Deoxyguanosine

All samples were assessed using a microplate reader spectrophotometer (InfiniteM200, Tecan, Austria). All the determinations were duplicated, and the interassay coefficient of variation was in the range indicated by the kit's manufacturer.
The malondialdehyde (MDA) levels were analysed spectrophotometrically using the modified thiobarbituric acid-reactive substance method to determine the amount of lipid peroxidation in plasma. The measurement of thiobarbituric acid-reactive substances (TBARS) by a commercial assay kit (Cayman Chemical, USA) allows a rapid photometric detection at 535 nm of the thiobarbituric acid malondialdehyde (TBAMDA) adduct, as previously reported [7 (link)]. A linear calibration curve was computed from pure MDA-containing reactions.
The protein carbonyl (PC) content, an index of protein oxidation, was determined utilizing a commercial kit (Cayman Chemical, USA) through the reaction of 2,4-dinitrophenylhydrazine (DNPH) and carbonyls. This reaction forms a Schiff base producing the correspondent hydrazone. The latter was analysed by spectrophotometry, reading the absorbance signal in the 360–385 nm range. Values were normalized to the total protein concentration in the final pellet (absorbance reading at 280 nm) to consider protein loss during the washing steps.
8-OH-2-deoxyguanosine (8-OH-dG), established as a marker of oxidative DNA damage, was assessed by using a commercially available enzyme immune assay EIA kit (Cayman Chemical, USA). The EIA employs an anti-mouse IgG-coated plate and a tracer consisting of an 8-OH-dG-enzyme conjugate, while the sample 8-OH-dG concentration was determined using an 8-OH-dG standard curve. Meanwhile, samples and standards were read at a wavelength of 412 nm.
Nitrite (NO₂⁻)+nitrate (NO₃⁻) (NOx) level determination was performed by the spectrophotometric method to Griess reagent, utilizing a commercial colorimetric assay kit (Cayman Chemical, USA).
Nitric oxide synthase (iNOS) expression was assessed by using a commercial assay EIA kit (cat no. EH0556; FineTest, Wuhan China). This assay was based on sandwich enzyme-linked immune-sorbent assay technology and carried out according to the manufacturer's instructions, while NOS2/iNOS protein synthesis was determined using a standard curve. Samples and standards were read at a wavelength of 450 nm.
Interleukin-6, interleukin-1β, and interleukin-10 (IL-6, IL-1β, and IL-10, respectively) levels were determined by using commercially available enzyme immune assay kits (R&D Systems, USA; Cayman Chemical, USA; and BioVendor, Czech Republic, respectively) following the manufacturer's instruction. The assays are based on a double-antibody sandwich technique. The signal was spectrophotometrically measured.

The Gaussian09 program package [28 ] was used for all the calculations based on density functional theory (DFT). The M06-2X/6-311++G(d,p) theoretical model (with polarization and diffuse functions included) was employed for the optimization of the structures of the coumarin derivatives, as suggested in [29 (link)]. The applied theoretical model is suitable for thermodynamic and kinetic analyses of various reactions [23 (link),24 (link),25 (link),30 (link),31 (link)]. The conductor-like polarizable continuum model (CPCM, water (ε = 78.36)) was applied to approximate the solvent effect in the experimental environment [32 (link)].
The radical mechanisms presented in this study were evaluated based on thermodynamic and kinetic considerations. This was consistent with the quantum mechanics-based test for overall free radical scavenging activity (QM-ORSA) methodology [22 (link)], commonly used to determine antiradical activity. After the calculation of the corresponding reaction Gibbs free energies (Δ_rG), kinetic calculations were performed for all exergonic (Δ_rG < 0) and isoergonic (Δ_rG = 0) reaction pathways. The rate constants (k) were calculated using transition state theory (TST) [33 (link)] or the Eyring equation, as well as Eckart’s method, which represents the special case of the zero-curvature tunneling approach (ZCT_0) [34 (link)]. The first theory is based on the laws of classical kinetics, whereas the second includes quantum effects, such as tunneling (Equation (1)): $$k_{ZCT\_0}=\sigma \gamma (T)\frac{k_{B}T}{h}\exp \left(\frac{-\Delta G_a^{\ne }}{RT}\right)$$
where k_B and h are the Boltzmann and Planck constants; T is the temperature in K (298.15 K); ΔG_а^≠ is the activation Gibbs free energy; σ represents the reaction path degeneracy accounting for the number of equivalent reaction paths; and γ(T) is the tunneling correction [35 (link)]. For these calculations, TheRate program was used [36 (link)].
Evaluation of the overall rate constant (k_overall) in a polar medium offers a comprehensive picture of the reactivity of the investigated compounds. The k_overall is the sum of the products of the molar fractions of acid–base species included in specific reactions and the total rate constant (k_tot). The k_tot comprises the sum of all kinetically favored reaction pathways for a particular species. A detailed explanation of the k_overall estimation, the process of quantifying molar fractions of acid–base species at physiological pH, is given in previous research [23 (link)]. Additionally, the equations for the estimation of reactivity towards a specific radical (r^T) relative to the reference standard antioxidant (Trolox), as well as relative amounts of products (%)—i.e., the branching ratios (Г_i)—are integral parts of a previous report [23 (link)].
The Ecological Structure–Activity Relationships program (ECOSAR V2.0) [37 (link)] was used to evaluate the acute and chronic toxicities (ChV, mg·L⁻¹) of the investigated compounds and their oxidation products towards aquatic organisms: green algae, fish, and daphnia. Acute toxicity was defined using EC₅₀ values (the concentration of the examined compound that affected the growth of 50% of green algae after 96 h of exposure) and LC₅₀ values (the concentration of the investigated compound that caused 50% mortality in fish and daphnia after 96 h) [38 (link),39 (link)].
The estimated k_overall values made it possible to determine the stability of the investigated compounds during their degradation initiated by HO^• radicals through the half-life (τ_1/2) using the following equation: $${\tau }_{1/2}=\ln 2/k_{\mathrm{overall}}\times \left[\mathrm{H}{\mathrm{O}}^{•}\right]$$
where [HO^•]_aq is the concentration of HO^• in an aqueous solution [40 (link)].
To examine the activity of the newly formed radical products (A₁–R^•, A₂–R^•, A₃–R^•) towards biologically essential macromolecules, interactions with three groups of building blocks were considered: model lipids, amino acid residues, and nucleobases, as depicted in Figure 2 [41 (link)]. The lipid model (LM) mimics unsaturated fatty acids as essential biomolecules. It is represented as a reduced linoleic acid (LA) model that retains its primary chemical reactivity characteristic: two allylic H atoms. Amino acids, as constituents of proteins, are modeled realistically. This model has been successfully used and is widely accepted as appropriate for investigating protein site reactions. The following residues, being the most susceptible to oxidative damage in proteins, were used in this study: cysteine (Cys), leucine (Leu), tyrosine (Tyr), tryptophan (Trp), methionine (Met), and histidine (His). 2′-Deoxyguanosine (2dG) was selected as a model for oxidative DNA damage because guanine (G) is the most easily oxidized nucleobase. Therefore, when one-electron oxidation of DNA occurs, it is primarily located at G sites. Consequently, if a chemical oxidant (radical species) can oxidize 2dG, it can cause oxidative damage to DNA. In contrast, if there is no potential to oxidize 2dG, the oxidant is considered harmless to DNA.