Barbara Crone (Presenter)
Institute of Inorganic and Analytical Chemistry
Bio: -PhD student in the 2nd year -at the Institute of Inorganic and Analytical Chemistry, University of Münster
Authorship: Barbara Crone (1), Julia Bornhorst (2), Michael Aschner (3), Tanja Schwerdtle (2), Uwe Karst (1)
(1) Institute of Inorganic and Analytical Chemistry, University of Münster, Münster, Germany (2) Institute of Nutritional Science, University of Potsdam, Nuthetal, Germany (3) 3Department of Molecular Pharmacology, Neuroscience, and Pediatrics, Albert Einstein College of Medicine, Bronx NY, USA
| Short Abstract Cisplatin is one of the most important and frequently used cytostatic drugs within treatment of cancer. The cytostatic effect is based on binding the DNA. The associated DNA deformations and the interference with DNA repair mechanisms lead to apoptosis in cancer cells. One important factor of these repair mechanisms is poly(ADP-ribose)-polymerase-1 (PARP-1). To analyze the bioavailability of Cisplatin in Caenorhabditis elegans, a method laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and total reflection x-ray fluorescence (TXRF) were developed. Therefore, L4 stage wildtype worms and poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutants were treated with Cisplatin. Loss of pme-1, the ortholog of human PARP-1, is resulting in a disturbed DNA repair mechanism. The influence of Cisplatin was analyzed for situations, when DNA damage already exists. |
Long Abstract
Introduction
Cancer is a leading cause of illness and death worldwide. Since its discovery in the late 1960s as a chemotherapy drug, cis-diamminedichloroplatinum(II) (Cisplatin) is one of the most commonly used anticancer drugs and is clinically proven to efficaciously combat various types of cancers. It is generally considered that the DNA is the critical target for Cisplatin cytotoxicity by inhibiting DNA synthesis and damaging DNA, whereby different DNA adducts of Cisplatin, including intra- and interstrand cross-links, DNA-protein cross-links and DNA monoadducts are formed. Since Cisplatin can induce apoptosis, the signaling pathways that regulate apoptosis have significant impact on regulating cellular responsiveness. The success of Cisplatin therapy is compromised due to dose-limiting toxicity in the kidney, cochlea and peripheral nerves, with nephrotoxicity being the most well described.
Caenorhabditis elegans (C. elegans) represents an established model organism for biomedical research. Characteristics that have been contributed to its success include the genetic manipulability, the well-characterized genome, the ease of maintenance and the small body size. It takes three days for an embryo to reach adulthood and reproduce. A single hermaphrodite has the ability to produce approximately 300 offspring, which enables high-throughput analyses. C. elegans is less complex than the mammalian system, while still sharing high genetic homology (60–80%). The model organism C. elegans has been previously used in a limited number of studies to identify interactions between Cisplatin and signaling pathways in vivo. Since DNA repair mechanisms are known to occur in the genetically amenable nematode, deletion mutants have been used to identify various genes of the DNA repair pathways that are protective against Cisplatin toxicity.
Herein, a LA-ICP-MS method for Cisplatin bioimaging in the larval 4 (L4) stage wildtype (WT) worms was developed to localize Cisplatin distribution in C. elegans. Additionally, bioimaging was performed in poly(ADP-ribose)metabolism enzyme 1 (pme-1) deletion mutant worms following Cisplatin exposure. Loss of pme-1, which is the C. elegans ortholog of human poly(ADP-ribose) polymerase-1 (PARP-1), was posited to decrease genomic stability due to a disturbance of DNA repair pathways. Additionally, we assessed the response to Cisplatin treatment to determine appropriate dosing for the bioimaging studies as well as Cisplatin toxicity and its effect on the nematode`s reproduction.
Method
Treatment of C. elegans was performed using 500 (survival, brood size) or 1500 (Cisplatin uptake) L4 stage worms. L4 nematodes were exposed to Cisplatin (50, 100 µg/mL) in siliconized tubes for 1 h or 2 h. For the lethality assay 30 - 50 worms were placed on OP50-seeded NGM plates, pre-counted and the number of surviving worms was scored 24 h post-treatment.
For brood size evaluations, worms were evaluated by counting the number of progenies from each individual worm day-by-day. Following Cisplatin treatment individual worms were kept on OP50-seeded NGM plates and transferred every day to a new plate until day eight of adulthood. The number of progenies was scored every day.
Imaging experiments were performed using a laser ablation system based on a Nd:YAG laser with a wavelength of 213 nm, coupled to a quadrupole-based inductively coupled plasma mass spectrometer. Besides the dry aerosol, a rhodium solution as the internal standard was simultaneously introduced, to improve plasma stability. Possible interferences of including [40Ar155Gd] were minimized in the kinetic energy discrimination mode (KED) with He as cell gas. The ablation of the nematodes was performed in a multiline scan. Laser parameters were optimized to obtain optimal spatial resolution and elemental information.
The quantification of Cisplatin by total reflection X-ray fluorescence (TXRF) was done after ashing Cisplatin exposed L4 stage worms and dilution of the ash in 0.5 mL HNO3.
Results
The elemental mapping of the platinum distribution shows that Cisplatin is predominantly located in the area of the intestine. Moreover, an accumulation in the head of the nematode could be observed. Overall, signal intensities were in the range of 500 cps to 1000 cps. A longer incubation time (2 h) led to an increased platinum intensity. Similar results for time- and dose-dependency could be detected incubating 50 µg/mL Cisplatin with an overall reduced signal intensity due to the lower incubation concentration. Cisplatin exposure of the pme-1 deletion mutants resulted also in a Cisplatin uptake, which was indistinguishable from the WT worms at the respective dose and exposure time.
Overall, the analyzed strains, determined by TXRF, showed a significant dose-dependent increase. Additionally, Cisplatin uptake in the pme-1 deletion mutants was indistinguishable from WT worms for each of the respective doses and duration of Cisplatin exposure, corroborating the LA-ICP-MS data.
In order to determine appropriate Cisplatin concentrations for dosing in the bioimaging study and to determine Cisplatin sensitivity, survival curves following Cisplatin exposure were assessed. The genetic deletion of pme 1 increased mortality compared to WT worms following 2 h Cisplatin treatment. Cisplatin treatment at 100 µg/mL (1 h), 50 µg/mL (2 h) or 100 µg/mL (2 h) resulted in a significant decrease in surviving pme 1 deletion mutants.
To determine whether the Cisplatin doses used within the bioimaging study caused lasting effects on C. elegans reproduction, brood size was examined following Cisplatin incubation. Overall, the analyzed strains showed a significantly decreased brood size following 100 µg/mL (1 h), 50 µg/mL (2 h) or 100 µg/mL (2 h) Cisplatin incubation.
Conclusion
The newly developed laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method requires a fast and simple sample preparation and can be used rapidly and easily to visualize the Cisplatin distribution in the model organism C. elegans. A spatially-resolved elemental analysis with respect to the anatomic structure is possible. The data suggest that Cisplatin is taken up dose- and time-dependently and that it is predominantly located in the area of the intestine and in the head of the worms. Studies in the genetically tractable worm also established that deletion of pme-1 is associated with increased sensitivity to Cisplatin compared to WT worms.
Taken together, spatially-resolved visualization of Cisplatin in a whole model organism opens up the possibility to correlate toxicological alterations with the amount of drug and might be helpful for a better understanding of Cisplatin pharmacokinetics and dose-efficiency studies of Cisplatin. Furthermore, the development of a quantification method might provide helpful additional information about concentration levels inside the worm.
References & Acknowledgements:
Crone, B.; Aschner, M.; Schwerdtle, T.; Karst, U.; Bornhorst, J.; Elemental bioimaging of Cisplatin in Caenorhabditis elegans by LA-ICP-MS, Metallomics, 07 1189-1195 (2015)
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