Bioanalytical strategies for the quantification of xenobiotics in biological fluids and tissues 3

Chapter 3 Biomonitoring of environmental organic pollutants in human ovarian tumor cyst fluids samples using principal component analysis... 3.1 Preface to Chapter 3 To assess a possible

Chapter 3 Biomonitoring of environmental organic pollutants in human ovarian tumor cyst fluids samples using

principal component analysis

3.1 Preface to Chapter 3

To assess a possible etiological role of environmental organic pollutants in ovarian cancer development on ovarian cancer patients, concentrations of different groups of organic pollutants were measured in 20 malignant and benign ovarian cyst fluid samples of women with ovarian cancer A total of 60 chemicals of six groups including heterocyclic aromatic amines, Low molecular weight organic acids, aromatic amines, N-nitrosamines, Polybrominated diphenyl ethers and halogenated flame retardants and organochlorine pesticides were assayed via porous membrane protected micro-solid-phase extraction followed by GC-MS detection and HPLC-fluorescence detection High performance liquid chromatography coupled with fluorescence detection was used to quantify heterocyclic aromatic amines and aromatic amines Gas chromatography-mass spectrometry was used to quantify PBDEs and halogenated flame retardants, and LMW organic acids Trace amounts of most of the chemicals were found both in benign and malignant cyst fluid samples The trend in their concentration in benign and malignant samples was projected by principal component analysis using R program The results reveal that the possible correlations in the concentration of chemicals with the malignancy of the ovarian tumour

3.2 Introduction

Ovarian cancer is the fifth most common cancer among women worldwide and

is the fourth most common cancer in Singapore [1] It causes more deaths than any other type of female reproductive cancer The risk for developing ovarian cancer appears to be affected by several factors Exposure to endocrine disruptive xenobiotics is recognized as an important environmental risk factor associated with development of cancer

Global epidemiologic studies have indentified environmental and occupational chemicals as potential carcinogens Many studies provide the direct association between these chemicals, especially EDCs with the development of different types of gynaecological cancer [2] Many environmental chemical contaminants, which may also be the metabolic intermediates, particularly those that are lipophilic and of relatively low molecular weight, can accumulate in tissue and body fluids The potential health effects of these contaminants on human are of great concern, making

it important to carefully monitor their levels and trends

Many methods have been developed for the exposure to carcinogens in human, through the detection of carcinogens or their metabolic derivatives in body fluids Biomonitoring studies, designed to assess the health implication of environmental chemicals, including carcinogens, are seriously negotiated by the lack

of quantitative exposure data for individuals in exposed populations Monitoring data

on levels of compounds in environmental media often represent the average population exposure is therefore the only quantitative factor that can be estimated In the present study, we evaluated the association level of wide range of environmental

contaminants in human ovarian cyst fluids with early stage (benign) and late stage (malignant) ovarian cancer The groups of EDCs, which are well known carcinogens, studied were heterocyclic aromatic amines (HAAs), PBDEs, OCPs and N-nitrosamines In addition, metabolic intermediates such as aromatic amines and low molecular weight (LMW) organic acids, which are potential xenobiotics, were studied

as well

OCPs are persistant in nature and non biodegradable Since they are highly lipophilic, they can bioaccumulate in fatty tissues getting up to metabolism through diet, especially foods of animal origin [3] Being a chlorinated compound, organochlorine pesticides strongly mimic estrogen in the body Due to their estrogenic activity, most OCPs were classified as “possibly carcinogenic to humans” (2B group); consequently they increase special attention in public health and epidemiology [4-6].

N-nitrosamines are classified as class 2A genotoxic chemical carcinogens and animal testing indicated mutagenic, carcinogenic and tetragonal effects They occur in the human diet and in environment, and can be formed endogenously in the human body [7] More than 90% of nitrosamines had shown to cause cancer in animals It had also been reported that with exposure to endogenously formed N-nitrosamines, there is a higher risk of tumor [8]

PBDEs are flame-retardant chemicals that are added to plastics and foam products to make them difficult to burn [9] They are environmentally widespread and human exposure to those compounds is logical Many studies have been reported that PBDEs have endocrine disrupting properties suggesting their potential role in hormonally related cancers such as ovarian cancer [10-13] Based on a study on mice

and rats, the US EPA has classified some of the PBDEs are a possible human carcinogen [14].

The carcinogenicity of aromatic amines and heterocyclic aromatic amines are well documented [15-17] Being an important class of industrial and environmental chemical, aromatic amines easily entered into biota and human metabolism Aromatic amines are converted in the hosted organism to arylhydroxamic acid or arylhydroxylamines derivatives which are thought to be the critical carcinogenic forms of those amines These derivatives stimulate tumors, usually in tissues distance from the site of administration [18] Cooking of protein-rich foods mainly from animal origin may stimulate the formation of a series of heterocyclic aromatic amines [19] They have also identified in cigarette smoke condensate and diesel exhaust [20, 21]

LMW organic acids can be found in the environment naturally such as in rainwater or soil They are important intermediate breakdown products between large biomolecules and the ultimate demineralization products CH4 and CO2 [22].Determination of organic acid concentrations is crucial in body fluids since abnormal levels of organic acids in the blood (organic acidemia), urine (organic aciduria), and tissues can be toxic and can cause adverse health effects [23] Moreover, it is significance to estimate the organic acid level variation in benign and malignant tumor cyst fluids as they have genotoxicity

Hence, monitoring these chemicals in ovarian tumor cyst fluids will be useful

to explore the carcinogenicity of such chemicals The objective of this study is to determine profile and quantify various xenobiotics (total sixty individual analyte of

six different groups) which mimic estrogens and estrogen metabolites from malignant and benign ovarian cyst fluid samples Further, from the results, their potential associated with malignancy associated with ovarian tumors was investigated The samples were preconcentrated using the micro-solid phase extraction (µ-SPE) which had proved to be a suitable technique for cyst fluid samples [25] Wide choice of sorbents makes this technique versatile for variety of group of analytes The determination was done by using liquid and gas chromatographic techniques The obtained data was processed by principal component analysis (PCA) to simplify the complex data system with focus on concentration patterns and correlations Measurements are made on twenty individual samples, they provide an indication not only of exposure to a given substance, but also of the amount absorbed and metabolically transformed to activated derivatives No previous study has directly investigated the presence of these toxic chemicals in the ovarian cyst fluids of human patients with ovarian cancer

body fluids and solvents used in this project were decontaminated according to standard biohazard disposal protocols All patients’ personal information was concealed to protect their identities The pathological information of the samples is listed in Table 3.1

Table 3.1

Age, Tumour marker CA-125* and Pathology of the samples

focal borderline change

borderline

*CA-125, cancer antigen-125, is a protein that is found at levels in most ovarian cancer cells that are elevated, compared to normal cells CA-125 is produced on the surface of cells and is released in the blood stream

3.3.2 Chemicals

BDE -47, -49, -99, -153 and -154 were bought from AccuStandard (New Haven, USA) (Figure 3.1) PEB was obtained from Sigma-Aldrich (Wisconsin, MO,USA) HAAs compounds studied were purchased from Eckert & Ziegler CNL Scientific Resources (Valencia, CA, USA) (Figure 3.2) Aromatic amine compounds studied were bought from Fluka (neu-Ulm, Germany) (Figure 3.3) Oxalic acid, fumaric acid and citric acid were purchased from Sigma Aldrich (Milwaukee, USA) whereas lactic acid came from Fluka (Buchs, Switzerland) (Figure 3.4) 3-methylglutaric acid, adipic acid and sebacic acid were purchased from Merck OCPs were were purchased from Polyscience (Niles, IL, USA) Q3/2 Accurel polypropylene hollow fiber membrane was purchased from Membrana GmbH (Wuppertal, Germany) The solvents used for HPLC detection (HPLC-grade methanol, acetone, triethylamine) were obtained from Tedia Company, Inc (Farfield, OH, USA) From Fisher Scientific (Loughborough, UK), HPLC-grade toluene, hexane, isooctane and dichloromethane were obtained HPLC-grade acetonitrile and ACS-grade sodium acetate, glacial acetic acid, bis (trimethylsilyl) – trifluoroacetamide (BESTFA) were bought From Merck Analytical grade Pyridine was obtained from J.T Baker (Philipsburg, NJ) Sodium chloride, sodium sulphate anhydrous and sodium hydroxide come from Goodrich Chemical Enterprise (Singapore) The water used was purified using a Milli-Q (Millipore, Bedford, MA, USA) water purification system

Figure 3.1 Chemical Structures and abbreviated names of PBDEs and PEB

Figure 3.2 Structures, names and abbreviated names of the 6 heterocyclic aromatic

amines

Figure 3.3 Structures and names of aromatic amines

Figure 3.4 Structures and names of LMW organic acids

3.4 Laboratory Methods

3.4.1 Preparation of µ-SPE device

3.4.1.1 µ-SPE device for endocrine disrupting chemicals

The preparation of the µ-SPE device has been described previously in chapter

2 Briefly, the device consisted of sorbent held within an envelope made from polypropylene membrane sheet of dimension 2 cm × 1.5 cm The edges were heat sealed Before use, each µ-SPE device was conditioned (ultrasonication for 10 min with 5 mL of acetone) and stored in the same solvent

The choice of sorbent for these four groups of EDCs was tested using different sorbent materials for better extraction efficiency For PBDEs, OCPs and N-nitrosamines, Hayesep A-C18 (1:1, 10 mg each) was selected as a suitable sorbent based on the peak area analysis The other combination tested were HayeSep A with

C8, HayeSep A with C2, HayeSep B with C18, HayeSep B with C8, HayeSep B with

C2 For HAAs activated alumina was selected as a suitable solvent since it shows higher extraction efficiency The sorbents which are tested for their suitability for HAAs are C2, C8, C18, Hayesep A, Hayesep B, activated alumina

3.4.1.2 µ-SPE device for metabolic intermediates

For the metabolic intermediates, to be exact, aromatic amines and organic acids, the material used for the micro extraction is similar to a µ-SPE device However, unlike the usual device whereby the sorbent was packed into a polypropylene membrane, the gold nanoparticles were coated onto the membrane

itself Polyethersulfone membrane was chosen as the support material as it is hydrophilic and can bind to the hydroxyethylcellulose (HEC) capped gold nanoparticles

Gold nanoparticles coated membrane were prepared by adding 1mM of gold (III) chloride to 10 ml of 15mg mL-1 of HEC The gold solution mixture was constantly stirred on a magnetic stirrer at 1200 rpm and maintained at a constant temperature of 75˚C for 2 hours to allow the formation of gold nanoparticles The color of the solution should change from yellow to brown purple in colour The gold nanoparticles were between 150 to 600nm (obtained from unpublished previous experiment)

Pieces of polyethersulfone membrane with dimensions 0.5 cm × 2.5 cm were added to the gold solution mixture for 2 hours to allow coating to take place The newly formed gold nanoparticles coated membranes were then removed and air-dried The gold nanoparticles coated membranes were then soaked in ultra-pure water followed by ultra-sonication in toluene for 20 min and stored in ultrapure water until use

3.4.2 µ-SPE procedure

For endocrine disrupting chemicals, previously reported µ-SPE procedure for

cyst fluids was employed for extraction [25] Briefly, the µ-SPE device after drying in

air for few minutes was placed in 10mL of sample solution The sample solution was agitated at 105 rad s−1 for 60 min to facilitate extraction After extraction, the device was taken out of the sample solution, dried thoroughly with lint free tissue and placed

in a 500 µL autosampler vial for desorption The analytes were desorbed from the

device to the solvent by 8 min ultrasonication Then µ-SPE device was removed from the desorption vial and the extract was kept in a water bath at 60˚C for 20 min Finally, 2 µL of extract was injected into the GC-MS or HPLC for analysis For GC-

MS analysis 100 µL of acetone and BSTFA mixture (5:1 ratio) was added after desorption and then undergo ultrasonication

For the extraction of metabolic intermediates i.e aromatic amines and LMW organic acids, the gold nanoparticles coated membrane was hanged using a fishing line and immersed into the sample This was to keep the membrane in suspension and

to prevent the stirrer from breaking the membrane The sample was stirred at 100 rpm for 50 min After extraction, the membrane, the membrane was inserted into a 250 µL micro vial containing 100 µL of acetonitrile as the desorption solvent The analytes were desorbed by ultrasonication for 20 min and the extract was later transferred to a clean 250 µL auto sampler viol for analysis The gold-PES membrane was cleaned by ultrasonication in acetonitrile for 10 min before reuse for the next extraction

Amines

Desorption solvent Acetone Hexane Dichloromethane Toluene Toluene

3.4.4 Analytical quality assurance

The optimized conditions were employed to evaluate the performance of SPE Under these conditions, Limit of detection (LOD) , Limit of quantification (LOQ), Relative standard deviation (RSD), Relative recovery of the current methods were measured To calculate LODs the sample was spiked with 10 µg L-1 ofstandards and three replicates were used LOQs at S/N = 10 were calculated and are listed in Table 3.3

µ-Table 3.3

Quality assurance data

QA data LOD (µgL -1 ) LOQ (µgL -1 ) RSD (%)

Relative recovery (%) Organic acids 0.002 - 0.003 0.007 - 0.009 16.1 - 20.4 81

Analysis of LMW organic acids, nitrosamines, OCPs, PBDEs were carried out

by a Shimadzu QP2010 GC–MS system equipped with a Shimadzu AOC-20i autosampler For all these compounds a DB-5 (J & W Scientific, Folsom, CA) fused

silica capillary column (30 m × 0.32 mm internal diameter, 0.25µm film thickness) was employed

For LMW organic acids, helium with 1.92 mL min-1 flow rate was employed

as the carrier gas Other conditions used are: initially 90˚C for 1 minute followed by the increase of 10˚C min-1 until 270˚C which is held for 1 minute 280˚C injector port, 90˚C column oven, 200˚C ion source and 280˚C interface temperature were used To determine the organic acids derivatives, SIM with high sensitivity was employed

For PBDEs, helium with 0.93 mL min-1 flow rate was employed as the carrier gas Other conditions used are: initially 50˚C for 2 minutes followed by the increase

of 20˚C min-1

until 100˚C, 10˚C min-1 to 200˚C and 20˚C min-1 until 300˚C which is held for 7.5 minutes 300˚C injector port, 200˚ ion source and 300˚C interface temperature were used Electron impact ionization (EI) mode with selected ion monitoring (SIM) was used for the spectrometer

For nitrosamines, the helium carrier gas was maintained at a constant flow of

0.5mL min−1.Injection volumes of 3mL were used The injection port was held at 230˚C and used in the splitless mode, applying a pressure pulse of 40 psi The GC temperature was programmed as follows: start temperature of 70˚C (held 3min) and increase to 140˚C at 15˚C min−1, then to 200˚C at 5˚ C min−1 and finally to 250 at 10˚C min−1.Ionization was carried out in the electron-impact (EI) mode

For OCPs helium was used at a flow rate of 1.5 ml min−1 and a split ratio of

20 Samples (2 µL) were injected in splitless mode with an injection time of 2 min The injection temperature was set at 250◦C, and the interface temperature at 280◦C The GC-MS temperature program used was as follows: initial temperature 50◦C, held

for 2 min, then increased by 10◦ C min−1 to 300◦C and held for 3 min OCP standards and samples were analyzed in selective ion monitoring (SIM) mode with a detector

voltage of 1.5 kV and a scan range of m/z 50 to 500

3.5.2 HPLC analysis

HAAs and aromatic amines were analysed by HPLC A Shimadzu CBM-20A system controller, DGU-20A5 degasser, Shimadzu binary HPLC pump LC-20AD coupled with Shimadzu fluorescence RF-10A was employed (Kyoto, Japan)

For the analysis of HAAs, a Zorbax Eclipse Plus C18 column (4.6 mm × 250

mm, 5 μm particle size) was employed The fluorescent detection was used with detection wavelength 310 to 400 nm and gain sensitivity medium The flow-rate of the binary mobile phase is 1 mL min-1, 0.01M with pH 3.65 solvent A (triethylamine phosphate), and solvent B (acetonitrile) The gradient program employed was 85% solvent A from 0 to 11 minutes, 80% solvent A from 11 to 17 minutes, 65% solvent A from 17 to 25 minutes, 85% solvent A in 5 minutes and 5 minutes of post run delay

For the analysis of aromatic amines, I.D MetaSil 5u ODS column (50 mm × 3.0 mm) (Varian, Palo Alto, CA) was employed The following conditions were used during the analysis: the fluorescent detection wavelength was 254 nm and gain sensitivity medium, mobile phase of 0.01M pH 3.5 acetate buffer and acetonitrile, flow rate of the mobile phase 0.3 ml/min, gradient programme of 85:15, v/v

3.5.3 Statistical analysis

PCA was applied in an attempt to reveal latent structures in the dataset, with focus on concentration patterns and correlations in cyst fluid analysis Chemical