Environmental Biomarkers For Human Pharmaceutical Contamination

Published Date: 02 Nov 2017

Richard Chan

BIOL/CHEM 465

Aquatic Toxicology

Introduction

As pharmaceutical companies continue to develop medications for use in treatment of human and animal diseases, the importance of monitoring environmental systems for contamination intensifies. These emerging toxins in the ecosystem have the potential to alter normal physiological processes in organisms, ultimately affecting behaviors and lifespan. In mammalian systems, many pharmaceuticals are metabolized by the hepatic enzymes for excretion or bioactivation (Christen et al., 2010). Fish have also been shown to have similar biotransformation and induction in the liver in response to synthesized drugs (Kleinow et al., 1987). Due to the resemblance to hepatic systems of mammals and ecological consequences, the objective of this paper is to demonstrate the relevance of monitoring piscine enzymes as biomarkers of contamination by pharmaceuticals.

Pharmaceuticals can enter the system by numerous methods. One main pathway is via excretion from the system of humans after application. The drug can enter as its complete form or as a metabolite with wastewater. Additionally, unused medications are also disposed in waste water or as runoff from landfills. The chemicals may then be released as effluent and contaminate bodies of water such as rivers, estuaries, and lakes (Fent et al., 2006).

Biotransformation of drugs primarily takes place over two phases within the liver. Phase I, or bioelimination, converts the parent compound into a more polar substance. Cytochrome P450 enzymes play a major role during the first phase via processes such as oxidation, reduction, and hydrolysis. These reactions can either reduce the effectiveness of the drug or potentially produce a more biologically active metabolite (Siroka & Drastichova, 2004). Substrates for Phase II reactions can involve products from Phase I or the parent compound, depending on its properties. This step conjugates the compounds with groups such as glucuronate and glutathione to further polarize compounds to allow elimination. Siroka & Drastichova (2004) define biomarkers as "measurable responses to the exposure of an organism to xenobiotics. They usually respond to the mechanism of toxic activity and not to the presence of a specific xenobiotic." In the environment, induction of these enzymes in fish can play a major role in biomonitoring as increased elimination may also be an indicator of tolerance to pollutants (Stegeman & Kloepper-Sams, 1987).

Pharmaceutical Effluents

The Patancheru, India is the site for a large pharmaceutical manufacturing region where wastewater is contaminated with many drugs (Larsson, de Pedro, & Paxeus, 2007). In a previous study conducted by Carlsson et al (2009), a 0.2% dilution of the effluent from the local water treatment plant was capable of reducing the growth of tadpoles by 40%. This prompted Gunnarsson et al. (2009) to investigate the effects of effluent from the same location on rainbow trout (Oncorhynchus mykiss). Their objective was to analyze differential gene analysis as a means of identifying biomarkers for exposure.

After exposure of rainbow trout to a 0.2% concentration of effluent samples for 5 days, liver tissue was removed from the fish and fractionated for microarray analysis and quantitative polymerase chain reaction (qPCR). Additionally, microsomal liver fractions were further analyzed for hepatic enzyme activity also using qPCR. Over 200 gene expression changes were seen from microarray analysis. The gene most significantly up-regulated was cytochrome P450 1A (CYP1A) with an increase of 5.5-fold compared to the control. CYP1A enzymatic activity was represented by ethoxyresorufin-O-deethylase (EROD) activity, which was 4-fold higher compared to the controls. Glutathione-S-transferase  (GST) was another gene significantly induced in the exposed fish. GST expression doubled the levels in the controls by microarray and qPCR. However, an interesting finding was that the total levels of glutathione in the liver did not change, indicating relatively similar enzymatic activity as the non-exposed fish.

The induction of CYP1A in fish with elevated EROD activity indicates enhanced detoxification processes in the liver, as CYP1A is a Phase I enzyme involved in drug metabolism. This has the potential to support its use as a biomarker for contamination. Additionally, GST expression may also represent contamination as it is an enzyme indicative of oxidative stress. However, due to the large mixture of drugs contained in the effluent as well as the possibility for other non-pharmaceutical contaminants from runoff to be present, the specificity of the test is low.

Non-steroidal anti-inflammatory drugs (NSAID).

Non-steroidal anti-inflammatory drugs are a class of medications used to reduce inflammation and pain. Levels as high as 1 μg/L have been reported in sewage (Ternes, 1998). An important contribution to the concentrations of NSAIDs is their sales as over-the-counter medications. The primary mode of action by NSAIDs is inhibiting cyclooxygenase (COX) enzymes, thereby preventing the synthesis of prostaglandins, which are potent inflammatory molecules. In a study by Mehinto et al. (2010), the metabolic effects of environmentally-relevant levels of Diclofenac, an NSAID, on rainbow trout were investigated using mRNA expression of COX enzymes, CYP1A1 and p53 as biomarkers.

Rainbow trout were exposed to varying concentrations of diclofenac (0, 1, 5, and 25 μg/L) for a period of 21 days.