Modern toxicity testing places a strong emphasis on in vitro systems that employ cultured cells and various assay chemistries to deliver data. We have investigated the cytotoxicity and reactive oxygen species (ROS) Keywords: cytotoxicity assessment, ROS assays, FESEM and TEM analysis. Reactive oxygen species (ROS) are chemically reactive chemical species containing oxygen. .. Radiotherapy also relies on ROS toxicity to eradicate tumor cells. Radiotherapy uses X-rays, γ-rays as well as heavy particle radiation such as.
Toxicity Assay. ROS
ROS levels decreased gradually after the first day Fig. After the fifth day, cell viability showed significant increases, reaching control levels at day 7. From 24 to 72 h, comparable with the data shown in Figure 3C , there is a high degree of overlap of nuclear and ROS staining.
At h, ROS staining is too weak to indicate with confidence whether it coincides with nuclear staining. The ROS staining disappears entirely at h. Cytotoxicity and persistence of ROS generation were further investigated for both aminophenol and hydroquinone. Immediately following treatment 0 h ROS staining does not appear to co-localize with nuclear staining; however, by 24 h, there is a high degree of overlap, which remains apparent through 72 h.
At h, ROS staining is no longer apparent. Figure 4 shows data at 24 h after a 1-h exposure. Responses were the same in AA8 and UV5 cells. In both cases, it appears that ROS production occurs in both nucleus and cytoplasm. At this level of magnification, ROS and nuclear staining are almost completely coincident.
D The highlighted area is shown at higher magnification in B. Values were divided by the amount of nuclei stain in the assessed region to attain the number of ROS per cells. The values are normalized to the control and showed as the percentage change relative to control.
The foregoing results did not reveal whether ROS were present within nuclei. To address this question, confocal microscopy was used to acquire images at 1. This finding might account for its presumptive genotoxic and cytotoxic effects. Protein carbonyls were also assayed as an indicator of protein oxidation. Assays were conducted independently on cytoplasmic and nuclear fractions.
Data from both assays are shown in Figures 5C—5F. Nuclear fractions generally gave lower values than cytoplasmic fractions but the overall pattern of responses to various treatments was similar.
All increases were statistically significant. Ascorbate alone neither reduced nor increased control values of TBARS or protein carbonyls significantly in both nuclear and cytoplasmic fractions. NAC only reduced nuclear protein carbonyl levels when applied alone. The treatment protocol used was the same as that described above, involving exposure to 3,5-DMAP for 1 h, after which cells were collected, washed, and re-plated in fresh medium.
After 24 h, treated AA8 cells Fig. This response was completely blocked by simultaneous exposure to 5-mM NAC. Caspase-3 activation was entirely blocked in the cells co-treated with 5-mM NAC. B Dose responses based on all data are plotted.
Top and right of the graphs on panel A indicate late apoptosis and below of right part indicates early apoptosis as also shown. Panel A shows the data including normal cells, necrotic cells, early apoptotic, and late apoptotic cells with histogram plotting. When the concentration of 3,5-DMAP is increased, cells are more late apoptotic. NAC seems to be protective for both of the cell types and protects the viability of the cells.
Apoptotic marker caspase-3 assay shows the inverse dose- A and time-response B relationship between 3,5-DMAP and caspase-3 activity.
NAC diminished these responses. Table 2 summarizes the results. The survival rate in each assay was calculated and normalized to untreated control. Responses of the two cell types to each treatment were essentially identical, indicating that NER DNA repair status had negligible influence on processes leading to cell death. Uric acid at 0. The cell treated with DMSO is defined as the negative control. The results are normalized to the control.
The data in Figure 7 demonstrated that 3,5-DMAP activates caspase-3 activity which may eventually lead to apoptosis. Significant differences relative to 3,5-DMAP sample: Here, we continue the experiments concerning cell viability after treatment of 3,5-DMA and its metabolites.
This work suggests a diverse profile of cytotoxic potencies in both AA8 and UV5 cells: Our results show that aminophenols are mechanistically unique as redox-active compounds and ROS production can be mentioned among the underlying factors of the cytotoxicity of 3,5-DMAP.
These findings prompted the more extensive characterization of intracellular ROS production by 3,5-DMAP, with the goals of characterizing the persistence of ROS production through cell growth and replication. The present results show that the persistent, indeed, increased the production of ROS at this time relative to the period immediately after dosing Fig.
In addition, this study extended the investigated time period to 7 days. ROS production continued for at least the first five days at which point cell survival became too low for meaningful data analysis.
These findings strongly indicate that intracellular ROS formation is an important, if not principal, mechanism by which aminophenols cause damage to DNA and to other cellular targets.
To our knowledge, this is the first study showing that antioxidants are capable of reducing the oxidative damage exerted by alkylanilines.
The effects of NAC on these two outcomes were dissimilar, however. Therefore, NAC may act partly as a nucleophilic scavenger, reacting with the quinone imine form of 3,5-DMAP through Michael addition to inactivate it with respect to transimination.
Additionally, NAC might serve the same function as GSH in maintaining the intracellular redox environment—it possesses the same redox couple—without the need for a reductase because NAC in the growth medium serves as a reservoir of free thiol that is very large relative to the intracellular pool.
The limited protection of GSH by NAC with high doses of 3,5-DMAP can be interpreted as the result of GR inactivation, which, if it occurs by electrophilic attack on the enzyme, is unlikely to be significantly mitigated by NAC because small molecules are generally poor competitors compared with proteins Skipper, Uric acid was highly effective at 5mM, but toxic at the highest dose tested. Although the antioxidant potential of uric acid was reported that high dose uric acid might act as a pro-oxidant Proctor, This phenomenon might explain the observation that high-dose uric acid caused cytotoxicity.
These results suggest three possibilities: The increased intracellular level of LP is a good indicator of intracellular oxidation Mateos and Bravo, Because quinones are also capable of binding to proteins through Michael addition of cysteine sulfhydryls , it was of interest to compare the structurally analogous 3,5-DMHQ with 3,5-DMAP which is capable of both transimination and Michael addition.
The reason might be that NAC is a stronger electron donor and it provides higher protection potential than uric acid Krasowska and Konat, The prolonged intracellular ROS production and concomitant cytotoxicity are a signal property of 3,5-DMAP and by extension, perhaps, other aminophenols. These compounds are cytotoxic and have been shown to produce DNA damage in the form of strand breaks. The concerted action of caspases is responsible for apoptosis Dang, When activated, the extrinsic pathway, which includes the action of initiator caspases 8 and 10, cleaves and activates the executioner caspases 3 and 7.
It was demonstrated that oxidative stress could activate caspase 8 Boatright and Salvesen, After 3,5-DMAP exposure, apoptosis was virtually the sole mechanism responsible for loss of viability, and the common response of the two cell lines with respect to apoptosis, DNA strand breakage, and protection by ROS scavengers is not protective, but rather favors oxidative DNA damage as one of the principal toxicity mechanisms.
Though it is not feasible to draw clear conclusions from the presented data, the results evoke oxidative damage as an important, if not predominant, mechanism underlying the apoptotic effect of 3,5-DMAP. Additionally, this mechanism is further supported by the results showing the effect of NAC in maintaining expression of pro-caspase 3 and pro-PARP and in inhibiting caspase-3 activity Fig.
Although it remains to be determined whether ROS produced by embedded aminophenol structures are actual toxicants, the apoptotic response reported herein is a further indicator of DNA damage. In conclusion, this is the first study showing that antioxidants, particularly NAC, are protective against oxidative potential of 3,5-DMAP.
This study also provides a more mechanistical approach for the cytotoxic potential of 2,6-DMA and its metabolites. The results presented herein emphasize the importance of antioxidants, with respect to the high probability of alkylaniline exposures and their toxic effects.
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Abstract Epidemiological studies have demonstrated extensive human exposure to the monocyclic aromatic amines, particularly to 3,5-dimethylaniline, and found an association between exposure to these compounds and risk for bladder cancer.
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In Vitro ROS/RNS Assay
Assay Oxidative Stress in 40 min in cell samples with ab Cited in > publications. For microplate reader or fluor. microscope or flow cyt. I know that primary mechanism to be investigated for NP toxicity is the ROS . I have conducted cell viability analysis using MTT assay, from that i observe the. I have tried to check LPS induced cytotoxicity on cells using MTT, where I plated x cells per well and treated with LPS at 7 concentrations (30ug/ml.