Studies on the reactions of formaldehyde with aminothiols and the antioxidative properties of the reaction products

Abstract

The simplest aldehyde, formaldehyde, is ubiquitous in the ambient environment, and can also be found in all cells. Formaldehyde is cytotoxic when above the threshold level. It reacts with nucleophiles in various biomacromolecules and small molecules to form adducts. Low-weight thiols, such as cysteine, cysteinylglycine (Cys-Gly), and glutathione (GSH), are one group of substances that formaldehyde reacts with. Their unique properties as nucleophiles and redox characteristics make them invaluable in detoxifying electrophiles that could damage cellular structures. Previously, there have been some studies on the in vitro reaction of low-weight thiols and formaldehyde, but none of them discuss the involvement of these formaldehyde-low-weight thiol conjugates in the biological system metabolic pathway systematically. Understanding the metabolic fate of formaldehyde in the biological system is extremely important to gain more knowledge of its detoxifying mechanism. It is also required in advance to use related metabolites as biomarkers for assessing the risk of disease development induced by oxidative stress and formaldehyde exposure. The present study is first initiated by quantitating the major conjugates of GSH, one of the low-weight thiols, and formaldehyde in cells with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope-dilution method. By this, thioproline-glycine (SPro-Gly) and (S)-1-((I-2-amino-3-(carboxymethylamino)- 3-oxopropylthio)methyl)-5-oxopyrrolidine-2-carboxylic acid (PGF), from the reaction of formaldehyde with GSH, had been revealed as major metabolites. As a result, a novel and effective mechanism of the metabolic pathway of formaldehyde in the biological system was unraveled. Notably, data revealed that these adducts had antioxidative properties, which can defend cells from oxidative damage. The results of this work are anticipated to shed light on the development of PGF and SPro-Gly as dietary supplements as well as the creation of non-invasive techniques to evaluate the health risks of formaldehyde exposure. In addition to identifying SPro-Gly and PGF in the reaction mixture of GSH and formaldehyde, thioproline (SPro), an adduct of cysteine and formaldehyde, is also produced during the reaction. SPro is shown to be one of the major degradation products of SPro-Gly and PGF in the biological system. Given the knowledge that SPro-Gly and PGF exhibited antioxidative effects and SPro performed as an efficient nitrile trapping agent, the antioxidative property of SPro was tested in this work. It was found that HeLa cells grown in SPro-supplemented culture media were more resistant to oxidative stress when compared with the control, indicated by a dose-dependent decrease in cell mortality. Results also showed that SPro acts as an effective antioxidant through sacrificial oxidation and incorporation into cellular proteins, which altered the protein level of treated cells. In addition to SPro-Gly and PGF, this work also illuminates the possibility of using SPro as a dietary supplement to protect humans from oxidative stress-related degenerative diseases. With the knowledge that SPro acts as an effective antioxidant, I have proposed a new supplement, γ-Glu-SPro-Gly, as to improve the absorption efficiency of reported thiol antioxidants, such as GSH and SPro. Using various strategies, such as mortality rate, GSH/GSSG ratio, and ROS formation assay, the newly synthesized γ-Glu-SPro-Gly has been shown to protect cells from oxidative insults. The supplemented γ-Glu-SPro-Gly will degrade into SPro and increase the availability of SPro in cells, which further increases cells’ tolerance to oxidative stress by sacrificial oxidation. In addition to determining the antioxidative property of the thiol-formaldehyde adducts, I also examine the feasibility of utilizing them as indicators for formaldehyde/oxidative stress exposure. The study is extended to detecting thiol-formaldehyde adducts in human urine samples after exposure to cigarette smoke. Smoking is reported as one of the main sources of formaldehyde exposure. With the newly developed clean-up and LC-MS/MS methods, SPro and SPro-Gly have been successfully detected in human urine samples. Smokers’ urine contains a higher level of SPro and SPro-Gly compared to urine from non-smokers. These two metabolites also increase with second-hand smoking exposure, and they rise to a maximal level in 3 to 6 h after the exposure. This work proved the feasibility of using the thiol-formaldehyde adducts as biomarkers for cigarette smoking exposure, either first-hand or second-hand smoking. Through this thesis work, I identified novel thiol-formaldehyde adducts, such as SPro-Gly and PGF, in physiological conditions and cells. As to further examine the feasibility of using thiol-formaldehyde adducts as the biomarker for oxidative stress/or formaldehyde exposure, the study is then extended to quantitating the thiol-formaldehyde adducts in smokers’ urine, as smoking is reported as one of the major sources of formaldehyde exposure. This work successfully shows that the level of chosen thiol-formaldehyde adducts increases with cigarette smoking exposure, which further sheds light on using novel thiol-formaldehyde adducts as the potential biomarker for formaldehyde exposure. Besides, their antioxidative effects and underlying mechanism have been investigated. The thiol-formaldehyde adducts mentioned above have proven to contain a certain degree of antioxidative ability. Thus, it states that the thiol-formaldehyde has the potential of being biomarkers for disease risk assessment together with providing antioxidative means to biological systems.

Date of Award	2023
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Wan CHAN (Supervisor)