ABSTRACT
This study aimed at investigating a possible early screening diagnostic tool for cancer. Cancer is ranked third among the leading causes of deaths in Kenya with an average of 75% of cancer patients being diagnosed at advanced stages of the disease. This late diagnosis is attributed to most patients only presenting themselves for check-ups when the disease has progressed. Information from various literature established that among the several causes of cancer, is prolonged exposure to toxic heavy metals and high concentrations of trace elements to the body. The study therefore aimed at evaluating the concentrations of Cr, Hg, Se, Pb, Cu, Zn, and Fe elements in the human body from esophageal and stomach cancer patients in Kenya. It was expected that this research work would identify a correlation between trace elements’ concentrations with esophageal and stomach cancers, which can be used as a prediction of the disease. This will allow for early intervention measures thereby reducing and mitigating the disease burden. It was a case-controlled study comprising 95 esophageal and stomach cancer patients and 31 non-cancer volunteers as a control group. Fingernail clippings were obtained by the researcher from newly diagnosed stomach and esophageal cancer patients at the Kenyatta National Hospital, Nairobi, Kenya who had consented to take part in the study. The study participants were recruited from adults aged between 32 to 65 years and were randomly selected from the health records at the Kenyatta National Hospital, Cancer Treatment Centre. The control group comprised of adult volunteers of matched age and sex and without any known cancer history, from orthopedic surgery, general surgery and ophthalmology wards at the Kenyatta National Hospital. Informed consent was obtained and written questionnaires administered by the principal investigator before participating in the study. The total number of esophageal cancer patients who participated in this study were 72, while 23 were stomach cancer patients and 31 non-cancer control group. An average of 50 mg of nail clipping samples were obtained from each participant using sterilized stainless nail cutters. Each of the nail samples were then cleaned by putting under continuous stirring according to sample washing procedure as suggested by International Atomic Energy Agency. Drying of the samples was done between filter papers for 24 hours and weighed before digestion. Wet acid digestion method using concentrated nitric acid was applied. The accuracy of the procedure was validated by analyzing three replicate samples; with Yttrium as the internal standard. Analysis of the samples was done using Total Reflection X-Ray Fluorescence technique for detection of the seven selected trace elements for the study; while R statistical software was used to analyze the data. Trace elements in finger nail clippings varied in concentrations. The mean concentrations of Cr, Fe, Cu, Zn, Se, Hg and Pb in esophageal cancer patients were 8.32 µg g-1, 212 µg g-1, 21. 8 µg g- 1, 211 µg g-1, 2.04 µg g-1, 2.24 µg g-1, 9.01 µg g-1respectively; and 11.6 µg g-1, 209 µg g-1, 18.3 µg g-1, 265 µg g-1, 1.70 µg g-1, 2.56 µg g-1, 10.1 µg g-1 respectively in stomach cancer patients; while in the non-cancer patients, the mean concentrations were 3.17 µg g-1, 213 µg g-1, 28.1 µg g-1, 258 µg g-1, 1.06 µg g-1, 1.39 µg g-1, 10.5 µg g-1for Cr, Fe, Cu, Zn, Se, Hg and Pb respectively. The specific objectives of the study were therefore achieved. These findings revealed that evaluation of Cu, Cr, Pb, Se and Hg in nail clippings using TXRF can be used as an early screening diagnostic tool for cancer. There were no correlations established for Fe and Zn concentrations. Further research on analysis of other heavy metals and trace elements using different analytical tools are recommended to ascertain the conclusions of this study.
Table of Contents
DECLARATION ii
DEDICATION iii
List of Figures viii
List of Tables ix
ABBREVIATIONS/ACRONYMS AND SYMBOLS x
OPERATIONAL DEFINITIONS xi
ABSTRACT xii
CHAPTER ONE
Introduction
1.1 Background 1
1.2 Problem Statement 4
1.3 Objectives 6
1.3.1 General Objective 6
1.3.2 Specific Objectives 6
1.4 Justification of the Study 6
1.5 Scope and Delimitations of the Study 7
CHAPTER TWO
Literature Review
2.1 Total Reflection Xray Fluorescence (TXRF) 9
2.2 Nail Bed Trace Elements Analysis for Early Cancer Detection 11
2.2 Trace Elements and Heavy Metals and Human Biological Systems 12
2.3 Concentration Trends of Trace Elements in Cancer Patients 14
2.4 Specific Trace Elements Associated with Different Cancers 17
2.5 Lifestyles and Regional Prevalence of Some Cancers in Kenya 19
CHAPTER THREE
Research Methodology
3.1 Study Design 23
3.2 Ethical Considerations 23
3.3 Study Site 23
3.4 Study Population 24
3.4 Sample Size Determination 24
3.5 Data Collection Procedures 26
3.5.1 Sample Carrier Preparation 26
3.5.2 Sample Preparation 27
3.5.3 Quality Assurance Protocols 27
3.6 Experimental Procedures and Instrumentation 28
3.8 Study Limitations 29
3.9 Study Population 29
CHAPTER FOUR
Results and Discussion
4.1 Results 30
4.2 Trace Elements Analysis 32
4.2.1 Concentrations of Selected Trace Elements in Nail Clippings 33
4.3.1 Copper 37
4.3.2 Chromium 38
4.3.3 Iron 38
4.3.4 Zinc 39
4.3.5 Selenium 39
4.3.6 Mercury 40
4.3.7 Lead 41
CHAPTER FIVE
Conclusions
References 45
Appendix 1 Informed Consent Form 50
Appendix 2 Questionnaire for the Cases 54
Appendix 3 Questionnaire for the Control Group 61
Appendix 4 Data Frame of Participants’ Cases and Trace Elements 67
Appendix 5 Boxplots 71
Appendix 6 Grouping the data to remove ‘PARTICIPANTS’ and ‘Cd’ Columns 76
Appendix 7 Filtering the Rows that have “CN” in “CLASS” Column to Obtain New Subsets 82
Appendix 8 Matching the Rows and Columns 88
Appendix 9 Data Summary 92
Appendix 10 Unpaired t-test Results at 95% Confidence Interval 93
Appendix 11 Ethics Approval 94
Appendix 12 Sample Spectra of Esophagus Cancer Patient 97
Appendix 13 Sample Spectra of Stomach Cancer Patient 98
Appendix 14 Sample Spectra of Non-Cancer Patient 99
List of Figures
Figure 1: X-ray Fluorescence Process 9
Figure 2: Schematic Working Principle of S2 PICOFOX TXRF Spectrometer 10
Figure 3: Typical Spectrum Showing Peaks of Several Elements 11
Figure 4: 2017 National Cancer Institute Report on Cancer Prevalence Per County 22
Figure 5: TXRF Spectra of Finger Nail Clippings Sample 28
Figure 6: Pie Chart Showing the Study Population 30
Figure 7: Age Distribution of Cancer Patients 31
Figure 8: Distribution by Gender of Cancer Patients 31
Figure 9: Distribution by County of Cancer Patients 32
Figure 10: Concentration of Heavy Metals and Trace Elements in Esophageal Cancer Patients 33
Figure 11: Concentration of Heavy Metals and Trace Elements in Stomach Cancer Patients 34 Figure 12: Concentration of Heavy Metals and Trace Elements in Control Group 34
Figure 13: Comparison in the Mean Concentrations of Copper in Test Groups 37
Figure 14: Comparison in the Mean Concentrations of Chromium in Test Groups 38
Figure 15: Comparison in the Mean Concentrations of Iron in Test Groups 39
Figure 16: Comparison in the Mean Concentrations of Zinc in Test Groups 39
Figure 17: Comparison in the Mean Concentrations of Selenium in Test Groups 40
Figure 18: Comparison in the Mean Concentrations of Mercury in Test Groups 40
Figure 19: Comparison in the Mean Concentrations of Lead in Test Groups 41
List of Tables
Table 1: Concentrations of Trace Elements and Heavy Metals in Hair and Nails of a Study Participants 16
Table 2: T-test Results 36
Table 3: Relationships and Differences on Elemental Concentrations 42
ABBREVIATIONS/ACRONYMS AND SYMBOLS
AAS - Atomic Absorption Spectroscopy DNA - Deoxyribonucleic Acid
EPA - Environmental Protection Agency IAEA - International Atomic Energy Agency
IARC - International Agency for Research in Cancer ICP - Inductive Coupled Plasma
NCCS - National Cancer Control Strategy NCI - National Cancer Institute
PC - Personal Computer
TXRF - Total Reflection X-ray Fluorescence WHO - World Health Organization
CE - Cancer of the Esophagus CS - Cancer of the Stomach CN - Control Group
OPERATIONAL DEFINITIONS
Cancer A group of diseases characterized by abnormal cell growth capable of spreading and invading other cells.
Heavy Metals Metals with high atomic density and atomic numbers
Trace Elements Chemical elements required in very low/minute quantities by living organisms
Esophageal Cancer Buildup of abnormal cells arising from the food pipe that runs between the throat and the stomach
Stomach Cancer Buildup of abnormal cells that develop in any part of the stomach and can spread from the stomach to other organs
CHAPTER ONE
Introduction
1.1 Background
Cancer is currently a global burden as it exerts too much pressure on demographic and health systems across all income levels in a population. Local and international news report that both the rich and the poor, rural and urban populations of all faiths and lifestyles are dying of one form of cancer or the other. According to Kenya Ministry of Health, 2017, cancer is ranked third among the leading causes of deaths, after cardiovascular and infectious diseases (Bray et al., 2018).
Moreover, estimated new cancer cases now stand at 47,887 reported annually with a mortality of 32,987. In the year 2012, it is reported that 28,500 people died of cancer and cancer related ailments (KMH, 2017). Clearly, there is as increase of the cancer epidemic.
As reiterated by Muinao and Co-workers, 2018, survival rates for cancer are typically lower due to late stage of diagnosis and lack of accessibility to quick and efficient as well as standard treatment. Therefore, correct and timely disease detection is critical for clinical diagnosis, proper toxicity monitoring, and ultimately ineffective cancer treatment (Muinao et al., 2018).
Moreover, the key obstacle in treatment for cancer is early-stage disease identification, which would significantly improve cancer treatment efficiency and survival. Consequently, minimally invasive tests are ideal for detecting early phase melanoma, and developing these techniques in clinical applications is desirable. Fortunately, recent efforts have been made to develop a variety of chemical tools for detecting cancer-related biomarkers with high sensitivity, such as protein molecules, nucleic acids, enzymes, organic molecules and cancerous cells (Muinao et al., 2018). Current diagnostic screening methods include the use of Cancer Protein Biomarkers, Enzyme- linked Immunosorbent Assay methods, Electrochemical and Electrical Detection methods, Optical methods, Enzyme-induced Conformational Change method, Electrophoresis-based methods and fluorescent methods (Muinao et al., 2018).
However, the future of cancer biomarker detection lies in the development of efficient screening platforms with highly sensitive and selective, smaller size, highly flexible, elevated, and the discovery of new biomarkers that explicitly state the need for earlier detection (Ferlay et al., 2012).
It is observed that among the major causes of cancer, are toxic heavy metals which have been confirmed to be carcinogenic (Karimi et al., 2012 & Mulware, 2013). Due to the physiological and chemical properties of these heavy metals, chronic exposures to them are almost unavoidable in daily life. Consequently, due to the use of these metals in, drug manufacturing, food additives, industrial applications, mining, manufacturing of semiconductors, cement-manufacturing plants and refining of metal ores, leading to the release of heavy metals into the environment. These in addition, raise the population's exposure to these metals, thereby contributing to the environmental contamination and also human body accumulation (Abo El-Atta, 2011).
Trace elements are dietary nutrients required in minute quantities of an organism’s mass; normally not up to 0.01%. They have a vital role in health maintenance, proper growth, development and are also components of enzymes in living organisms. They are inorganic micronutrients involved in many cellular functions; hence their deficiency may cause malfunctions, diseases and possibly result in death (Mehri & Marjan, 2015).
According to Chitturi et al. (2015), there are nineteen trace elements known which are categorized as either essential, probably essential or potentially toxic elements.
Requirements for essential elements in humans per day, which include Co, Cu, F, Fe, Zn, Se, Mn, Mo and I ranges from 50 µg to 10 mg. Their imbalance in the body is considered in many diseases as a risk factor. Probably essential elements have very little or no beneficial function in the humans’ life process and very little is known about them and they include Sn, Ni, B, and V; while potentially toxic elements’ excessive concentrations are considered hazardous to human health and can also inhibit growth in plants. There may however be some possibility with essential functions for these elements, and they include Au, Al, Pb, Cr, Cd, and Hg (Chitturi et al., 2015).
Hence, in the bodily concentration of trace elements, a balance needs to be maintained for proper maintenance of life and health of living organisms (Mehri & Marjan, 2015).
Heavy metals on the other hand, are elements that occur naturally with high atomic mass and weight. They are considered toxic in the human body even at low concentrations because they are
understood to cause numerous organ harm, even at minimum levels of exposure. They are widely dispersed throughout the environment as a result of their numerous applications in agriculture, household life, industry, and medicine. This factor gives rise to worries about their possible negative consequences on human health. Their toxicity is dependent on the method of exposure, the dosage, the chemical species, as well as the exposed person's weight, age, gender, and nutritional status. Due to their high toxicity levels, As, Cd, Cr, Pb, and Hg are designated as priority metals of concern for public health (He et al, 2005).
According to Liang and his Co-workers, 2017, Hg, Pb and Cd represent significant health concerns and are categorized as heavy metal pollutants. This is mainly due to their ability to induce adverse health effects, more serious one being their role in carcinogenesis. Trace metals on the other hand such as Se, Zn, Fe and Cu are essential micronutrients, but at concentrations higher than the amount required by the body, they become toxic just as heavy metals whose roles are unknown in living organisms; making them toxic no matter how low their concentration, for example Cadmium, Mercury and Lead. They are therefore non-essential micronutrients. This poses serious risks to human health and ecosystem (Liang et al, 2017).
However, due to their occurrence in small amounts in the environment, heavy metals are also regarded as trace elements. (Kabata, 2001).
Hence, assessing the concentrations of heavy metals and trace elements is crucial, in order to check their potential health risks.
Consequently, in studies involving these elements’ status in the body, nail measurements have demonstrated to be useful. For instance, in assessing Se status, toenail Se level has always provided a time-integrated and a more superior measure than other biomarkers. In a certain case-control study on investigation of trace elements, nail specimens were employed as biomarkers. The study examined Fe, Co, Zn, Ca and Cr in association to cancer of the upper digestive tract. The results showed that persons who got certain upper aerodigestive tract carcinomas and those who did not, consumed different amounts of minerals. Thus, measuring the concentration of heavy metals and trace elements in nails continues to be vital in clinical research (Janbabai et al., 2018).
This study only used nail clippings because most of the body tissues are in a flux state due to metabolic activities, except the nails and hair. For example, after the nail formation, what follows is its complete expulsion from the nail-bed before its isolation from the continuing metabolic activities of the body. Therefore, nails represent the body’s exposure or intake during the past few months or so; thus, one millimeter of the nail sample could correspond roughly to one month of the body’s nutritional status (Abdulrahman, 2012). It then follows that analysis of heavy metals and trace elements in the nails can provide information about the body intake for a given period of time.
The current study therefore, used using nail clippings of cancer patients and a non-cancer control group in evaluating the concentration of selected heavy metals and trace elements in esophageal and stomach cancer patients. These patients were those being attended to at the Kenyatta National Hospital. The effort of this work was to find a non-invasive and method of fostering early cancer detection, diagnosis and treatment.
1.2 Problem Statement
In Kenya, breast, prostate, cervical, esophageal, stomach and colorectal cancers are the leading new cancer type cases in both males and females across all ages; most of which have no family histories of cancer. Approximately, 70-80% of the Kenyan cancer patients are discovered when the disease is quite advanced, and is nearly impossible to cure (Bray et al., 2018).
In Kenya, there is a rising demand for cancer treatments, but the capacity for detection and treatment is extremely constrained. This presents the government with major health-care policy difficulties. The fundamental issue arises from the fact that the number of patients has been steadily increasing and is predicted to do so going forward, particularly with regard to malignancies of the esophagus, prostate, cervix and breast. However, access to equipment and facilities continues to be a major obstacle. (Wambalaba et al, 2019).
Between January 1999 and September 2007, all pathology-confirmed cancers identified in Tenwek Hospital, Bomet County, Kenya, were examined retrospectively. The study found that the stomach, esophagus, prostate, cervix, and colorectum were the five most frequent cancer locations since 1999. 914 of the 2643 newly diagnosed cancer cases, with a growing tendency both inside and outside the catchment region, were esophageal cancer cases. The youngest patient was 14 years old at the time of diagnosis, and 58 (6.3%) patients were under 30 and 9 (1%) were under
20. (Parker et al, 2010).
The study in Tenwek further revealed that cancer of esophagus was the most prevalent cancer seen in Western Kenya affecting even younger generations; and hence highlighted the need for additional research on the environmental and genetic predispositions to esophageal cancer.
According to a study carried out by Lodenyo and Co-workers, 2018, information about stomach cancer remains low in developing countries and especially in Africa; yet it a major killer across the globe. According to the study, 990,000 people worldwide are diagnosed with stomach cancer each year, and 938,000 of them pass away as a result of the illness. Aside from that, this condition has one of the highest burdens of cancer in terms of years of life lost with a disability. Multiple studies have found that nutrition has a key role in stomach cancer, especially in Africa, where incidence rates have continued to increase (Lodenyo et al, 2018).
In cancer inhibition and development, heavy metals possess a complex character in their roles; thus, creating a lot of concerns due to their importance to human health and potential toxicity. Their carcinogenic capability depends mainly on their chemical structures and oxidative states. Thus, the complexes they form catalyze redox reactions within DNA, thereby oxidizing the DNA. This leads to DNA damage, promoting the onset of carcinogenesis in most cases (Mulware, 2013).
Heavy metals and trace elements have also been shown to have beneficial effects in biological systems notably in some enzymes involved in metabolism, detoxification, and damage repair, as well as in cellular organelles and portions such as the nucleus, cell membrane, lysosomes, and mitochondria. (Wang, 2001).
However, Beyersmann & Hartwig, 2008 observed that the metal ions engage in interactions with nuclear proteins and DNA that make up cells. Due to DNA damage and mutations brought on by this, cell cycle regulation, carcinogenesis, or apoptosis may result. Their research also showed that oxidative stress and the generation of reactive oxygen species are crucial factors in the toxic and carcinogenic nature of metals such As, Cd, Cr, Pb, and Hg (Beyersmann & Hartwig, 2008).
Having experienced very rapid growth in industrialization and economic development in the urbanization process over the past few years, Kenya has with no doubt pollution issues. These environmental changes affect human health. There is absolutely no research in Kenya that has biologically monitored heavy metals or trace elements levels in humans which can be a direct indicator of the cancer disease in the human body. Hence, the current study focused at determining the concentration of these elements in the human body and establishing their association to the growth of esophageal and stomach cancer cells.
1.3 Objectives
1.3.1 General Objective
To evaluate the concentrations of Cr, Fe, Zn, Cu, Pb, Se, Hg in esophageal and stomach cancer patients in search of a non-invasive method of early cancer screening in Kenya.
1.3.2 Specific Objectives
1) To determine concentrations of selected trace elements in nail clippings of esophageal cancer patients at the Kenyatta National Hospital.
2) To determine concentrations of selected trace elements in nail clippings of stomach cancer patients at the Kenyatta National Hospital.
3) To evaluate the monitoring of concentrations of the trace elements as an early cancer screening method.
1.4 Justification of the Study
International Agency for Research in Cancer, 2012 classified, among other heavy metals, As, Cd, Ni and Cr as group one carcinogens. Ironically, some of the trace elements such as Zn and Cu are biological co-factors for enzymes that are necessary for many intracellular processes. They also have DNA-binding domains (Mulware, 2013). Thus, it is important to critically analyze and quantify, despite their essential biological functions, specific concentration levels in the human body and their effects on the growth of cancerous cells in cancer patients (IARC, 2012).
In the year 2017, Kenya Ministry of Health highlighted the estimated cancer cases, in Kenya in 2012 as 37,000 cases and 28,500 deaths. The highlight informed the basis of drawing the National Cancer Control Strategy 2017-2022 in which the first of the documented five priorities is prevention, early detection and screening of cancer and the second includes diagnosis. However, even though it is curable, most of the cancer patients in Kenya are diagnosed while at advanced stages. The findings of this study will therefore help the health professionals in early presentation, fasten referrals, diagnosis and treatment as revealed by the levels of various elements in the body. Through early detections, most cancers may be optimally cured with complete surgical removal. This is because, at late diagnosis when the symptoms occur, the cancer is no longer localized making surgery not an option.
In addition, no study has been carried out in Kenya that used biomarkers to correlate the levels of trace elements in human population with the prevalence of cancer, hence, this study will be a gateway to environmental monitoring and earlier diagnosis of the cancer.
1.5 Scope and Delimitations of the Study
The current study only focused on trace elements. This is due to the fact that in daily life, it is almost unavoidable not to expose any human population to heavy metals. Thus, analysis of their intake and concentration levels in the body is of great importance. In addition, levels of exposure to these elements can be potentially modified. Consequently, the study only compared the levels of these elements among esophageal and stomach cancer patients with a control group.
Since the elements; Cr, Fe, Zn, Cu, Pb, Se and Hg have been identified among other trace elements by IARC (2012) as having an impact on cancer, they were the only ones analyzed in this study.
In sample collection, cancer patients who had been on chemotherapy treatment for more than one month and those who would have undergone any form of esophageal or stomach surgery were excluded from the study. In addition, any participant engaged in any drug and substance abuse was also be excluded. These measures were meant to make the study more objective.
The study was conducted using nail clippings as the samples since they are bio-accumulators of toxic heavy metals and trace elements. As compared to other tissues, they accumulate elements over longer periods of time without any changes thereby serving as surrogate in measuring the status of critical body organs. Moreover, these samples don't need special storage conditions, allowing analysis to be performed securely without element loss. In addition, the collection of samples is non-invasive; a factor that is expected to increase the participation rate of the target population. The collection is also easy and economical; which made the study to be time and cost effective.
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