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Abstract
MC-LR has become a global health concern, being produced by cyanobacteria, or blue-green algae, in freshwater environments around the world. Harmful algal blooms are increasing in frequency and severity annually. MC-LR has become well-known for its hepatotoxicity, having been reported to cause liver damage and even liver failure in humans. While safe human exposure levels to MC-LR have been established by the World Health Organization, it is alarming that these guidelines have been extrapolated from healthy animal studies. This fails to account for the large global populations of individuals with pre-existing disease conditions, who may be more vulnerable and susceptible to MC-LR toxicity.
In order to provide a convenient model for studying MC-LR toxicity in an in vitro setting of pre-existing liver disease, we have designed a novel in vitro model of NAFLD, given that NAFLD is one of the most common forms of liver disease globally. We believe that this model will not only allow for robust and efficient investigation of MC-LR toxicity, but also drive other research endeavors that could benefit from a new, convenient in vitro model of NAFLD.
Another urgent concern is that there are currently no established methods for the diagnosis of MC-LR hepatotoxicity. In order to begin addressing this issue, we provide a thorough investigation into whether ALT and ALP are suitable as diagnostic biomarkers, given that ALT and ALP are two of the most commonly used liver biomarkers used clinically for the diagnosis of a wide range of liver diseases and disorders. As part of this investigation, we utilize our new in vitro model of NAFLD in parallel with a well-established Leprdb/J mouse model of NAFLD. We report that, following thorough investigation, ALT and ALP are unsuitable as diagnostic biomarkers of MC-LR-induced liver damage. However, we have identified three genes of hepatotoxicity that are commonly upregulated in vivo and in vitro that are associated with serum markers previously used for the diagnosis of other diseases. These provide potential candidates that will need to be further investigated in future studies.
While the majority of MC-LR research focuses on its effects within the liver, it is important not to neglect its potential effects within other organ systems. Taking into account that MC-LR is first absorbed through the intestines following oral ingestion and shows high levels of bioaccumulation within the intestines, we were the first to report that MC-LR appears to have limited effects in the colon in healthy settings, but has severe toxicity in the setting of pre-existing colitis. Given that IBD is one of the most common GI diseases around the world, this again stresses the heightened susceptibility of individuals with pre-existing disease conditions to MC-LR toxicity.
Further studies have revealed that CD40 appears to play an important role in driving MC-LR exacerbation of colitis, as knocking out CD40 ameliorates many of MC-LR’s effects. Similarly, TLR2 seems to also play a role, although to a lesser extent. Noting that CD40 and TLR2 are not the sole drivers in MC-LR exacerbation of colitis, we sought to use a high throughput, unbiased approach to identify a unique signature characterizing MC-LR’s effects and identify specific antagonists to reverse its effects. Having observed the recruitment of macrophages into colonic tissue exposed to MC-LR and observing in vitro that MC-LR initiates inflammatory responses in macrophages, we used the PamGene to profile kinome signatures, allowing us to identify differential peptide phosphorylation levels and differential kinase activity levels. Further bioinformatic analysis allowed for the identification of a candidate inhibitory compound, doramapimod. We have confirmed that doramapimod can effectively suppressed inflammatory responses in macrophages as a result of MC-LR exposure.
