ROS-induced oxidative damage in lymphocytes ex vivo/in vitro from healthy individuals and MGUS patients: protection by myricetin bulk and nanoforms

Abstract

We investigated the protective role of myricetin bulk and nanoforms, against reactive oxygen species (ROS)-induced oxidative stress caused by hydrogen peroxide and tertiary-butyl hydro peroxide in lymphocytes in vitro from healthy individuals and those from pre-cancerous patients suffering with monoclonal gammopathy of undetermined significance (MGUS). The change in intracellular reactive oxygen species was measured once cells were treated with myricetin bulk forms and nanoforms with and without either hydrogen peroxide or tertiary-butyl hydro peroxide co-supplementation. The direct and indirect antioxidant activity of myricetin was spectrofluometrically measured using the fluorescent dye 2′,7′-dichlorofluorescin diacetate and using the Comet assay, respectively. Hydrogen peroxide (50 µM) and tertiary-butyl hydro peroxide (300 µM) induced a higher level of reactive oxygen species-related DNA damage and strand breaks. Addition of myricetin nanoform (20 µM) and bulk (10 µM) form could, however, significantly prevent hydrogen peroxide-and tertiary-butyl hydro peroxide-induced oxidative imbalances and the nanoform was more effective. Glutathione levels were also quantified using a non-fluorescent dye. Results suggest that myricetin treatment had no significant effect on the cellular antioxidant enzyme, glutathione. The current study also investigates the effect of myricetin on the induction of double-strand breaks by staining the gamma-H2AX foci immunocytochemically. It was observed that myricetin does not induce double-strand breaks at basal levels rather demonstrated a protective effect. Keywords Myricetin bulk forms and nanoforms • TBHP • hydrogen peroxide • Lymphocytes • MGUS patients • γ-H2AX

Anticancer potential of myricetin bulk and nano forms in vitro in lymphocytes from myeloma patients

“…In our previous study (Akhtar et al 2020a , b ), we found no relationship among the confounding factors (age, ethnicity, smoking) regardless of the treatment groups and these factors also do not seem to be contributing in this study towards DNA damage.…”

“…The effects of MYR B and MYR N on the viability of healthy lymphocytes were compared to those of the untreated group. Neither of the concentrations of MYR B and MYR N reduced viability less than 80% in the healthy group (Akhtar et al 2020a , b ). However, MYR B and MYR N at 40 µM reduced the viability to 68% and 51%, respectively, after 48 h of treatment in lymphocytes from multiple myeloma patients (shown in Fig.…”

“…Hence, the current study was conducted using the non-toxic concentrations of MYR B and MYR N so that any of the results obtained are not due to the artefact of toxicity. The concentrations of MYR B (10 µM) and MYR N (20 µM) and hydrogen peroxide (H 2 O 2 ) (50 µM) used throughout the current in vitro study were determined by dose response studies using the comet assay in our previous study (Akhtar et al 2020a , b ).

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“…Myricetin was purchased from Sigma Aldrich, UK, and was dissolved in excipient mixture to produce its bulk and nano forms. The particle size of myricetin, stability and constitution of excipient mixture are provided in our previous study (Akhtar et al 2020a , b ). The primary antibodies against P53, Bcl-2, Bax and GAPDH were purchased from Abcam, Cambridge, UK.…”

Ex vivo/in vitro protective effect of myricetin bulk and nano-forms on PhIP-induced DNA damage in lymphocytes from healthy individuals and pre-cancerous MGUS patients

“…The suspensions were stored in amber glass bottles at 4 ℃ for the research duration. The concentrations of myricetin bulk (MYR B) and NP (MYR N) forms, used for this research study were 10 µM and 20 µM, respectively (data used from previous study) (Akhtar et al 2020 ). The concentration of PhIP used throughout the study was 100 µM.…”