c. Carbonyl Protein Assay
Carbonyl protein content is important in determining the extent of oxidation towards the protein backbones and amino acid residues such as proline, arginine, lysine, and threonine by ROS molecules. Protein oxidation leads to loss of critical sulfhydryl groups in addition to modification of amino acids, leading to the formation of carbonyl and other oxidized moieties. The oxidized proteins can be quantified using the 2,4-dinitrophenylhydrazine (DNPH). DNPH reacts with carbonyl proteins, forming a Schiff base to produce dinitrophenylhydrazone products, the levels of which can be analyzed spectrophotometrically at 375 nm and correlated to the levels of oxidized proteins [8].
Glutathione Peroxidase Activity
The presence of ROS can be countered by the production of antioxidants in our body through a series of redox reactions, forming what we call as redox homeostasis. This balance is disturbed by the excessive production of free radicals and ROS that have deleterious effects on our body functions. Thus, quantifying a specified antioxidant’s amount in an organism is a measure of the oxidative stress extent. One of these is the glutathione peroxidase (GPx), an enzyme that catalyzes the reduction of hydrogen peroxide and lipid peroxides to water and their corresponding lipid alcohols via the oxidation of reduced glutathione (GSH) into glutathione disulfide (GSSG). Activity was determined spectrophotometrically by coupling the oxidation of glutathione and NADPH using glutathione reductase (GR). GPx catalyzes the oxidation of glutathione by cumene hydroperoxide (for selenium-independent GPx) or hydrogen peroxide (for selenium-dependent GPx). The oxidized glutathione is later reduced by exogenous glutathione reductase causing the coenzyme of the reaction, NADPH, to become oxidized into NADP+. The change in the absorbance can then be read spectrophotometrically at λ = 340 nm [9].
Analysis of 1-NP
There’s a need for 1-NP to be separated out from the bound body tissues and due derivatization before it is injected to high performance liquid chromatography (HPLC). HPLC is an important qualitative and quantitative technique, generally used for the estimation of pharmaceutical and biological samples thru proper separation. Depending on the molecular size and polarity effect, 1-NP moves differently from the rest of the molecules in the stationary phase when the mobile phase is being passed through. Thus, optimization should be set first to maximize sensitivity and selectivity of its resulting chromatogram [10]. This is then detected using mass spectrometry wherein the instrument generates multiple ions from the sample under investigation, it then separates them according to their specific mass-to-charge ratio (m/z), and then records the relative abundance of each ion type. In a setup by Bacolod, et. al., detection limit (LOD) of 1-NP was 2 ng/kg in fish and 0.002 ng/L in water with recoveries from 95 to 98% [11].
Separation of 1-NP can be done by thorough extraction of its freeze-dried homogenized soft tissues with 3:1 (v/v) dichloromethane-methanol prior to the addition of hexane-water mixture to the liquid extract. Significant amounts of 1-NP are transferred towards the hexane layer during successive extraction. The dehydrated hexane layer containing 1-NP is loaded into silica gel column, which serves as the stationary phase, where the target chemicals are eluted with the mobile phase in the form of 60% acetone-hexane (v/v). Eluate was spiked with 100 μL of 100 ng/mL 1-nitropyrene-d9 as an internal standard before changing solvent to 1.5 mL of ethanol. Derivatization was conducted by reduction with 3 mL 20% NaHS at 95 °C for 60 min and acetylation with acetic acid anhydride at 65 °C for 45 min. The acetylated 1-NP was extracted with 10 mL dichloromethane and the extract was evaporated to dryness under a gentle nitrogen stream. The residue was dissolved in 0.1 mL of 80% acetonitrile. The resulting 1-NP solution was measured using high-performance liquid chromatography with mass spectrometry detector in positive electron scanning ionization mode.
Biomarker Induction
In the study by Bacolod, et. al., there’s an observed positive correlations (r > 0.49) between the 1-NP concentration exposure of the Oreochromis niloticus to the levels of thiobarbituric reactive substances (TBARS), GPx, carbonyl protein, 8-OHdG, MN and NA which suggests occurrence of oxidative stress and genotoxicity. Increase of 8-OHdG in the fish serum served to be the primary toxic effect of the 1-NP exposure thus increasing DNA lesions are then expected for this organism. Generally, 1-NP induces the ROS that attacks the target sites especially in DNA. However, intracellular levels of 8-OHdG may be initially hindered due to the presence of 8-OHdG repair enzymes. In the study by Kim, et. al., results suggested that the repair mechanism of human oxoguanine glycosylase (hOGG1) and AP endonuclease (APEX) prevented an 8-OHdG formation increase in cellular DNA of A549 human lung adenocarcinoma cell line below 250 μM 1-NP, and that excessive generation of ROS exceeds the capacity for DNA repair, resulting to slight increase in 8-OHdG [12]. Thus, it is imperative that the lack of significant 8-OHdG level increase for a certain organism in an increasing 1-NP exposure would not necessarily conclude the absence of DNA oxidative damage.
When 1-NP is metabolized inside the organism, electrophilic hydroxyl amino metabolites are formed wherein it transforms into DNA adducts, the primary form of which is N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP), as shown in Figure 4. The production of dG-C8-AP has been observed to be present in several in vivo and in vitro studies. DNA damage induced by the metabolites of 1-nitropyrene produced mutations, primarily frameshifts in bacterial systems and base substitutions (mostly GC→TA) in mammalian cells. Standard slippage models explained the frameshifts, and polymerase misincorporation models explained the base substitutions. In addition to gene mutation, 1-nitropyrene also caused chromosomal mutation, such as micronucleus formation, and morphological cell transformation. In contrast to the mutation spectrum of 1-nitropyrene in mammalian cells in vitro or in bacterial cells, oncogenes in 1-nitropyrene- induced tumors in rodents have primarily AT→GC mutations, as well as altered patterns of expression [13]. In general, 1-NP not only induced oxidative stress and the formation of reactive oxygen species, but also inflammatory proteins and apoptosis in mammalian cell systems and rodents. These mechanisms, together with its direct genotoxicity, could contribute to the carcinogenicity of 1-nitropyrene.