It is fantastic to have Dr. Kleiner back, and it's an honor to host my colleague, one of my mentors, Dr. Andrew Stoltz. For those of you who don't know, Andrew obviously is well-known in the Dillon, but he has a very specific interest in anabolic steroids, and when I met him, he was doing actually research in prostate cancer, and he was really into this whole steroid stuff. So this, I think, is a very natural fit for him. It's a very complicated chemical structure, and he's really interested in it. So today's case-based discussion is going to center around a patient who presents with cholestasis after taking anabolic steroids, and we're going to talk about the presentation of Dillon in this context. We're going to review the pathology with Dr. Kleiner, and I'm going to go ahead and introduce our speakers so we can get started. So the first speaker is Dr. Andrew Stoltz. Andrew's a professor of medicine at the University of Southern California. He trained in New York and went to Albert Einstein, where he got his medical degree, did an internship in the Bronx at Montefiore and residency at Mount Sinai, and came to UCLA, like I did, because he wanted to work with Neil, I mean, LA. I went to USC by that time, and Neil was there. So he worked with Neil for many years and eventually started his own lab. For a first period of his scientific career, he was involved in, sorry, I apologize. He was involved in, turn this off, research into prostate cancer, and specifically, Andrew has a lot of interest in genetics. He eventually became the PI on, a site PI for our Dillon U01, and also worked on the alcohol, NIH alcohol network from the NIAAA. He is the co-chair of the herbal supplements and dietary supplements induced of the Dillon, and he has mentored so many of us here at USC. He mentors a lot of the faculty, and we all appreciate him very much, and yeah, I'm trying to go through your CV because you didn't send me your bio. He's multiple publications and is really interested in drug-induced liver injury. David Kleiner is well known to us. This is, I think, the fourth time, David, you're presenting one of these case-based conferences. He trained at the University of Chicago. After residency in anatomical pathology at the NCI, then he stayed at the NCI, and he's the senior research physician and chief of post-mortem section at the Laboratory of Pathology, and his expertise relies in hepatic pathology. His research focus has been in drug-induced liver injury, liver cancer, and histological responses to therapy, and he is the pathologist on the NASH CRN, the Dillon, and the Hepi Research Network. He's co-authored many, many articles and review chapters and has contributed a lot to the Dillon. So it is my pleasure to welcome you both. Andrew, thank you for joining us while you're at the LCN meeting currently in DC. Please take it away. Okay. Well, Louis, thank you very much for that very kind introduction. Let me just get my slides up and put this in presentation mode. Okay. Continue to share. Okay. So what I thought I would do today is really give you an opportunity, using a case that was actually enrolled into the Dillon approximately 10 years ago, but this case really highlights some of the features that we see in anabolic steroid-associated drug-induced liver injury, and it's an opportunity to talk about the clinical presentation, as well as also, in this patient developed renal insufficiency, and we'll review for you as well some recent data from an animal model that might hope to show a potential cure, a potential treatment for this as well, and if time is permitting, to also review some of the genetics that we've identified as such. So first, this is the first slide, and let me just get my pointer here to annotate with you, and this is really a very simple slide just to recall that the effects of androgens is mediated in part through testosterone, which is the major androgen which is in the bloodstream, bound to a sex-alignment binding protein. The testosterone itself then enters into the cell through diffusion, and one of the issues with testosterone is that through aromatization, you can therefore generate estrogen, and so this is one of the issues in terms of using testosterone that is a concern in terms of patients that are using this. They don't want to get the excess effects of estrogens. Now once the testosterone enters into the cell, it then binds to the androgen receptor shown here, which then dimerizes and then binds to androgen-responsive elements, which then leads to the expression of those genes that are regulated by androgens per se. The more potent androgen actually is dihydrotestosterone, and this is mediated by the enzyme 5-alpha reductase, and the expression of this is limited, and a lot of the secondary effects of testosterone, which one thinks about it, are more the androgenic, that is those features that define more the male sex characteristics, are mediated in part by DHT, and therefore the localization of the enzyme, which is responsible for generation of DHT, it will also be those tissues that are associated with secondary sex characteristics, and the 5-alpha reductase responsible for this is in the limited number of tissues, including the liver, the skin, the prostate, and other organs involved with the testes formation, sperm formation as well, as well as the kidney and the brain as well. Now one issue then is to consider is that the anabolic steroids that are then used predominantly by people who are looking for muscle fitness and for performance of appearances as well, is therefore to try to identify an agent which will preferentially produce more the anabolic effect than the androgenic effect as well, and this is just to show you these are medications that are available, that are used commercially for treatment, and notice that testosterone, which has approximately a one-to-one ratio of anabolic to androgenic effect, in fact as you get to some more of these agents which have been used inappropriately or has been available through the internet, for example, has shown in fact that there is much greater formation of the anabolic effect per se than the androgenic effect, and in part this is because of this desire not to have the effect of estrogens being formed from testosterone, and therefore there are modifications that are undertaken. And in addition, some of these are medications that can be taken orally, which then provides an advantage as opposed to being given by IM injection, and our desire is such then to prevent their metabolism by the liver to allow them to be more effective. So the presentation that I'm going to present today really gives an example of what we have found in our experience in the villain as to those people that are exposed to anabolic steroids. So we have a 35-year-old male, previously healthy, who has been in a history of asthma, who has been treated PRN with albuterol, so an asthma agent, and he presents with new onset fatigue and jaundice in January 2015. And this occurred after starting an oral anabolic steroid supplement twice a day which he purchased online, referred to as trimethyloxtreme by EPG, and this was in the end of November. So we have a patient that presents after starting these medications about six or eight weeks earlier, who then presents with a profound cholestatic syndrome as well. Now as I'll describe to you later on, this patient actually has a lot of the clinical features that we see in those patients that in fact consume anabolic steroids, that being male, and also with a desire for increased fitness and the anabolic effects of associated muscle build. Now interestingly enough, if we look at the patient's past medical history, there are other clues that we see that are similar to those other patients that we've enrolled. First of all, there's a long history of working out in the gym four to six times a week and a prior history of taking bodybuilding supplements. So these are important clues in those patients that present in this kind of clinical pattern is to get a pertinent past medical history that will then look for the exposure to potentially other bodybuilding supplements. And in fact, this patient in January of 2014 started taking another agent that he bought off the internet called OneXXD tablet twice a day. And the reason why he took it was this was reported to increase energy and serum testosterone. Now on the right here, you see what's in here. And in fact, there are no, as reported at least on the label, there are no steroids present and just various botanical agents that are associated here that are supposed to increase the serum testosterone level. In this case, that was not the case, and he stopped taking these medications. So when did he present, initially he was noted new onset of fatigue. He had a decreased ability to work, his workouts, and he was stopped taking the supplement in the beginning of January. And as often as the case, his episode of jaundice were not noticed by the patient, but rather by colleagues and friends that noticed that he was becoming jaundiced. An initial blood test done in the beginning of January revealed a little over a seven and a half, a significant elevation of the AST and the ALT. And there was no alkaline phosphatase performed. And we found that, at least in our cases in the Dillon, that this was a feature that was associated initially with the initial presentation as often the transaminases were elevated more in proportion with the ALT phosphatase not being as elevated. Patient noted an acholic stool, severe paritis, and a darkened urine, so all consistent with a cholestatic picture. He continued to be seen by his primary medical care physician, and they had follow-up labs which continued to show a rising bilirubin, as you can see, back in January 21st of 24 with a slight reduction in his ALT and AST and a relatively normal alkaline phosphatase. And as his disease progresses, bilirubin increased, the transaminases became further decreased, and the alkaline phosphatase increased as well. So subsequently, he was then admitted in the mid-February with a bilirubin of 48.9, minimal elevations of the AST and ALT with an elevation of the alkaline phosphatase. And of note, there was a creatinine of 3.36, so this was something, although we don't have prior creatinine levels on this patient, this was clearly abnormal and led to it being admitted. Of note, he also had a 25-pound weight loss, he had severe paritis, nausea, and had painless jaundice. His symptoms were those of severe paritis and insomnia, and physical exam noted a deeply jaundiced male, multiple excoriations, and there was no evidence of chronic liver disease, there was no spiders, hepatosplenomegaly, ascites, or peripheral edema as well, suggesting that there was no chronic liver disease in this patient. Now, the workup at that time done to exclude other causes of post-stasis included a P-ANCA, a C-ANCA, ANA, anti-mitochondrial, anti-smooth muscle that was negative, his hepatitis serologies were all negative, and a cerebral plasma of 47. So there was nothing to suggest some other potential etiology for this disease as well. And he underwent imaging, which included an ultrasound, which was no evidence for cirrhosis, nor biliary duct obstruction, which was obviously an important consideration in this patient. A contracted gallbladder was noted, likely due to the patient eating prior to the exam. There was no evidence of cholelithiasis or acute cholecystitis. And it was noted that there was bilateral renal echogenicity, which may be related to the renal disease. And as I noted previously, the patient did have a Fragment of 3.36, and a prior MRI or MRCP, looking for other evidence of biliary obstruction, was reported as without any evidence of dilatation as well. Now the elevation in the creatinine led to a nephrology consult, and the determinants was a non-oligarchic acute kidney injury. And the UA had minimal protein, and no RBCs, or cellular cast, or negative for eosinophilia. So this sort of essentially, the differentiation was an acute tubular necrosis, versus a association with a nephritis, possibly due to some agents, that was unclear. But there was no evidence of any glomerular dysfunction as well. It's important to note that anabolic steroids have been shown to be associated with focal segmental glomerulosclerosis, but in the absence of an active sediment, this makes this unlikely. And the patient was then treated for metabolic acidosis. This just sort of shows you, over the course of this patient, showing his initial hospitalization with a creatinine of 3.36, and then over time, was discharged, and it took a significant amount of time before the creatinine improved in this patient. In fact, oh, okay, so I'm going to show you a little bit later about the mechanism, presumably. But given the fact that the patient presented such severe cholestasis, as well as no evidence of obstruction, the patient underwent a liver biopsy. And the report from our local pathologist was a cholestatic hepatitis with duct damage and focal duct loss, so there was absent to mild portal fibrosis, focal mild iron deposition, predominantly in Kupfer cells, and there was no alpha-1 antitrypsin lobules. So I will stop sharing, and then allow David to review at least this, the biopsy of this specific patient. Andrew, this was a County patient or a Keck patient? It was a Keck patient. David, you're muted. Sorry about that. So we got recuts of the biopsy to review in the Dillon. So I think it was somewhat smaller than the one that they had. What you can see is that there's a fairly mild lymphocytic infiltrate in the portal areas. Very little in the way of interface hepatitis. There's not much happening around the edge. There's some histologic artifact, which is all of this sort of horizontal cracking lines, which can make some of the features harder to see. But really, the inflammation was quite mild. The cholestasis is actually quite subtle in this case compared to some of the other cases that I will show later. So this is the central vein here. And the cholestasis in drug-induced and many other types of jaundice appears first in this area. And if I use the arrows to sort of highlight where the little foci are, you can see these little canalicular cholestatic plugs. So you'd look for these dilated canaliculi, which in this patient were very small. And you can see this material, which isn't all that pigmented. It's still more pink than it is green. But looking around, once you sort of train your eye to see it, then you can find some larger things. You can see it's kind of fluffy in this patient, not as solid and gloppy as I would like. But it was easy to find once you sort of train your eye to see what's there. And so this is the keratin 7. The fact that there are two things here, actually. So there's the staining of hepatocytes out in the middle of the parenchyma. This is a change that you see in cholestasis. So when you get bile accumulation with those canalicular cholestatic rosettes, you can start to see ductular hepatocytes out in the parenchyma. And this patient also had some hepatocytes that were positive next to the portal area. So this goes along with the duct injury and possible duct loss, some chronic cholestatic features. It's a little bit different meaning. There is a little bit of ductular reaction around the edges, but not very much. So canalicular cholestasis with minimal inflammation, so-called a static hepatitis pattern, consistent with the anabolic steroid-related injury. And so that's his biopsy. And now I will turn it back over to Andrew. Andrew, I think you're muted still. Yeah, thank you. Yeah, so the biopsy, as David showed, sort of greeted sort of what we saw here with the whole stasis noted in the pericentral area. So I wanna go over then to sort of show you the pattern of liver tests that this patient had throughout this period. And to sort of point out that the AST and ALT ratio initially, and this is from the day of onset, so when the patient presented back in January when the initial enrollment occurred, as you can see that the AST and the ALT, but only the ALT is much more elevated in the initial phase of the injury, whereas the alkaline phosphatase tended to rise later after the presentation of the injury, much higher levels occurring in the mid, approximately a month after initial presentation with this generally decreasing in time and associated with a significant increase in the bilirubin as well over time, although recognizing that when the patient was admitted to the hospital, he also had significant renal insufficiency, which is likely then to have also driven the increase in the bilirubin. And of note, the INR was normal at this time as well. So this is sort of the pattern that we have at least seen in this patient, and as I'll show you later in reviewing our experience in the Dillon is what we found in other patients as well. So this is one of the unique features of these cases. Another unique feature that we found both in this patient and other patients was the gamma-glutamate transpeptidase. In fact, despite being severely cholestatic, at least by clinical, actually the GGT was often normal or minimally elevated. And as you see on enrollment, the GGT was within normal limits. There was a slight increase later on, and then later on at the end of this patient's presentation, the GGT had gone down further as well. So this was something that we found that uniquely occurred in these patients as well. And I'll show you, this was also our experience previously, and suggested sort of a different mechanism of injury than we might see in normal cholestatic injury. Another thing that we noticed was that the, as been reported previously as one of the complications of anabolic steroids is that there's elevations in the lipid panel as well. And as you can see in this patient, now a month after presenting in the admission, there was an elevated total cholesterol and LDL cholesterol. And then as the liver injury resolved, you can see a nice resolution of the total cholesterol, the triglycerides, although the LDL cholesterol calculation is still elevated, there's a significant improvement as well. And this has also been well-recognized with anabolic steroids and is associated with chronic cholestatic disorders as well. So I wanna sort of review the features that we then saw in our 44 patients that we reported back in 2019. And I'll also compare this with the Spanish, and hopefully I've seen some of the Spanish investigators as well, and it'd be interesting then to look at their experience that they found with their cohort of patients. So first of all, in these patients, they were all males. They were all relatively young. Oh, I have to turn on my marking here. Hold on. So they were all males. They were relatively young. In our population, they were predominantly Caucasians as well. Their weight in the BMI was reasonably normal, though there was a wide range. There was a fair amount of alcohol use in these patients, and this was something that we had also noted in a prior publication as well, is that those patients using anabolic steroids had a high incidence of alcohol use as well. There were some of those who were HCV positive, and it took about, in our experience, about 60 to 70 days in order to, by the time of presentation after being exposed as well. DRONS was a feature in all our patients as well as pruritus, so suggesting, again, a very full static pattern of injury as well. And there was some nausea noted as well in these patients as well. Let me just go back. I need to move the screen, okay. And so what about the laboratory features in these patients as well? So oftentimes, on initial presentation, there was mild elevations of the ALT. The AST was not as elevated as much. The alkaline phosphatase was relatively within normal limits. The bilirubin was elevated in all these patients. And when an R value was calculated at the initial presentation, we found that only about a third of these were presented with cholestatic, another third presented with mixed, and another third, 40%, presented with hepatocellular pattern of injury. So this sort of suggests that the initial elevation of the AST and ALT were responsible then for the elevations in the R value. And then if we look sort of at the peak levels, the AST and ALT are elevated in some cases in some patients quite dramatically, and the alkaline phosphatase is elevated as well, but not to the same degree. And again, with the significant elevations in the bilirubin, as well as the INR, not being noted to be elevated as well. And then these patients had a profound period of cholestasis with an elevated bilirubin for almost greater than three months, another clinical feature of this disorder. And one of the problems that we had is that these patients often didn't return for their six-month follow-up. And so the follow-up was actually quite poor in these patients. So the length of chronic liver disease per se is difficult to assess in this population as well. So this slide then highlights one of the patterns over time of the R value and the transaminases that we observed in our population. So again, shown here in the blue, the ALT is often elevated at the initial presentation. The bilirubin is elevated as well, and the alkaline phosphatase in red is minimally elevated. And over time, you can see that as the bilirubin then increases over time, that there tends to be an increase in the alkaline phosphatase as noted by the red bars here. And then with resolution over a long period of time, there is eventual resolution of these abnormalities as well. So at least in our experience with those patients with exposed anabolic steroids, we see that there is an initial had a cellular injury followed by a more colostatic period with not that significant elevations in the alkaline phosphatase. Interestingly enough, the group from Spain also reported their experience, and there are a number of differences in their experience as compared to ours. And one has to do with the identification of the specific agent responsible for the injury, where we in the United States, as I'll show later, were not as, the actual agent used was much harder to actually determine than as compared to the cases reported, the 25 cases reported in the Hispanic, in this study from Spain and from Latin America. And they also divided these both based on a had a cellular and colostatic appearance, in part based on their elevations of the total bilirubin as they presented as well. Most of their patients presented with jaundice, as I've noted, there were hospitalizations in about a significant number of the patients. And the elevations in the ALT were much greater in those presented with the hepatocellular pattern as compared to those with the colostatic. And conversely, the bilirubin was more elevated in the colostatic as well. And interestingly enough, they also noted that there was a greater chance for renal insufficiency in the colostatic, much like we observed in this patient in particular. And again, the time of resolution was quite large as we also observed in our case as well. So an interesting thing that the Spanish group noticed was this association between the elevation of the bilirubin and the association with renal insufficiency as shown here in this slide. And so I suspect that this was part of the, was driving the elevated bilirubin, in fact, was also the elevation in the renal insufficiency associated with damage associated with the anabolic steroid exposure as well. So we can see here that the significant increase in the fragment as well, whereas those in which the bilirubin was not as elevated had a much lower incidence of having actually renal insufficiency as well associated. So that was of interest to note that association. So I thought what I would just do now is just sort of briefly then describe to you what are the possible mechanisms for the cholemic nephropathy that has been associated with described with anabolic steroids. And this is well-known and it's also big, it can be associated with cholestatic liver injury as well. And in part, some of the association with the anabolic steroids may in fact be compound related. And there have been previously noted associations in anabolic steroid use with renal injury and even association with occasional renal carcinoma. The association is not as well for those. As I mentioned before, the glomerulosclerosis has also been identified with anabolic steroid use as well. So I've just taken this as a slide which looks at the causes of renal insufficiency in patients who present with jaundice. And I really wanna focus here on the bile acids per se as a potential mechanisms, because we recognize that in those patients who present with cholestatic injury, particularly in these patients, that there is elevated serum bile acid levels. And therefore the bile acids are likely, or certainly in this instance in particular, are likely to be a major cause of injury in these patients. So just to sort of review for you what the kidney does with regards to bile acids is that as the blood is entering into the glomerulus and there is ultrafiltration being formed in the tubules of the kidneys then, we start seeing is that in those patients, we start seeing injury in the proximal tubules as well. And eventually this can lead to the sloughing of these cells and then further obstruction in the more distal parts of the nephron as well, thereby thought to be leading to renal insufficiency. And so this is sort of one of the accepted mechanisms. And I'm gonna review for you now a paper actually looking at a mass model of renal insufficiency associated with bile duct injury that suggests that there may be a possible role or inhibition of bile acid uptakes by the renal cells in order to prevent getting renal injury. And I'm not gonna go through the details of this, but I will show you then a summary slide and I'm referring to the studies shown in this journal of hepatology paper from I believe it's June of last year in which they looked at the inhibition of the renal apical sodium dependent bile acid transporter to prevent cholemic nephropathy in mice with obstructive cholestasis. So just as a reminder, and we can still look at a summary slide. If we look at sort of what the potential implications of what is thought to be in this the bile duct induced the renal injury in these mice is to recognize that in the hepatocyte, bile acids are taken up by the NTCP, they enter into the bile acid. We assume that an anabolic steroid associated cholestatic injury that there's a accumulation of bile acids which in the liver that then leads to being excreted back into the sinusoid predominantly by MRP3 and MRP4. This then appears in the blood, which then in the kidney then allows for the glomerular filtration as the bile acids will need to be reaccumulated so as not to be lost. And it turns out in the kidney in the proximal tubule that the same stone dependent bile acid transporter that we see in the terminal ileum is present also in the renal cells as well. This then is taken up by the renal cell in the normal conditions and then again, it's pumped back out to reattrieve those bile acids that are taken up and thereby allowed to be reabsorbed, excuse me, it's reabsorbed through the same transporter in the terminal ileum that's present as well as MRP3 to go back into the renal capillary and thereby to reserve the bile acid so as not to be lost in the urine. So in the study with the mice, what they proposed is actually using a systemic IBAT inhibitor. So unlike the ones that we use clinically, which only are inhibiting the IBAT in the distal ileum, so the inhibition occurs only in the intestine, there are other IBAT inhibitors which in fact can enter into systemically and can inhibit the IBAT, the same transporter in other sites. And so in this mouse model, what they showed was that when they gave this inhibitor referred to as ASO369, they inhibited therefore the uptake of the bile acid in the epithelial and the renal epithelial cells, thereby reducing the accumulation of bile acids, which thereby is thought to prevent the injury of the renal epithelial cell. Therefore there's less appearing back in the bloodstream of the bile acids being transported out of the epithelial cells by the OST alpha-1 and the MRP3 and also thereby promoting significant amount of urinary excretion. Now, obviously there are other sites within the body, such as the biliary epithelium, which also contain this transporter. And so the effects of that would need to be considered. But at least in this mouse model, it suggests that by inhibition of the uptake of bile acids into the nephrons, one can be able to then prevent renal injury because there's not accumulation of bile acids and also provides a way of markedly increasing bile acid excretion. So that this could in theory with obviously clinical results being looked in clinical patients, this could in theory be used as a potential treatment for these patients. So what about this agent that this patient took? Turns out it's manufactured by this Extreme Product Group in Las Vegas. It's available on the internet, on retail stores or on gyms. And there are three case reports that thereby triggered an FDA warning back in April 13th. And this is just to show you what's in these comp, what's reported to be in these, in this anabolic steroid preparation. And I've shown you here the structures of these different agents, and you can see related to testosterone, their differences in part, these are being done because although some of these agents have been developed to be given IM, these are all being given orally and therefore their actual efficacy may be unknown. But as you can see, they share a lot of these structural features testosterone as one would expect given the association, the ability to activate the antigen receptor and thereby lead to antigen receptor dependent gene expression. So this is the FDA warning that was released. And so the importance then of this identification of these patients then led to this warning and led to the product being removed from the marketplace. So then the next section, I just wanted to briefly review what our experience has been in terms of looking at those anabolic, those compounds that were taken by these 44 patients and to highlight the difficulty that's involved in actually being able to determine specifically what's in them. And to notice how there is a tremendous amount of disagreement between what's written on the label and what is actually detected, as well as also the presentation of hidden compounds that are not listed and how these compounds are known anabolic steroids that have actually been prohibited from the different leads involved with either the DEA or the WADA or the associated as well. So we can see is that of the 44 patients, we had 57 products that we analyzed. Of those, there were 17 that actually met the label requirement. Those not meeting were about 70% and approximately half the products also contained hidden ingredients that were not listed per se. So if we look specifically at the steroid anabolic products that we had 29 of them, only about 14% of four products actually met the label requirements. The rest did not meet it. And importantly noted that there was over two thirds had some sort of hidden product as well. And when we looked at what actually was detected, a lot of these products in fact were prohibited compounds as noted or by the DEA and actually been removed from the market as well. And also hidden compounds of which there were 12 were noted again, a large number of these were products that were deemed to be prohibited by the Doping Association and also by the DEA. So I just wanna sort of go through, go explain just to go over what are some of those things that were detected on the label and some that were not detected. And as you can see, a lot of these contained products which in fact were prohibited by various different organizations and yet were available mainly sold through the internet. So obviously this is not appropriate and it's actually illegal in order to contain this. And also more importantly, as well as the fact that there were hidden compounds as well noted in about 16 products as well. And again, these are products which are recognized to be anabolic steroids and should not be included. Let's just go review for you again then those that are associated with the anabolic steroids and number that were identified, a small number. Those that only had anabolic steroids, only two of them had met the label requirement and a significant amount also had hidden ingredients. And there were also others with other ingredients included as well. And here again, only a small percentage actually met the label requirement. So the assumption is that if you see a patient that has this and then presents with their bottle that the label is likely not to accurately reflect what they've actually been consuming. And so just to go through in detail what are some of those examples and it may be difficult to read this. So the label claimed certain anabolic steroids. Sometimes they were found, occasionally they were not found but there was another unknown steroid that was noted. In some cases, a steroid was identified and then another unidentified anabolic steroid was noted as well. And so there are a lot of example where you see both the label can be accurate and can either contain or not contain an unrecognized anabolic steroid as well. So this is just to go through the list and to show to you that there is obviously a disconnect between what's written on the label and what's turns out to be associated with the actual compound when analyzed. And again, here's another example where sometimes the label were correct but oftentimes they were not and there were other anabolic steroids noted as well in these compounds as well. And these analysis were performed in Mississippi as all our herbal and dietary supplements are analyzed and they were able then to also look for these anabolic steroids as well. And this is just further showing examples where both the compound was not found and where there was adulteration with other steroids and occasionally there were some labels in fact that did match what was observed as well. So the label is really a poor indicator of what is actually contained in the herbal and dietary supplement as well. So I just sort of want to then review then briefly what are some of the recognized complications of androgenic steroids that include not only the liver but in male, both in male and females shown here, males in the green and females in the purple is that there's also a lot of other concerns in terms of heart disease associated with the left ventricular hypertrophy. There are other effects that can be seen. There can be renal effects as well. There can be testicular atrophy because of the inhibition of the normal pathways that are involved in testosterone secretion and hepatotoxicity we've mentioned as well, and there are also other manifestations that we can see on injury that can occur in the liver. And I'm now going to have David then show you some of the other complications that we've seen as well in the Dillon of those patients who've had anabolic steroid exposure. So I'll stop sharing my slides and allow David then to share his. We see your screen. Yep. So there are different patterns or different manifestations of anabolic steroid injury. The cholestasis is clearly ahead of everything else. These other manifestations are more rare, but we have seen some of them. And usually with the cholestasis, you do get some inflammation. There have been cases of vanishing bile duct syndrome reported with anabolic steroids. And then you get, there are vascular injuries too, and they manifest in one of two ways, either sinusoidal dilation or peliosis, and then nodular regenerative hyperplasia. And then finally, we don't have any examples of this in the Dillon, but tumors have been reported in the liver, most of them hepatic adenomas, which are otherwise fairly rare in men, but also case reports of hepatocellular carcinoma and angiosarcoma, an extremely rare tumor. So this is a more typical appearance of the cholestasis, where you can actually kind of see the color of the bile and the canaliculi are quite dilated. So there's no trouble seeing the cholestasis, even from low magnification, little bit of inflammation here. And you can't have basically no portal inflammation at all, which was true in this case, and you get kind of this bland cholestasis. But more often there is a little bit of inflammation in the portal areas, generally very mild, but you can clearly see that there are lymphocytes here, and here there's a little duct injury to this small duct here. But again, not much in the way of sort of interface hepatitis or what we would be concerned about thinking about some of the other patterns of liver injury. So even in the parenchyma, you can see more inflammation. So these are other cases that we had where you can see here the bile plugs pretty clearly, a few extra inflammatory cells, mostly histiocytes in the sinusoids. But over here on the right, there's not only many more foci of inflammation, but also giant cell transformation. So you get hepatocyte giant cell formation in this patient, which is an unusual thing that we do see in 1% of our Dillon cases. This is an example of peliosis. It was the only one amongst the cases that I reviewed for the network. And this is peliosis here. It's not an artifact. It's not just sinusoidal dilation, because you can see that we've lost the endothelial cells. There are no endothelial cells around this area. It's basically just a blood lake. They're usually very irregular, and they can be very large and can cause hemorrhage outside the liver. And then finally, we have nodular regenerative hyperplasia. Again, this was the only case that we had in the network. But you can see the variation in the plate width here. So here, the liver cell plates are wide. Some of them are two cells thick. But the hepatocytes are normal to large in size. And then around the edges of this nodular area, the hepatocytes are small. And they're only in one cell thick plates. And this was another part of the biopsy. Again, you can see wide plates here and narrower, more condensed plates around the edge. So both of these are variations on vascular injury that you can see with anabolic steroids. So just in summary, canalicular cholestasis with minimal to mild inflammation. That's the most common thing you see. Vascular injury with a variety of manifestations, which is much less frequent. And then tumors, which are even less frequent than that. So we'll stop and let Andrew finish up. Well, I think we have about 10 minutes. So we can open up for questions and comments. And I'd be, technically, because I see some of the Spanish people are in terms of their experience, what they found in terms of their 25 patients with regards to this, you know, if they observed similar pattern with increased transaminases in the beginning, which then over time seemed to reduce with a mild increase in their alkaline phosphatase. So. Yeah, Raul, I know, I know you're on. I was wondering that also, like, is it a timeframe thing? Do you also see the alkaline phosphatase rise late, or do you see patients that are purely hepatocellular? Yes. First of all, congratulations to Andrew and David for this great talk and excellent overview. Excellent overview. Well, it is something that happened in the last year that we are not, right now, not seeing really cases admitted to the hospital with endogenic anabolic steroid hepatotoxicity. There are, right now, very rare. There was some kind of epidemic in Spain in 2014, 2013, but right now it's really, really rare. I think that's the case for us too, right, Andrew? The numbers have dropped off. Yeah, the numbers have come down. I mean, it's interesting that this has also been reported with those that are taking modulators of the androgen receptor, the SARMs. It can also present with a similar, suggesting that the activation of the androgen receptor is essential for this to occur. The mechanism, though, is not clear as to what's causing, why that's occurring. You think the supplement makers are using different things? That's been described. There was a recent review in Australia that noticed some taking these SARMs, selective androgen receptor modulators, are thereby causing, also presenting with a blank all-static pattern of injury, suggesting, again, that the androgen receptor is essential. Dr. Boyer, did you want to ask a question or say something? Sure, thanks. Great talks. You didn't present any data on bile, and I'm wondering whether you had them or not. And my other question has to do, did anyone look at the urine to see whether there were bile casts or bile granules? Because for cholemic nephropathy, you sometimes can diagnose that from looking at the urine sediment. Yeah. So, great question. So, the first one was to do in terms of bile acids levels in these patients? Yes. Yeah. So, we have noted in a genetic study that we did that there are markedly elevated primary bile acids in these patients as well. In terms of the cholemic patient, we did, I don't think that they noticed anything on the urine analysis of that. And we don't typically, we require urine for these patients, but we don't typically analyze them for the casts and things like that. In our population, I think we had six out of the 44 that had a creatinine greater than 1.5. You know, anybody with severe cholestasis, probably should, that should be done. The other comment I want to make is that you mentioned that the GGT was quite low. And I'm wondering if that isn't because bile acids are not getting transported across to the canalicula. And so, they're not chopping off the apical membrane which contains this enzyme. And that's probably because the drugs are inhibiting the bile salt export pump. Although, I don't know, does anybody ever measure that? I was thinking about that as you talked. In terms of whether testosterone is inhibiting the bile salt export pump. I think that's the speculation, but I don't know that it's ever been specifically measured. Measured for that. I know that some of the estrogens can do that, but I'm not sure about the testosterone per se. And oftentimes, these are chemical modifications that, you know, may not get pumped out. But we assumed, as you suggest, that was the reason that GGT is so normal is that the bile acids are not getting, the main problem is that they're being, the excretion of them to the canalicula domain is being affected. And that may be why the canalicula size is so small that David showed us. Very interesting. Nazia, do you want to talk about your patients and what you normally see? Sure. So, I see a lot of these patients over here in Long Island. And typically, it's the same age group. The age cohort is around like late thirties to greater than 40. They're inevitably have, to the extent that I've noticed it persistently is polycythemia. They are polycythemic and they are, their creatinine is also increased. So, this is what I see on a consistent basis, but it's a group, it's a cohort, which is very married to the anabolic steroids. They get them at the gym as an injection, they get them. So, I've, you know, it's very hard to persuade people to stop this. They don't follow up. It's a very kind of a specific pattern of behavior. They don't follow up. They don't want to hear that it's the anabolic steroids. And I also, I'm a little nervous kind of recommending that they stop it suddenly because, you know, there's this potential of withdrawal and, you know, there's talk of suicidality if they stop anabolic steroids suddenly, et cetera. I usually recommend that they should just, the providers are usually not, they're not medical professionals. So, I ask them to taper it down. And then, you know, I've even recommended switching to a, you know, a patch. Is that right? Is that the way to go to in terms of management if they're absolutely refused to stop it? I mean, certainly you bring up an important point, which I didn't mention, which was the psychological effects of the testosterone and whether or not weaning through a patch, I have no personal experience with that in terms of trying to reduce the dependency. I think you have to send them to endocrine, right? Because like they need to monitor their testosterone and figure out how to wean them off. Well, even monitored, I mean, if it's not, if it's one of these alternate compounds, is the testosterone level going to be- It may not be, it may be suppressed as well. So, the mechanism, I wonder if Jim could comment about the iBAT, the role of iBAT, systemic iBAT inhibitor in these cases, whether that would make a sense in terms of the potential treatment to reduce the bile acid level and potentially protect the kidneys. Absolutely. I mean, you, there's been a review of this following a paper from the same group, I believe, showing the mechanism again that you showed so well. I think the iBAT's a really coming drug. We don't have them for adults yet, haven't been approved for adults, and this systemic one has not been approved as far as I know. But the pathophysiology there, as you went through very nicely, is there and has to be a systemic one, has to be absorbed. You made that point, so it's not going to be like the other iBATs. But, yeah. The other thing that iBATs do in general is they markedly lower the bile acid pool size. You know, the primary endpoints have been pruritus and the trials, but I'm much more enamored with their ability to improve liver function. And the reason I think they do that is because they're lowering the bile acid pool size, and so the stimulus from inflammation and liver injury is diminished markedly. But, hi, it's Skip. Jim, this is Jim Boyer. In that paper, I was thinking if you block it getting into the kidney, would you worry that it won't do that? I was worried that the bile acid's pool or exposures to the liver might go up because you're cutting off the kidney's ability to excrete it. Did I… Andrew, is that… It enhances the urinary excretion of bile acid, because they don't get reabsorbed in the normal process. So, the block doesn't allow the bile acid into the cell. To come back into the bloodstream. ASBT is on the luminal side of… Okay. Yeah. I got it. Okay. Thanks. Thanks for clarifying. From the ultrafiltrate, it doesn't get reabsorbed, so you're going to spill a lot of bile acids. Okay. Got it. Thanks. It would provide a way to reduce the serum bile acid level. Okay. Clearly. It's interesting how evolutionarily we've evolved to hang on to all our bile acids. It's so interesting. Absolutely. Great talk, Andrew. I'm just curious about whether… I mean, there are probably large numbers of people out there taking these things. Right. So, is there any evidence that there are any genetic polymorphisms of the transport, you know, the canalicular transporters that could predispose? Yeah. So, that was our hypothesis, and I think I didn't show the genetic data that… But what our genetic data suggests is that there are variations, predominantly in the ABCD11 gene that are associated with it in about 8% to 10% of patients. So, it's not like all the patients have this, which I would have thought maybe might have been the case that they are likely to have it. But there seems to be the functionality of it, whether or not it's a genetic variant in the transporter itself or in the regulation or of its function, as a consequence of the metabolic effects of the androgens per se, it looks like the inhibition of the transporter function is what's driving this. Nice. Very interesting. Nazia's adding that methylene blue is the new drug on the block. I mean, we use methylene blue in endoscopy all the time. Carbon dioxide poisoning, yeah. Yeah. So, they add it as drops to beverages? What does it do? I think maybe for the euphoric effect. Bobby Kennedy uses it. So, it became very popular around that. Interesting. I will keep an eye. Is it the same type of person, like the young male? No, it's just anybody, really. This is for the alpha male euphoric effect. I don't know why they use it. I'm still looking into it, but I've seen, you know, a couple of people have mentioned it. Long Island is an interesting place, Nazia. Yes, it is. Okay. Are there any other questions? What a wonderful talk. Andrew, thank you for summarizing that. And I learned a lot. I didn't know the mechanism of kidney injury. We always talk about how kidney injury results in high bilirubins, but I didn't really. Thank you for explaining that mechanism so nicely. David, thank you for the talk. It was a very nice study. I highly recommend reviewing that study in Journal of Hepatology. Yeah. Being a head of the autopsy service, I've seen a lot of, you know, patients die with very high bilirubin levels, and they all get these bile-stained casts in their kidneys. It's pretty common. And, you know, but, and we all think or know that it causes other kinds of liver injury, but that's what you see. Dr. Matani-Rohit, you wanted to ask two questions before we log off? Feel free to ask them on mute. Consider corticosteroids for the treatment? Yeah, one we considered. I don't particularly recommend it, although it's commonly given. There is some, you know, anecdotal data that suggests that it might help, but there have been no studies that I've come across that review that. And its effect may be more on transporters, per se, in terms of the bilirubin levels. Yeah, so I don't know which country you practice or if you're in the U.S. We generally don't treat dilly with steroids unless you're dealing with, you know, the ICIs or some sort of autoimmune phenotype. Mm hmm. Sometimes you just. Yeah, yeah. Hi, I'm from India. So recently I had a case with anabolic steroid-induced, drug-induced liver injury and the bilirubin was around 42 milligrams per deciliter. He had a stage 1 AKI creatinine of around 1.5, 1.6 milligrams per deciliter. So we did a liver biopsy. There was cholestasis along with lymphocytic infiltrates in the portal traps and some spillover in the lobules with eosinophils also. So a lot of complementary medicine intake is also there in India and the patient had taken complementary medicines also along with the anabolic steroids. So he had significant symptoms of pruritus and with a VASc score of around 8 to 9. So we started him on steroids of 1 mg per kg and after 4 weeks of steroids, his bilirubin is now down to 2. And before coming to us, he had been to multiple hospitals being managed conservatively symptomatic with the anti-pruritic treatment, but there was no response. So even with the viral hepatitis-induced prolonged cholestasis steroids significantly helps in the reduction of pruritus. So I was just wondering what are your thoughts on using steroids? Dr. Boyer, are you meaning to share your screen? No, I'm not. No. Sorry. We don't routinely do that. I understand that. But there are some other effects of steroids that might promote other transporters that maybe are, you know, helping to reduce the bilirubin per se. Oh, okay. Okay, yeah. And Dr. Boyer, were you saying that you do use steroids? No, no, no. I was saying ursodiol is one. You know, everybody who's cholestatic gets ursodiol. Whether, how effective it is, you know, in drug-induced cholestasis, you know, well, it's not clear. We don't use it, but I've seen a lot of Europeans use it. Very common in Europe. Also, it's like, they're like, oh, it's not that harmful. So what's the downside? Yeah. And colostomy was given to the spatial response as well. Yeah, absolutely. Rohit, thank you. Thank you for joining. We always love hearing from our friends in India and Asia and learning from your experiences because you have different patient populations and you see different things. And it's always very, very helpful to get your perspective. Thank you. Thank you. Okay, everyone, another successful Heptoxic meeting. We will- Thank you, David. Thank you, David. Thank you, Andrew. Get back to your meeting. Thank you. Congratulations again. Yeah, thank you. Thank you. Bye.

anabolic steroids

liver injury

cholestasis

Dr. Andrew Stoltz

USC

renal complications

bodybuilding supplements

liver pathology

mislabeling

treatment