On the Question of CO’s Ability to Induce HO-1 Expression in Cell Culture: A Comparative Study Using Different CO Sources

Abstract

With the recognition of the endogenous signaling roles and pharmacological functions of carbon monoxide (CO), there is an increasing need to understand CO's mechanism of actions. Along this line, chemical donors have been introduced as CO surrogates for ease of delivery, dosage control, and sometimes the ability to target. Among all of the donors, two ruthenium− carbonyl complexes, CORM-2 and -3, are arguably the most commonly used tools for about 20 years in studying the mechanism of actions of CO. Largely based on data using these two CORMs, there has been a widely accepted inference that the upregulation of heme oxygenase-1 (HO-1) expression is one of the key mechanisms for CO's actions. However, recent years have seen reports of very pronounced chemical reactivities and CO-independent activities of these CORMs. We are interested in examining this question by conducting comparative studies using CO gas, CORM-2/-3, and organic CO donors in RAW264.7, HeLa, and HepG2 cell cultures. CORM-2 and CORM-3 treatment showed significant dose-dependent induction of HO-1 compared to "controls," while incubation for 6 h with 250−500 ppm CO gas did not increase the HO-1 protein expression and mRNA transcription level. A further increase of the CO concentration to 5% did not lead to HO-1 expression either. Additionally, we demonstrate that CORM-2/-3 releases minimal amounts of CO under the experimental conditions. These results indicate that the HO-1 induction effects of CORM-2/-3 are not attributable to CO. We also assessed two organic CO prodrugs, BW-CO-103 and BW-CO-111. BW-CO-111 but not BW-CO-103 dose-dependently increased HO-1 levels in RAW264.7 and HeLa cells. We subsequently studied the mechanism of induction with an Nrf2-luciferase reporter assay, showing that the HO-1 induction activity is likely due to the activation of Nrf2 by the CO donors. Overall, CO alone is unable to induce HO-1 or activate Nrf2 under various conditions in vitro. As such, there is no evidence to support attributing the HO-1 induction effect of the CO donors such as CORM-2/-3 and BW-CO-111 in cell culture to CO. This comparative study demonstrates the critical need to consider possible CO-independent effects of a chemical CO donor before attributing the observed biological effects to CO. It is also important to note that such in vitro results cannot be directly extrapolated to in vivo studies because of the increased level of complexity and the likelihood of secondary and/or synergistic effects in the latter.

On the Question of Uncatalyzed CO Insertion into a Hydrazone Double Bond: A Comparative Study Using Different CO Sources and Substrates

“…None of our results support the feasibility of uncatalyzed CO insertion into a hydrazone double bond as a way to sense CO. Just as important to demonstrate the improbable nature of the originally proposed CO-insertion reaction, the problems associated with ruthenium-based CORMs (and some other CORMs) as CO surrogates should also be emphasized. It is important to note that four independent laboratories have shown that these ruthenium–carbonyl complexes do NOT produce a meaningful amount of CO in buffer and cell culture media unless there is an added nucleophile, sodium dithionite. ,,,, The case study described here is another example that these ruthenium-based CORMs (CORM-2 and CORM-3) should not be used to study CO biology or CO chemistry and should not be used to further develop new CO probes.…”

“…It is important to note that four independent laboratories have shown that these ruthenium–carbonyl complexes do NOT produce a meaningful amount of CO in buffer and cell culture media unless there is an added nucleophile, sodium dithionite. 14 , 33 , 34 , 44 , 45 The case study described here is another example that these ruthenium-based CORMs (CORM-2 and CORM-3) should not be used to study CO biology or CO chemistry and should not be used to further develop new CO probes.…”

“…One can safely say that this publication by Michel, Lippert, and Chang started a wave of work on designing new small-molecule fluorescent probes for CO with various designs, including the use of Pd-catalyzed carbonylation and Pd-mediated deallylation for fluorophore activation in CO detection (Table S2). , We are very interested in such work because of our interest in carbonylation chemistry, CO detection, CO biology, , and CO donors as potential therapeutic agents. , Our interest in CO largely stems from the fact that CO is an endogenously produced molecule in mammals with demonstrated pharmacological effects. − …”

Fe(III)-Based Fluorescent Probe for High-Performance Recognition, Test Strip Analysis, and Cell Imaging of Carbon Monoxide

“…Carbon monoxide (CO) is primarily considered to be explosive and have mammalian toxicity. , Nevertheless, recent studies revealed that CO can be generated endogenously by heme oxygenase (HO) in the heme of the human body and act as an important gasotransmitter in various physiological and pathological processes. − The level of CO in cells and blood is always altered by pathological syndromes, such as lung disease, septicemia, diabetes, and cancer. , Thus, the real-time monitoring of CO in living cells and blood is an important alternative to diagnose a variety of diseases. Despite advances in CO detection, convenient and functional monitoring methods are still urgent to be developed. − …”