AbstractEndogenous and exogenous factors cause DNA damage through chemical changes in the genomic DNA structure. The comet assay is a versatile, rapid, and sensitive method for evaluating DNA integrity at the individual cell level. It is used in human biomonitoring studies, the identification of DNA lesions, and the measurement of DNA repair capacity. Despite its widespread application, variations between studies remain problematic, often due to the lack of a common protocol and appropriate test controls. Using positive controls is essential to assess inter-experimental variability and ensure reliable results. Hydrogen peroxide (H2O2) is the most commonly used positive control, while potassium bromate (KBrO₃), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), and etoposide are used less frequently. However, differences in concentrations and exposure durations prevent the confirmation of test method efficacy. This study investigates the dose–response relationship for H2O2, KBrO3, MMS, EMS, ENU and etoposide in the comet assay for 30 and 60-minute exposure durations in 3T3 cell lines. Accordingly recommended concentrations and exposure durations were found to be 50 μM 30 minutes (H2O2); 500 μM 60 min. (MMS); 10 μM 30 min. (Etoposide); 0.2 mM 30 min. and 2 mM 60 min. (EMS); 2 mM 30 min. (ENU); 500 μM 30 min. and 50 μM 60 min. (KBrO3). Our findings will contribute to reducing inter-laboratory variability by offering guidance on selecting doses and exposure durations for positive controls in the in vitro alkaline comet assay.

01 Jan 2024·Heliyon

Health risk assessment of potassium bromate in bread in Ghana

Article

Author: Gordon, A ; Gmakame, J ; Otu, P N Y ; Bonah, D A ; Gedza, W E ; Andorful, I A ; Ayembilla, J A ; Owusu-Ansah, L ; Whyte, B K ; Quarcoo, A

IntroductionPotassium bromate (KBrO₃) is an oxidizing agent added to flour to improve bread quality. However, KBrO₃ is nephrotoxic, and a class B carcinogen banned in most countries, including Ghana.AimThis study aimed to determine the residual KBrO₃ concentration in bread and to estimate the chemical and carcinogenic risk that is associated with the consumption of these breads in Ghana.MethodsA total of 285 samples of bread, consisting of 97 tea breads, 95 sugar breads, and 93 butter breads were randomly sampled from bakeries across ten regions of Ghana for this study. The bromate contents were qualitatively and quantitatively determined spectrophotometrically at 515 nm, based on the redox reaction between KBrO₃ and promethazine. The change in colour from colourless to pink indicated the presence of KBrO₃ in the sample. The data were analyzed with Microsoft Excel 365 Apps for Enterprise and GraphPad Prism and the map was plotted with QGIS.ResultsOut of the 285 bread samples analyzed, 12.28% (35/285) tested positive for KBrO₃ with concentrations ranging from 0.46 to 26.67 μg/g (mean 6.13 μg/g). This exceeded the permissible dose (0.02 μg/g) by 23-1334 times, respectively. Except Ashanti and Volta regions, all the other 8 regions had at least one bread type testing positive for KBrO₃. There was no statistically significant difference in the concentration of KBrO₃ between the bread types analyzed at p < 0.05. The hazard ratio ranged from 0.166 to 0.435 and the chronic hazard quotient ranged from 0.0037 to 0.00141.ConclusionBreads from 8 regions of Ghana tested positive for KBrO_3, but their concentrations may not pose chemical or carcinogenic risks after regular consumption.

01 Aug 2021·Archives of ToxicologyQ2 · MEDICINE

Validation of the in vitro comet assay for DNA cross-links and altered bases detection

Q2 · MEDICINE

Article

Author: Sanz-Serrano, Julen ; Muruzabal, Damián ; Olsen, Ann-Karin ; Azqueta, Amaya ; Sauvaigo, Sylvie ; Treillard, Bertrand ; López de Cerain, Adela ; Vettorazzi, Ariane

AbstractMechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.

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Breast Cancer	Preclinical	US	VA Medical Center	01 Jul 2017

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