were determined, if someone was saying, I'm not going to cut meat out of my diet, then I think that compromise would be, well, then this is an intake range where you're at least at lower risk, a takeover if substitutions are made with legumes and whole grains and the kind of plant component of diet and make sure that vitamin E is in there somewhere. So that's really useful. We have a situation that if someone currently consumes high intakes of unprocessed red meat, maybe because their position has been, well, it's unprocessed, it's not necessarily negative or the rest of my diet is okay. The implication is here, well, if you're consuming a high level, it's probably worth limiting, limiting that where we can have confidence where you would see that risk reduction is getting it to below that say 100 gram per day level. If someone is below that level, we can confidently say there's a lower risk there compared to higher intakes. And then where it becomes a bit more tricky than is saying, going beyond that point, if someone did continue to reduce that further, would there be continued risk reduction and how far that would extend? Maybe one thing that could fit in with that is if someone did want to, let's say, theoretically reduce all possible risks that red meat unprocessed moderate might contribute. Of course, then the option is to reduce that to zero if they wish. But again, that is different from concluding that maybe based on current evidence that we can say that would be the only safe level for someone without reducing risk. We're a bit away from that. But again, at an individual level, someone could do that and say, I want to reduce the possibility of it contributing to that, absolutely zero. And so that is maybe as much as we can conclude, given these numbers we're seeing. Yeah. Yeah, I think so. I think so. Excellent. I think this has been a really useful episode and hopefully a lot of you listening have enjoyed it. Alan and I will be back with another episode coming soon. We have some, what we think are really interesting topics that we're going to be discussing in the future. So hopefully you join us for that for those of you who are sigma nutrition premium subscribers, you can get a set of detailed study notes that goes through everything that we have discussed in today's episode as well as an edited transcript that will be linked up in the description box. And if you're listening on the public feed of the podcast, then click into the description box for more information about premium subscriptions or just anything related to today's episode. So that is that Alan and I will be back another episode soon. Please join us for that. And in the meantime, have a great week. Stay safe and take care.