Synergistic effects of UV radiation and polycyclic aromatic hydrocarbons (PAH) in the development of squamous cell carcinoma - mechanistic study

Status:

completed 03/2022

Aims:

In this project, a possible syncarcinogenesis of UV radiation and benzo[a]pyrene (B[a]P) in two different skin models (KeratinoSens^TM-/U937 cells and human ex vivo skin) should be investigated from a mechanistic point of view. For this purpose, the skin models were exposed to different B[a]P concentrations with/without UV radiation. DNA damage and repair, possible underlying mechanisms such as oxidative stress, and changes in cellular metabolic pathways (metabolomics) were investigated. A benchmark dose (BMD) calculation was performed to quantify the onset of an adverse effect.

Activities/Methods:

Exposure: Both skin models were exposed to a range of B[a]P concentrations with/without UV irradiation to map a possible transition from an adaptive to an adverse cellular response.

Methods:

In vitro

1. time- and dose-dependent uptake and metabolism of B[a]P by fluorescence spectrometry
2. normalization of results to living cells
3. assessment of the activity of various enzymes (CYP [cytochrome P450)], NQO1 [NAD(P)H quinone dehydrogenase]), viability (LDH [lactate dehydrogenase]), cellular metabolism (MTT), energy generation (MMP [mitochondrial membrane potential])
4. oxidative stress (ROS [reactive oxygen species]; ratio reduced/oxidized glutathione; lipid peroxidation; activation of Keap1-Nrf2-ARE pathway in KeratinoSens^TM), and DNA damage/repair (γH2AX [DNA repair]; Comet [DNA damage]
5. data modeling (BMD).

Ex vivo:

1. CYP450, NQO1
2. LDH, MTT
3. red./ox. glutathione; lipid peroxidation
4. γH2AX
5. BMD.

Metabolomics:

1. identification of regulated metabolites by untargeted gas chromatography-mass spectrometry (GC-MS) analysis in vitro as an indication of altered metabolic pathways in response to exposure
2. biostatistical/informatic analysis (MetaboAnalyst).

Results:

In vitro: In both cell lines, B[a]P alone had a minor effect on the measured parameters, whereas combined UV and B[a]P exposure showed a partial synergistic effect. BMD values for most endpoints ranged between 0.005-0.05 µM B[a]P. BMD values for genotoxic endpoints were at significantly lower B[a]P concentrations. Induction of DNA repair (γH2AX) was the most sensitive endpoint with BMD values of 0.0006 µM B[a]P (KeratinoSens^TM) and 0.001 µM B[a]P (U937).

Ex vivo: No synergistic effect occurred in the measured parameters. Combined B[a]P/UV exposure resulted in lower γH2AX values compared with B[a]P treatment alone and higher values compared with UV irradiation alone. The BMD value for γH2AX after B[a]P treatment alone was approximately 0.13 ng/cm².

Metabolomics: Combined exposure results in increased regulation of metabolites at lower B[a]P concentrations than B[a]P exposure alone. Statistical analysis indicated a clear separation of the regulated metabolites as a function of exposure. In KeratinoSens^TM cell, glutathione, adenosine monophosphate, and L-aspartate were the metabolites that most discriminated the irradiated and exposed cells from control cells. Glutathione is the major antioxidant defense, and adenosine monophosphate and L-aspartate are involved in DNA synthesis, among other functions.

In both models, the oxidative balance was stressed, but significantly less in the ex vivo skin. With regard to DNA damage, the responses in the models were different. In the ex vivo skin, B[a]P exposure alone had the strongest effect on DNA repair. In contrast, B[a]P + UV exposure had lesser effects, indicating no synergistic effect on DNA damage under the experimental conditions studied. Further investigations are necessary in this field.

Last Update: