Case–control study of male cancer patients exposed to arsenic-contaminated drinking water and tobacco smoke with relation to non-exposed cancer patients

Abstract

The investigated data indicated that inorganic arsenic in drinking water is associated with increased mortality from different types of cancers. In the present study, biological samples (blood and scalp hair) of male subjects having lung and bladder cancers and non-cancerous subjects belonging to arsenic (As)-exposed area of southern parts of Pakistan were analysed for As contents. The As levels in drinking water of understudy area showed that sections of understudy population are exposed to arsenic concentrations, which was 3–15-fold higher than the permissible level (<10 μg/L). For comparative purposes the biological samples of matched male cancer patient, as referent patients belonging to big city (Hyderabad) who had used municipal treated water with low arsenic levels <10 μg/L, were also collected. The exposed cancer patients have 2–3-fold higher level of As in both biological samples compared to non-exposed case-matched cancerous male subjects. This study is compelling evidence in support of positive associations between arsenic-contaminated water, food and cigarette with different types of risks of cancer.

Keywords

arsenic cancer blood scalp hair tobacco water

Introduction

Arsenic has been found in drinking water at high levels in many parts of the world, including Bangladesh, India, Argentina and Chile, and it has been shown to cause numerous health effects.^1–3 Humans are exposed to arsenic through water, air, food and beverages.^4–7 Sea foods contain a high level of arsenic which is predominantly in its organic forms.^8,9 Sources of exposure to arsenic through ingestion include drinking water, drugs used to treat leukemia and psoriasis and arsenic-contaminated wine.^10,11

Arsenical compounds are transported into the environment mainly by water from wells drilled into arsenic-rich geologic strata or by ambient air during the smelting and burning of coal.^10,12 However, the main route of arsenic exposure for the general population is drinking water.^10,13 Epidemiologic studies have documented that long-term exposure to inorganic arsenic (iAs) is associated with an increased risk of cancer of the lung, skin and probably other anatomic sites.^14,15 The iAs is also one of major risk factors for blackfoot disease, a unique peripheral vascular disease identified in endemic areas of arsenicosis in Taiwan, where residents had used high arsenic-tainted artesian well water for more than 50 years.¹⁶

Exposure to iAs causes different types of cancers (bladder, lung, skin, kidney, prostate and liver) as well as cardiovascular disease, diabetes, developmental and reproductive effects.^17–19 Increased mortality from lung and bladder cancers has previously been reported in Chile as compared with the rest of the country. These cancers have also been associated with high levels of arsenic in water supplies in Taiwan^15,20–22 and Argentina.^23,24 The reports from Chile confirmed that the elevated cancer rates in the other countries were likely to be attributable to arsenic, and in 2002, the working group of the International Agency for Research on Cancer classified arsenic in drinking water as a cause of lung and bladder cancers, along with skin cancer.²⁵

A number of established bladder cancer risk factors, such as smoking and occupational exposures, may have contributed to these increased rates.²⁶ Tobacco smoke has been related to an elevated risk of bladder cancer.^27–29 For non-smokers, environmental tobacco smoke is a source of arsenic exposure, that is, arsenic is a constituent of tobacco smoke.³⁰ Very few researches have been done for assessing the relationship between tobacco smoke and bladder cancer.^31–33 Many investigations have been carried out about the relationship between active smoking and occupational exposure to As. It strongly indicates that these two exposures act synergistically on the rate of lung cancer.^34–37

In different areas of Pakistan we are facing the As-related severe public health disasters as are present in neighboring countries.³⁸ Hence, it has become extremely important to recognize the need of assessing drinking water quality for As contamination. In few areas of Pakistan, As levels were high in ground and surface water used for drinking purpose.³⁹ Based on the monitoring program of groundwater quality, the Pakistan Council of Research in Water Resources (PCRWR) and UNICEF reported that As-contaminated groundwater was found (10–200 μg/L) in many areas of Punjab. In Sindh province, 16–36% of population has been exposed to As-contaminated water with over 10–50 μg/L.⁴⁰ The survey revealed hot spots of As enhancement in parts of the Indus alluvial basin.

In our previous studies, we measured the total As levels in surface and ground water, fish, agricultural soil, vegetables, grain crops and tobacco samples collected from the vicinity of Manchhar Lake (26°3′N: 67°6′E), 15 km apart from Sehwan Sharif, Sindh Pakistan.⁴¹ In understudy area the people are using contaminated lake water, which is the main source of water for domestic and irrigation purposes. The population of these areas is economically not well developed and they are affected by long-term exposure of As via consuming water, food and fish produced in same areas. The understudy population smoked locally made cigarette (made from tobacco grown in understudy areas irrigated with arsenic-contaminated water).⁴² The tobacco contains fourfold higher As content as compared to As found in tobacco of branded cigarette.⁴² As the rate of mortality due to different cancers is also increased in Pakistan even though an extensive list of risk factors has been well characterized in its pathogenesis, but exposure to toxic metal especially arsenic via drinking water and tobacco smoking are considered as the most important causes of different cancer.^4–6 Very limited data are available in Pakistan to show the association of different cancer with As. The principal objective of this study was to emphasize the severity of the arsenic contamination in the inhabitants of villages situated in southern parts of Pakistan. The levels of As were determined in whole blood and scalp hair of exposed male cancer patients (EXP), having lung and bladder cancers and referents (EXR) of same age group, from same villages. For comparative purposes, the biological samples of matched male cancer patient as referent patients (NRP) and controls (NEC) belongs to city (Hyderabad, Pakistan), who have used municipal treated water with low arsenic levels <10 μg/L were also collected.

Experiment

Study population

The understudied male cancer patients and non-cancerous subjects belong to villages of south western part of Pakistan. They were residents for long time in these villages, used surface and underground water which have high level of As and did not utilize alternative water sources (e.g. bottled water). For study purposes, 175 As exposed and non-exposed males cancer patients with bladder and lung admitted in Nuclear Institute of Medicine and Radiotherapy Jamshoro (NIMRA) during 2007–2009 were selected (Table 1). The data was obtained by collecting files and extracting important information. Initially, the 14 patients were excluded from study because they were non-smokers. The median age of the patients was 47 years, with a maximum of 65 and a minimum of 35 years. The major criteria of inclusion for the present study is histologically confirmed lung and bladder cancer patients at primary and advanced stages such as primary diagnosed tumor before any treatment and after treatment (chemotherapy, radiotherapy and surgery). The criteria for selection of controls for both groups were those who have not taken any mineral supplement and belong to same socio-economic status and dietary habits. Lifetime residential, occupational and smoking histories were obtained from the patients themselves and next of kin of patients (smoking habits before and after diagnosis). The entire exposed population has spent most of their lives in their respective villages. For comparison purposes, 83-matched male cancer patients (lungs and bladder) as referent cancer patients or non-exposed patients (NEP) and 95 non-exposed referents (NER) belonging to big cities were recruited, who drank municipal treated water having low levels of As (<10 μg/L) and smoked branded cigarette having low levels of As.⁴² We obtained verbal consent and provided information on study objectives, procedures and implications to participants. At the start of the study, the participants' weight, height and blood pressure were noted. The biochemical parameters were analyzed in the pathological laboratories of the hospital. Persons who did not complete or refused to answer the questionnaire were excluded from the study. The study was approved by the Higher Education Commission of Pakistan.

Table 1.

Demographics of subjects under study^a

	Exposed subjects	Non-exposed subjects
Referents	98	95
Patients with cancer	78	83
Lung cancer	52 (66.7)	55 (66.3)
Bladder cancer	26 (33.3)	28 (33.7)
	Farmer (55%)	Office worker (30%)
	Fishermen (45%)	Industrial workers (70%)

^a Values within parentheses are percentage (%) of total.

Chemicals and reagents

Ultrapure water obtained from ELGA Labwater System (Bucks, UK) was used throughout the work. Concentrated nitric acid (65%) and hydrogen peroxide (30%) were purchased from Merck (Darmstadt, Germany). Standard solutions of As were prepared by dilution of certified standard solution (1000 mg/L) Fluka Kamica (Bush, Switzerland). Dilute working standard solutions were prepared immediately prior to their use, by stepwise dilution of the stock standard solution with 0.2 M HNO₃. Stock standard solution of chemical modifiers, Mg (NO₃)₂ (5.0 g/L) and Pd (3.0 g/L) were prepared from Mg (NO₃)₂ (Merck Ltd., Poole, Dorset, UK) and Pd 99.99% (Aldrich, Milwaukee, WI, USA), respectively. All solutions were stored in polyethylene bottles at 4°C. For the accuracy of methodology, certified reference materials (CRMs), of human hair BCR 397 (Brussels, Belgium) and clincheck control-lyophilized human whole blood (Recipe, Munich, Germany) were used. All glassware and plastic materials used were earlier soaked for 24 hours in 2 M nitric acid, washed with distilled water and finally rinsed with ultrapure water, dried and stored in class 100 laminar flow hoods.

Instrumentation

A Perkin-Elmer model A. Analyst 700 (Norwalk, CT, USA) atomic absorption spectrometer equipped with deuterium background correction was used in the study. The hollow cathode lamp of As was run under the conditions suggested by the manufacturer. The analytical wavelength was set at 193.7 nm, operated at 7.5 mA with a spectral bandwidth of 0.7 nm. Magnesium nitrate and palladium: 5 μg Mg (NO₃)₂ + 3 μg Pd (10 ml + 10 ml modifier in each case) used for As. Portions of both, standard or sample and modifier were transferred into auto-sampler cups, and 20 μl were injected. Argon gas (200 ml/min) was used as the purge gas except during the atomization step. The graphite furnace heating program was set for different steps: drying, ashing, atomization and cleaning as temperature range °C/time (s) (80–120/15, 300–600/15, 2000–2100/5 and 2100–2400/2), respectively. A Pel (PMO23) domestic microwave oven (maximum heating power of 900 W) was used for digestion of the biological samples. Acid washed polytetrafluoroethylene (PTFE) vessels and flasks were used for preparing and storing solutions.

Microwave-assisted acid digestion

Duplicate samples of scalp hair (200 mg) and 0.5 ml of blood samples of each subject (exposed and non-exposed patients and referents) were directly placed into PTFE flasks (25 ml in capacity). About 2 ml of a freshly prepared mixture of concentrated HNO₃–H₂O₂ (2:1, v/v) was added to each flask and kept for 10 min at room temperature. Then the flask was placed in a covered PTFE container and heated following a one-stage digestion program at 80% of total power (900 W). Complete digestion of blood samples required 2–3 min, while 4–5 min was necessary for scalp hair samples. After digestion, the flasks were left to cool and the resulting solution was evaporated to semidried mass to remove excess acid. About 5 ml of 0.1 M nitric acid was added to the residue and filtered through a Whatman no. 42 filter paper and diluted with deionized water up to 10 ml in volumetric flasks. Blank extractions were carried out through the complete procedure. Blanks and standard solutions were prepared in a similar acid matrix. The validity and efficiency of the microwave-assisted digestion method was checked with certified values of CRMs of biological samples.⁴³

Statistical analysis

The arsenic level in blood and scalp hair samples of referents and patients was logarithm transformed to stabilize the variance and to cause the distribution to approach normality. We determined the associations between study variables by Pearson’s product-moment correlation coefficients (r).We performed linear regression analysis to examine the effect of arsenic concentration in whole blood and arsenic in drinking water. The level of statistical significance was p < 0.05.

Results and discussion

The mean and standard deviations of arsenic contents in whole blood and scalp hair samples of exposed and non-exposed referents as well as patients are presented in Table 2. The demographic data of the subjects recruited from arsenic-contaminated area as exposed cancer patients (EXP), exposed controls (EXC), matched male non-exposed cancer patients (NEP) and non-exposed controls (NEC) are shown in Table 2. They belong to an urban area of Hyderabad city with a consumed water permissible limit of As (<10 μg/L).

Table 2.

Comparison of As in whole blood and scalp hair samples of exposed patients with cancer (EXP) and referents (EXR) vs. non-exposed patients with cancer (NEP) and referents (NER)

Study subjects	Blood (μg/L), $\bar{x}$ ± SD		Scalp hair (μg/g), $\bar{x}$ ± SD
Study subjects	EX	NE	EX	NE
Referents	3.29 ± 0.64	1.28 ± 0.55	2.82 ± 0.32	0.824 ± 0.12
Cancer patients
Lung cancer	8.82 ± 4.13	3.54 ± 1.28	5.82 ± 2.43	2.94 ± 0.88
Bladder cancer	6.82 ± 3.36	2.68 ± 1.04	4.98 ± 2.36	2.08 ± 0.74

EX: exposed, NE: non-exposed.

From the obtained data, the concentrations of arsenic in the biological samples of male cancer patients (lung and bladder) were found to be significantly higher than those in the biological samples of EXR and NER of each case, using paired t test.

In scalp hair samples, the level of As was significantly higher at 95% confidence interval in exposed lung patients (ELP) [CI: 4.92, 6.65], bladder patients (EBP) [CI: 3.74, 6.22] and cancer patients vs. EXC [CI: 2.70, 2.94]. While NEP and NEC have significantly lower level of As (twofold) in their scalp hair samples (p = 0.001–0.0002) as compared to exposed patients and controls (Table 2, Figure 1).

Figure 1.

The As levels in scalp hair of exposed and non-exposed referent (ER and NER), exposed and non-exposed patients with lung cancer (EL and NEL) and exposed and non-exposed patients with bladder cancer (EB and NEB) are shown.

The average lifetime of inorganic arsenic in the blood is very small and it is cleared quickly from blood by the liver and kidney. Hence, the high blood arsenic level is a clear indication of prolonged chronic exposure from drinking, smoking and dietary sources. In blood samples, the level of As was significantly higher at 95% confidence interval in ELP and EBP [CI: 7.15, 10.1] and [Cl: 5.47, 8.04] vs. EXC [CI: 3.12, 3.42]. While the NEP and NEC have significantly low level of As (~threefold) in their blood samples (p < 0.001) as compared to their corresponding exposed patients and controls (Table 2, Figure 2). The differentiation of As in exposed and non-exposed cancer patients and referents also resolves a scientific dispute regarding the admissibility of blood arsenic levels as a biomarker. It is reported that the blood arsenic does not correlate well with arsenic exposure in drinking water, particularly at low levels⁴⁴; however, some investigations showed significant correlation between arsenic levels in drinking water and total arsenic in hair and blood samples.⁴⁵ They also reported that there is a positive correlation between the different cancerous symptoms, the blood arsenic level and the arsenic intake through drinking water.⁴⁵ It is unclear as to how the people having same life style and arsenic exposure, differ in their blood arsenic levels and overall response to the arsenic exposure. During the study period, 28 exposed cancer patients (36% of total) died among which 75% had lung cancer. While the mortality rates in non-exposed cancer patients were low (n = 17) corresponding to 20.5% of the total. Among the exposed and non-exposed cancer patients, lung cancer cases were 52 and 55, respectively. Logistic regression analysis revealed a clear trend. In exposed lung cancer, patients odds ratios showed evidences of synergism between cigarette smoking and ingestion of arsenic in drinking water. The odds ratio for lung cancer mortality among ELP was 3.05 (95% CI = 1.26–7.36) as compared to NELP (p < 0.001).

Figure 2.

The As levels in blood of exposed and non-exposed referent (ER and NER), exposed and non-exposed patients with lung cancer (EL and NEL) and exposed and non-exposed patients with bladder cancer (EB and NEB) are shown.

None of the published studies have assessed the carcinogenic effects of arsenic exposure from drinking water and tobacco smoke together on human. In the present case–control study the As levels were determined in whole blood and scalp hair samples of cancerous male patients (lung and bladder) who belong to arsenic exposed and non-exposed areas with relation to non-cancerous controls. The blood may be chosen as the biological indicator of arsenic exposure to estimate better relation between As exposure and the resultant physiological disorders. The intake of As through water is rapidly transported by the blood to other organs, such as the liver, kidneys, lungs, intestines and skin within 24 h.⁴⁶ Although 90% of the ingested As in blood is rapidly cleared.⁴⁶ However, with chronic and continuing exposure, steady-state concentrations in blood and urine are achieved; these have the potential to serve as biomarkers of past exposure.⁴⁷ Arsenic tends not to be accumulated in the body but is excreted naturally; if ingested faster than it can be excreted. As accumulate in the hair and fingernails.⁴⁸ As accumulates in hair and nails, due to its affinity for the abundant sulfhydryl groups in keratin; thus As concentrations in these slow growing tissues are considered to be a good measure of past exposure.⁴⁹ Clinical manifestations of As poisoning begin with various forms of skin disease, and progress via damage to internal organs ultimately to cancer and death.⁵⁰ High levels of As in water lead to health problems, such as melanosis, leuko-melanosis, hyperkeratosis, blackfoot disease, cardiovascular disease, hepatomegaly, neuropathy and cancer.⁵¹ The estimations of total As intake was reported in our previous study for the population of understudy area, based on the sum of As ingested from different food items (vegetable, grains and fish) in addition to drinking water, consumed by adults during 24-h period. The estimated daily intake of total As in the adults of understudy area was found in the range of 9.7–12.2 μg/kg body weight/day.⁴¹ In general, iAs is the more toxic form than organic forms and is present in water, which is readily absorbed by the animal and human body.⁵² Chronic arsenic exposure in the range of 10–40 μg/kg/day has been associated with skin cancer in Taiwan; respiratory cancers in Montana; bladder cancer in Finland; increased mortality from hypertensive heart disease, nephritis, nephrosis and prostate cancer in Utah; increased incidence of all cancers in Taiwan; late fetal mortality, neonatal mortality and postnatal mortality in Chile; cytogenetic damage in Mexico.⁵³ A study found some association between arsenic and bladder cancer risk and provided evidence for synergistic effects of smoking and nutritional factors with As.⁵⁴

A study on cell-type specificity of arsenic-induced bladder cancer in Taiwan showed that the As concentration correlated with the development of transitional cell carcinomas of the bladder, kidney and urethra as well as adenocarcinomas of the bladder in males, but not with squamous cell carcinomas of the bladder or renal cell carcinomas in the kidney.⁵⁵

Tobacco-related cancer constitutes 16% of the total annual incidence of cancer cases and 30% of cancer-related deaths in the developed countries. In populations where cigarette smoking has been common for several decades, 90% of lung cancers and 15–20% of other cancers are attributable to tobacco.⁵⁶ Cigarette smoke contains >4000 chemical components, including over 30 heavy metals.⁵⁷ Environmental exposure to cigarette smoke has been epidemiologically associated with different types of cancers.⁵⁸ The investigated data indicates that smokers could receive significantly higher exposures to various toxic and carcinogenic metals from locally produced tobacco.⁴² Tobacco smoke has been related to an elevated risk of bladder cancer.^27–29 For smokers, environmental tobacco smoke is also a source of As exposure, because As is a constituent of tobacco smoke.³⁰ This also affected non-smokers equally. Some investigation has been done for assessing the relationship between tobacco smoke and bladder cancer.³¹ In mammals arsenic causes lipid peroxidation, protein and enzyme oxidation and glutathione depletion.⁵⁹ It was also reported in literature about the interaction of arsenate with glutathione, several enzyme systems are involved, including arsenate reductase and a glutathione S-transferase, with the resultant formation of a complex consisting of three molecules of glutathione with a single atom of arsenic [(GS)3As].⁶⁰ This arsenic–glutathione complex undergoes rapid biliary excretion. The lack of glutathione is believed to result in the occurrence of an oxidative stress due to the decrease in adequate antioxidant protection within cells.

Therefore, arsenate in cigarette smoke may contribute to the oxidative stress that is produced in lungs, resulting in tissue damaging effects. The understudy population (patients and referents) of exposed area are 100% smokers. In addition to drinking As contaminated water, smoking locally made unbranded cigarettes made of tobacco grown in agricultural land irrigated with As contaminated lake water also have high risk of health hazard. The amount of As in raw tobacco leaves and non-branded cigarette made of tobacco was 3–4 folds higher than branded cigarette.⁴² The high level of arsenic was observed in blood and scalp hair samples of exposed and non-exposed male cancer patients as compared to their respective healthy referent male subjects. It indicated the strong association of As with both type of cancer.

Most of the studies reported markedly higher risks of lung cancer mortality or incidence in arsenic endemic areas as compared to the general population or a low As exposed reference group.⁶¹ Epidemiological studies of populations in Taiwan, Bangladesh and Argentina with high levels of As in drinking water have shown elevated risks of cancers of the urinary tract, lung and skin, and, less consistently, cancers of the colon and liver.^62,63

As noted the mortality rate of lung cancer patients belonging to exposed area was high as compared to matched non-exposed cancer patients, due to the consumption of As-contaminated water and tobacco. In addition to lung cancer, cancers of the upper aero digestive tract (UAT), lower urinary tract and pancreas are causally related to tobacco smoking.⁶⁴ Also the risk of cancers of the kidney, stomach, liver, colon, esophagus and bone marrow are increased in smokers.⁴⁹

Due to poverty and unawareness, about 40% of cancer patients of exposed area were diagnosed and admitted in Cancer hospital (NIMRA) at last stage of disease due to poor basic health facilities in these areas, so a proportion of morbidity and mortality of these patients belonging to As exposed area may be increased. It was also observed in our study that the understudy cancer patients were not regularly visiting the hospital for treatment (radiography and chemotherapy), so the mortality rate is very high in these cases. It was observed that malnutrition and the associated mortality are prevalent in the exposed cancer patients due to poor energy intake and an inadequate balanced diet before and after treatments.

Conclusion

The results of this study indicate that arsenic is a significant environmental toxicant that increases the risk of different type of cancer. We found an association between arsenic ingestion from drinking water and tobacco smoking with increased risk of cancer. The arsenic exposure through drinking water was confirmed by the analysis of biological samples. Some clinical features of arsenic toxicity such as weakness and muscle cramps, respiratory problems, anemia and gastrointestinal problems were also noticed in non-cancerous exposed control subjects. The concentrations of arsenic in biological samples were clearly increased in exposed cancer patients consuming drinking water which contain high arsenic concentration as compared to those people consuming municipal treated water of low level of As. Our results showed a good correlation between arsenic concentrations in biological samples (hair and blood) of cancerous and non-cancerous subjects and intake of As-contaminated drinking water. Our results are consistent with the reported literature studied about the interaction of arsenic-contaminated water and evidences of different cases. The mortality rate is manifold higher in exposed cancerous subjects.

Footnotes

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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