Identification of markers of cancer in urban sewage through the use of a suspect screening approach
Graphical abstract
Introduction
Growth in the incidence of cancer in the human population has led to an increase in the use of chemotherapy drugs with a further rise in the use of these drugs foreseen in the coming years [1]. As a result, greater attention needs to be paid to the occurrence of anticancer drugs in the environment and any potential ecological consequences. Although chemotherapy drugs are typically administered in hospitals, 75% of the treatments are outpatient therapies [2] and therefore cytostatic compounds can reach the aquatic environment via hospital or domestic wastewater with wastewater treatment plants (WWTPs) being the ultimate source [3], [4] It is also important to take into account that cytostatic compounds, as with many other pharmaceuticals, are excreted as both parent compound and as metabolites that may have the same mode of action as the parent cytostatic compound or potentially even greater activity. This is the case for 4-hydroxytamoxifen, a metabolite of tamoxifen, which is a more potent estrogen receptor antagonist than the parent compound [5]. Likewise, specific biomarkers produced by the body whilst suffering from cancer and during oncologic treatments, such α-fenotroin (prostate cancer), inositol (hepatocellular carcinoma), normethanephrine (hepatocellular carcinoma) [6], among other compounds, are present at higher or lower levels in urine. For instance α-fenotroin is present at concentrations of up to 50 ng/mL in the serum/urine of patients with liver cancer [6]. Since these substances are concomitant with chemotherapy drugs they have the potential to provide useful information about the prevalence of different types of cancer as well as about the chemotherapy drugs consumed in the population served by a WWTP.
The high cost of chemotherapy pharmaceutical reference standards, often based on a challenging and expensive synthesis, and especially their particular hazard to health or safety, makes conventional target analysis of these compounds difficult in most of the environmental laboratories due to the special and expensive safety conditions required for their handling (analyst training for cytotoxic handling and spills, personal protective equipment, biological safety cabinet or similar category hood, specific containers for residues, etc). Nevertheless different analytical methodologies without the use of standards have recently been developed for the screening of micropollutants in water samples as a qualitative mode of assessment. In general, these novel approaches are based on high-resolution mass spectrometry (HRMS) using time-of-flight (TOF) or Orbitrap mass spectrometers often coupled to liquid chromatography (LC) [7], [8]. LC-HRMS allows the wide-scope screening of organic pollutants without the pre-selection of analytes and without using authentic reference standards [9]. Known compounds suspected of being present in environmental samples can be retrospectively screened following data acquisition [10].
In this work, a “suspect screening” approach was applied to wastewater samples collected from Oslo, Norway for the purposes of detecting and identifying (bio-)markers associated with prostate cancer and breast cancer.
Oslo is an ideal sampling location because Norway has a strictly controlled system for the prescription and dispensing of medications that is well-managed by the Norwegian Institute of Public Health (NIPH) through the Norwegian prescription database (NORDP) (www.nordp.no). This database provides accurate information on the use of therapeutic agents in any given region/city.
Prostate cancer and breast cancer were selected as the targets because a review of the World Health Organization databases reveals that these two cancers are the most frequently diagnosed in Norway (www.who.com).,
Section snippets
Chemical reagents
LC-MS grade water and acetonitrile were supplied by Merck (Darmstadt, Germany). Reagents, such as formic acid 98% (HCOOH), were supplied by Sigma-Aldrich (HPLC-grade) and leucine encephalin and ethylenediaminetetraacetic acid disodium salt (EDTA) was supplied by Sigma-Aldrich (Germany).
Sampling campaign
The urban area selected for this study, Oslo, has a population of approximately 647,000 residents. Both VEAS and Bekkelaget WWTPs, which are located on the Oslofjord, receive the urban wastewater from the city
Data processing and evaluation
Eight substances that are directly linked with the chemotherapeutic treatment of prostate and breast cancer were tentatively identified (Table 1).
Two antineoplasic hormones, megestrol (MGT) and medroxyprogesterone (MDG), were identified in 3 and 7 samples respectively (Table 1, Fig. 3, Fig. 4 respectively). One antiviral and antitumor agent, atazanavir (ATZ) [29], [30], was identified in 7 samples (Table 1 and Fig. 5). One liver tumour biomarker, inositol (INO), was identified in 4 samples (
Conclusions
This is the first time that a suspect screening paradigm has been applied to the identification of pharmaceuticals and biomarkers related to chemotherapy in wastewater.
A list of suspect ions ( > 1420 compounds) was generated from a review of the chemotherapeutic agents, mitotic inhibitors, anti-metabolites, hormones and immunotherapeutic agents identified within the NORDP databases. Dispensing rates and limitations in the analytical technique reduced this suspect list to approximately 300
Acknowledgments
This work has been supported by Spanish Ministry of Economy and Competitiveness (project CTQ2010-21776-C02), co-financed by the European Union through the European Regional Development Fund (ERDF) and supported by Generalitat de Catalunya (Consolidate Research Group: Catalan Institute for Water Research 2014 SGR 291). Laura Ferrando-Climent gratefully acknowledges the Yggdrasil grant for young visitor researchers from the Research Council of Norway. The authors would further like to thank Jose
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