Past, Present and Future of the Section "Molecular Toxicology".

'Forever' chemicals that unintendedly in the long run pollute the environment, climate change, COVID; life continuously faces all sorts of unforeseen challenges that are an inevitable side product of 'progress' [...].

Unraveling the Antioxidant Activity of 2R,3R-dihydroquercetin.

It has been reported that in an oxidative environment, the flavonoid 2R,3R-dihydroquercetin (2R,3R-DHQ) oxidizes into a product that rearranges to form quercetin. As quercetin is a very potent antioxidant, much better than 2R,3R-DHQ, this would be an intriguing form of targeting the antioxidant quercetin. The aim of the present study is to further elaborate on this targeting. We can confirm the previous observation that 2R,3R-DHQ is oxidized by horseradish peroxidase (HRP), with H2O2 as the oxidant. However, HPLC analysis revealed that no quercetin was formed, but instead an unstable oxidation product. The inclusion of glutathione (GSH) during the oxidation process resulted in the formation of a 2R,3R-DHQ-GSH adduct, as was identified using HPLC with IT-TOF/MS detection. GSH adducts appeared on the B-ring of the 2R,3R-DHQ quinone, indicating that during oxidation, the B-ring is oxidized from a catechol to form a quinone group. Ascorbate could reduce the quinone back to 2R,3R-DHQ. No 2S,3R-DHQ was detected after the reduction by ascorbate, indicating that a possible epimerization of 2R,3R-DHQ quinone to 2S,3R-DHQ quinone does not occur. The fact that no epimerization of the oxidized product of 2R,3R-DHQ is observed, and that GSH adducts the oxidized product of 2R,3R-DHQ on the B-ring, led us to conclude that the redox-modulating activity of 2R,3R-DHQ quinone resides in its B-ring. This could be confirmed by chemical calculation. Apparently, the administration of 2R,3R-DHQ in an oxidative environment does not result in 'biotargeting' quercetin.

Flavonoids Seen through the Energy Perspective.

In all life forms, opposing forces provide the energy that flows through networks in an organism, which fuels life. In this concept, health is the ability of an organism to maintain the balance between these opposing forces, which creates resilience, and a deranged flow of energy is the basis for diseases. Treatment should focus on adjusting the deranged flow of energy, e.g., by the redox modulating activity of antioxidants. A major group of antioxidants is formed by flavonoids, a group of polyphenolic compounds abundantly present in our diet. The objective here is to review how the redox modulation by flavonoids fits in the various concepts on the mode of action of bioactive compounds, so we can 'see' where there is overlap and where the missing links are. Based on this fundament, we should choose our research path aiming to 'understand' the redox modulating profile of specific flavonoids, so we can ultimately rationally apply the redox modulating power of flavonoids to improve our health.

Delocalization of the Unpaired Electron in the Quercetin Radical: Comparison of Experimental ESR Data with DFT Calculations.

In the antioxidant activity of quercetin (Q), stabilization of the energy in the quercetin radical (Q•) by delocalization of the unpaired electron (UE) in Q• is pivotal. The aim of this study is to further examine the delocalization of the UE in Q•, and to elucidate the importance of the functional groups of Q for the stabilization of the UE by combining experimentally obtained spin resonance spectroscopy (ESR) measurements with theoretical density functional theory (DFT) calculations. The ESR spectrum and DFT calculation of Q• and structurally related radicals both suggest that the UE of Q• is mostly delocalized in the B ring and partly on the AC ring. The negatively charged oxygen groups in the B ring (3' and 4') of Q• have an electron-donating effect that attract and stabilize the UE in the B ring. Radicals structurally related to Q• indicate that the negatively charged oxygen at 4' has more of an effect on concentrating the UE in ring B than the negatively charged oxygen at 3'. The DFT calculation showed that an OH group at the 3-position of the AC ring is essential for concentrating the radical on the C2-C3 double bond. All these effects help to explain how the high energy of the UE is captured and a stable Q• is generated, which is pivotal in the antioxidant activity of Q.

The Flow of the Redox Energy in Quercetin during Its Antioxidant Activity in Water.

Most studies on the antioxidant activity of flavonoids like Quercetin (Q) do not consider that it comprises a series of sequential reactions. Therefore, the present study examines how the redox energy flows through the molecule during Q's antioxidant activity, by combining experimental data with quantum calculations. It appears that several main pathways are possible. Pivotal are subsequently: deprotonation of the 7-OH group; intramolecular hydrogen transfer from the 3-OH group to the 4-Oxygen atom; electron transfer leading to two conformers of the Q radical; deprotonation of the OH groups in the B-ring, leading to three different deprotonated Q radicals; and finally electron transfer of each deprotonated Q radical to form the corresponding quercetin quinones. The quinone in which the carbonyl groups are the most separated has the lowest energy content, and is the most abundant quinone. The pathways are also intertwined. The calculations show that Q can pick up redox energy at various sites of the molecule which explains Q's ability to scavenge all sorts of reactive oxidizing species. In the described pathways, Q picked up, e.g., two hydroxyl radicals, which can be processed and softened by forming quercetin quinone.

Connecting West and East.

Despite their similarities, Western medicine and Eastern medicine are very different because they are built on different fundamentals. The general idea has arisen that we will benefit by connecting Western and Eastern medicine. First, both the merits as well as the limitations of both types of medicine are discussed. It was concluded that to create a bridge, we should focus on similarities that inspire the further unravelling of the molecular mechanism of the mode of action and toxicity of Traditional Chinese Medicine. It is suggested that the energy perspective provides a basis to integrate Eastern and Western medicine.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

The potential of flavonoids in the treatment of non-alcoholic fatty liver disease.

The contemporary pathophysiological model of non-alcoholic fatty liver disease (NAFLD) comprises multiple parallel pathways with a dynamic cross talk that cumulate in steatosis and inflammation, and ultimately fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma. So far, no pharmacological treatment has been approved. A major impediment of drugs, in general, is that they are intended to act on one single target in the pathology of a disease. However, the multitude of pathways involved in the pathogenesis of NAFLD underpins the need for treatments that address these various pathways. Interestingly, flavonoids have been found to have positive effects on lipid metabolism, insulin resistance, inflammation, and oxidative stress, the most important pathophysiological pathways in NAFLD. This puts flavonoids in the spotlight for the treatment of NAFLD and prompted us to review the existing evidence for the use of these food-derived compounds in the treatment of NAFLD.

The Screening of Anticholinergic Accumulation by Traditional Chinese Medicine.

Many Western drugs can give rise to serious side effects due to their ability to bind to acetylcholine receptors in the brain. This aggravates when they are combined, which is known as anticholinergic accumulation (AA). Some bioactives in Traditional Chinese Medicine (TCM) are known to block acetylcholine receptors and thus potentially cause AA. The AA of TCM was screened by quantifying the displacement of [³H] pirenzepine on acetylcholine receptors in a rat brain homogenate. We used a new unit to express AA, namely the Total Atropine Equivalents (TOAT). The TOAT of various herbs used in TCM was very diverse and even negative for some herbs. This is indicative for the broadness of the pallet of ingredients used in TCM. Three TCM formulas were screened for AA: Ma Huang Decotion (MHD), Antiasthma Simplified Herbal Medicine intervention (ASHMI), and Yu Ping Feng San (YPFS). The TOAT of ASHMI was indicative for an additive effect of herbs used in it. Nevertheless, it can be calculated that one dose of ASHMI is probably too low to cause AA. The TOAT of YPFS was practically zero. This points to a protective interaction of AA. Remarkably, MHD gave a negative TOAT, indicating that the binding to the acetylcholine receptors was increased, which also circumvents AA. In conclusion, our results indicate that TCM is not prone to give AA and support that there is an intricate interaction between the various bioactives in TCM to cure diseases with minimal side effects.

Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells.

Glutathione transferases (GSTs) are often overexpressed in tumors and frequently correlated to bad prognosis and resistance against a number of different anticancer drugs. To selectively target these cells and to overcome this resistance we previously have developed prodrugs that are derivatives of existing anticancer drugs (e.g., doxorubicin) incorporating a sulfonamide moiety. When cleaved by GSTs, the prodrug releases the cytostatic moiety predominantly in GST overexpressing cells, thus sparing normal cells with moderate enzyme levels. By modifying the sulfonamide it is possible to control the rate of drug release and specifically target different GSTs. Here we show that the newly synthesized compounds, 4-acetyl-2-nitro-benzenesulfonyl etoposide (ANS-etoposide) and 4-acetyl-2-nitro-benzenesulfonyl doxorubicin (ANS-DOX), function as prodrugs for GSTA1 and MGST1 overexpressing cell lines. ANS-DOX, in particular, showed a desirable cytotoxic profile by inducing toxicity and DNA damage in a GST-dependent manner compared to control cells. Its moderate conversion of 500 nmol/min/mg, as catalyzed by GSTA1, seems hereby essential since the more reactive 2,4-dinitrobenzenesulfonyl doxorubicin (DNS-DOX) (14000 nmol/min/mg) did not display a preference for GSTA1 overexpressing cells. DNS-DOX, however, effectively killed GSTP1 (20 nmol/min/mg) and MGST1 (450 nmol/min/mg) overexpressing cells as did the less reactive 4-mononitrobenzenesulfonyl doxorubicin (MNS-DOX) in a MGST1-dependent manner (1.5 nmol/min/mg) as shown previously. Furthermore, we show that the mechanism of these prodrugs involves a reduction in GSH levels as well as inhibition of the redox regulatory enzyme thioredoxin reductase 1 (TrxR1) by virtue of their electrophilic sulfonamide moiety. TrxR1 is upregulated in many tumors and associated with resistance to chemotherapy and poor patient prognosis. Additionally, the prodrugs potentially acted as a general shuttle system for DOX, by overcoming resistance mechanisms in cells. Here we propose that GST-dependent prodrugs require a conversion rate "window" in order to selectively target GST overexpressing cells, while limiting their effects on normal cells. Prodrugs are furthermore a suitable system to specifically target GSTs and to overcome various drug resistance mechanisms that apply to the parental drug.