Molecular hydrogen (H2) was long regarded as non-functional in mammalian cells. We overturned the
concept by demonstrating that H2 exhibits antioxidant effects and protects cells against oxidative stress. Subsequently,
it has been revealed that H2 has multiple functions in addition to antioxidant effects, including antiinflammatory,
anti-allergic functions, and as cell death and autophagy regulation. Additionally, H2 stimulates
energy metabolism. As H2 does not readily react with most biomolecules without a catalyst, it is essential to identify
the primary targets with which H2 reacts or interacts directly. As a first event, H2 may react directly with
strong oxidants, such as hydroxyl radicals (•OH) in vivo. This review addresses the key issues related to this in
vivo reaction. •OH may have a physiological role because it triggers a free radical chain reaction and may be
involved in the regulation of Ca2+- or mitochondrial ATP-dependent K+-channeling. In the subsequent pathway,
H2 suppressed a free radical chain reaction, leading to decreases in lipid peroxide and its end products. Derived
from the peroxides, 4-hydroxy-2-nonenal functions as a mediator that up-regulates multiple functional PGC-1α.
As the other direct target in vitro and in vivo, H2 intervenes in the free radical chain reaction to modify oxidized
phospholipids, which may act as an antagonist of Ca2+-channels. The resulting suppression of Ca2+-signaling
inactivates multiple functional NFAT and CREB transcription factors, which may explain H2 multi-functionality.
This review also addresses the involvement of NFAT in the beneficial role of H2 in COVID-19, Alzheimer’s
disease and advanced cancer. We discuss some unsolved issues of H2 action on lipopolysaccharide signaling,
MAPK and NF-κB pathways and the Nrf2 paradox. Finally, as a novel idea for the direct targeting of H2, this
review introduces the possibility that H2 causes structural changes in proteins via hydrate water changes.