Superoxide mediated photomodification and DNA damage induced apoptosis by Benz(a)anthracene via mitochondrial mediated pathway

https://doi.org/10.1016/j.jphotobiol.2014.11.006Get rights and content

Highlights

  • Superoxide mediated photomodification of Benz(a)anthracene under sunlight exposure.

  • Photosensitized Benz(a)anthracene induced single and double strand breakage as well as CPDs formation.

  • p21 regulated apoptosis in HaCaT keratinocytes through up-regulation of pro-apoptotic Bax gene and protein.

  • Benz(a)anthracene induce apoptosis through mitochondrial mediated pathway.

Abstract

Benz(a)anthracene (BA) is an ubiquitous environmental pollutant of polycyclic aromatic hydrocarbon’s (PAHs) family. We showed superoxide (O2radical dot) catalyzed BA photo modification and apoptosis in HaCaT keratinocytes under sunlight exposure. O2radical dot generation was confirmed by quenching through superoxide dismutase (SOD). BA induced photocytotoxicity were investigated through MTT and NRU assay. We proposed DNA insults such as single and double strand breakage and CPDs formation which results in cell cycle arrest and apoptosis by photosensitized BA. BA induced apoptosis was caspase dependent and occurred through a mitochondrial pathway. Reduction of mitochondrial membrane potential, translocation of Bax to mitochondria and cytochrome c release favors involvement of mitochondria in BA phototoxicity. AO/EB double staining and TEM analysis also support apoptotic cell death. We propose a p21 regulated apoptosis via expression of Bax, and cleaved PARP under sunlight exposure. Thus, we conclude that it is imperative to avoid solar radiation during peak hr (between 11 A.M. and 3 P.M.) when the amount of solar radiation is high, in the light of DNA damage which may lead to mutation or skin cancer through photosensitized BA under sunlight exposure. Concomitantly, investigation is urgently required for the photosafety of BA photoproducts reaching in the environment through photomodification.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants, to which humans are largely exposed. PAHs have grasped much attention due to their carcinogenic potential and ubiquitous presence in the environment [1]. Non smokers are mainly exposed to PAHs through food and air. PAHs are significantly present in food due to heat processes such as smoking, grilling and smoke-drying. Food can also be naturally contaminated by an accumulation of PAHs in the food chain, due to their lipophilic properties [2]. In 2008, the scientific opinion of the panel on contaminants in the food chain [3] concluded that the current system based on monitoring benzo(a)pyrene alone is not a suitable indicator of the occurrence of total PAHs in food while including Benz(a)anthracene and other PAHs are also most suitable indicators of PAHs in food. According to [4], inadvertent dust exposure is responsible for 11% of non-dietary total PAHs exposure in adults and as much as 42% in young children. PAHs in indoor dust are derived from many origins including domestic burning, power generation, cooking, tobacco smoking, etc. [5]. Vehicular traffic, both diesel and petrol induced, continues to be the major problem for Delhi, as it has the highest number of vehicles among Indian cities [1]. Benz(a)anthracene concentration at traffic sites in Delhi ranged from 48.94 μg/kg−1 to 520.77 μg/kg−1, while in rural site the observed values were 0.1–47.21 μg/kg−1 [1]. Earlier studies including measurements of several PAHs species in ambient aerosol in Ahmedabad, New Delhi, Mumbai and Nagpur show that total PAHs concentration in Indian cities are 10–50 times higher than those reported internationally, in which Benz(a)anthracene range 1.2–358 ng m3 [6].

PAHs are very stable molecules, which generally nontoxic in dark, whereas they show phototoxicity under ultraviolet radiation of sunlight [7]. Most of these PAHs are carcinogenic and may additionally generate deleterious singlet oxygen inside the dermis when skin is exposed to UVA [8]. Several studies have found that exposure to sunlight changes the chemical structure of PAHs and enhance its toxicity. Photomodification generally occurs via an oxidation reaction, leading to the formation of new compounds which are in many cases more toxic than their parent compounds [9]. Our previous studies have reported that anthracene (PAHs) as well as its photoproduct viz. anthrone is phototoxic under sunlight/UV exposure [10], [11]. Highly reactive photoproducts can induce a spectrum of DNA damage including DNA adducts, oxidation of DNA bases and DNA strand breaks which are closely associated with mutation and carcinogenesis [9]. As photoproducts are likely to accumulate in the environment allowing for more realistic analysis of PAHs loads.

UVA irradiation decreased the mitochondrial activity of cells when the extracts contained PAHs [8]. Our previous finding suggested that PAHs under sunlight generates ROS like 1O2 which resulted in DNA damage, cell cycle arrest and finally cell death [11]. Tattoo inks contain PAHs such as Benz(a)anthracene at a concentration of 1.6 μg/g and after exposure of UV radiation generates singlet oxygen which affect cellular integrity [8]. Benzo(a)pyrene exposed to solar simulated light induced phosphorylation of histone H2AX (γ-H2AX) which was recently identified as an early event after the induction of DNA double strand breakage [9].

Oxidative stress is implicated widely in apoptotic cell death depending on the extent and the cell type [12]. Major sources of intracellular ROS include cytoplasmic and mitochondrial. When ROS production either by mitochondria or cytoplasmic becomes excessive, the natural cellular antioxidant defence system is over-whelmed, resulting in oxidative stress. One mechanism of cell death is by apoptosis also called programmed cell death. Apoptosis is a tightly regulated form of cell death, which can be initiated by two different types of signals: intracellular stress signals (intrinsic pathway) and extracellular ligands (extrinsic pathway) [13].

In this study, we examined the pattern of UV radiation in sunlight and its impact on environmental PAHs (BA) for photomodification and phototoxicity. Photosensitized BA induced oxidative stress mediated DNA damage induced apoptosis via mitochondrial mediated pathway.

Section snippets

Radiation source and dosimetry

The UV-irradiation system comprised an array of 1.2 m long UV-R emitting tubes manufactured by Vilber Lourmat (France). The intensity of emitted light was measured by a microprocessor-controlled RMX-3W radiometer (Vilber Lourmat) equipped with calibrated UV-A, UV-B and UV-C detecting probes. The spectral emission of UV-A source ranged from 320 to 400 nm with a peak at 365 nm, whereas the spectral emission of UV-B source ranged from 290 to 320 nm with a peak at 312 nm. The radiation dose was measured

O2radical dot mediated BA photomodification

(Fig. 1a–d) BA showed strong absorption maxima (λmax) in UV-B (281 and 292 nm) and UV-A (347 nm). BA (1 μg/ml) was exposed under sunlight for various time intervals from 0 to 7 h. BA was photodegraded under sunlight for up to 7 h and was time dependent. Spectrophotometry result of BA photodegradation was confirmed through GC–MS analysis, which also showed time dependent BA photodegradation. BA (dark control) without irradiation had a retention time of 15.4 min. After 1 h of sunlight exposure one

Discussion

The aim of our study was to assess the environmental stability and phototoxicity mechanism of BA under natural environment. BA showed a strong absorption maxima (λmax) under UV-B radiation. BA generates O2radical dot under sunlight exposure and further leads to photomodification which was confirmed through spectrophotometer and GC/MS analysis. This is the first report which showed O2radical dot catalyzed formation of Benz(a)anthracene-7,12-dione as a photomodified product of BA under natural sunlight exposure. BA

Conclusion

This is the first report which showed O2radical dot catalyzed photomodification of BA under natural sunlight exposure. O2radical dot mediated DNA damage induced apoptosis through mitochondrial mediated pathway. Thus, we conclude that photosensitized BA pose a threat to human beings in the light of skin cancer as well as flora and fauna due to the severity of DNA damage under natural sunlight exposure as well as its photoproduct formation which increases PAHs load in the environment.

Conflict of interest statement

There are no conflicts of interest.

Acknowledgements

The authors wish to thank the Director, CSIR-IITR for his support. We gratefully acknowledge the financial support provided by INDEPTH BSC 0111, CSIR, New Delhi, India. IITR-Communication No. 3167.

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