Background

Bladder cancer is a great health problem all over the world. It ranks the ninth worldwide in the incidence of cancer (Public health impact of schistosomiasis 1993). It represents 16.2% of male cancer and considered the first cancer among males (Khaled 2005). The incidence in males of rural areas in Egypt is about 32 per 100,000 (Amal and EL Sebai 1983).

The main etiology of bladder cancer is still obscure. Many risk factors have been involved in the pathology of bladder cancer, which include cigarette smoking (American Cancer Society 2006); synthetic nitrogen fertilizers (Mensing et al. 2003); organophosphate-based pesticides (Webster et al. 2002); aromatic amines (Xifeng et al. 2007); pelvic irradiation, A cyclophosphamide, chronic cystitis, and schistosomiasis (American Cancer Society 2006); human papilloma virus (LaRue et al. 1995); and some occupational disease and genetic factors (American Cancer Society 2006).

The importance of these risk factors in the occurrence of bladder cancer differs according to the populations. Risk factors like family history, pesticide exposure, consanguinity between parents, and chronic cystitis play an important role than bilharziasis and smoking in the development of bladder cancer in our country (Zarzour et al. 2008). During the twentieth century, fertilizers and insecticides have been very popular to be used due to the increase of the population to minimize the loss of plants and to increase the yields of the crop. Pesticides is the most widely used method of controlling the majority of agricultural pests (Hunter 1989). Since the 1960s, the amount of pesticides in Egypt and Africa has increased about fivefold. During the last 40 years, about 1 million tons have been injected into the Egyptian environment (Amr 1999).

Persistence of organochlorine compounds in the environment, and their accumulation in the living organisms, is a major factor in causing the bad effects on specific organs. The risks to all populations from the use and misuse of pesticides are one of the most important health and environmental problems mostly in the third world countries (Amr 1999).

RAS gene family is performed from four functional genes [Harvey ras (H-ras), Kristen ras (K-ras) A and B, and Neuroblastoma ras (N-ras)] that encode closely related proteins which are localized in the internal part of the cell membrane and have intrinsic GTPase activity, which regulates their cellular activity. The main action of these family proteins is to activate the downstream kinases belonging to mitogen-activated protein kinase pathway, which lead to continuous mutation signals (Nanda et al. 2010).

Some studies have detected mutation of RAS mutations of different types in human bladder tumors (Cattan et al. 2000; Jebar et al. 2005; and Zhu et al. 2004). These studies show different types and range of mutation frequencies. It is not clear whether these differences are related to the exposure to pesticides or not.

P53 protein is almost undetectable because it is rapidly degraded. Once activated, the protein escapes the degradation and accumulates in the nucleus. Simultaneously, it is turned from a latent one to an active by some changes which activate its capacity to transactivate target genes. Many genotoxic and non- genotoxic agents may lead to this activation of P53. These agents include those which lead to single- or double-strand breaks in DNA. Once the activation occurs, it can produce several cellular changes (Zeimet et al. 2000 and Tokino and Nakamura 2000).

According to that, the aim of the present study is to investigate the frequency of specific point mutations of the RAS gene family in a group of Egyptian patients suffering from bladder cancer who have been directly exposed to organophosphates. A secondary aim is to check if indirect exposure would cause the same mutation in the k-ras gene or not. The third aim is to find the relation between direct and indirect exposures to pesticides and level of P53 and the bladder cancer.

Research methods and techniques

Type of the study

This is a prospective case-control analytic study.

Outcome measures (primary/secondary)

-Detection of organophosphates in patients with bladder cancer as a risk factor (by questionnaire to evaluate the exposure and analysis of cholinesterase enzyme in blood to confirm the exposure to organophosphates)

-Evaluating the level of P53 as an indicator for cancer in the blood of the patient

-Detection of K-ras gene and incidence of its mutation in tissue of all patients with bladder cancer

-Comparing the incidence of K-ras mutation between those exposed to organophosphate and those who were not exposed to it

Sample size analysis

Sample size will be calculated by using the Epi Info program.

Patient suffering from bladder cancer and asking for treatment in South Egypt Cancer Institute in a period of 1 year was asked to share in the research after taking informed consent from them and to fill a questionnaire. Questions focused on occupation and lifestyle (including any method for organophosphate exposure) and duration of employment or duration of exposure.

Method

This is a case-control hospital-based study measuring the correlation between the exposure to organophosphorus compounds (by history, and confirmed with the level of cholinesterase enzyme) with different pathological types of bladder cancer occurring in patients from different upper Egypt governorates, and level of P53 and K-ras gene. The matching control group was selected to match for residence, age and sex. They were selected from those with nonmalignant lesion in bladder as (bladder polyp).

This study included 100 cases newly diagnosed with histopathologically proven bladder cancer and 200 controls who attended the outpatient clinic. An informed consent was obtained from all participants after the explanation of the aim of the study. A well-structured questionnaire asking about the exposure to organophosphates was offered to the patients. Diagnosis of the cases was based on histopathological examination of the tumor. Patients diagnosed with bladder cancer who have received radio- and/or chemotherapy prior to the study were excluded. Personal interview was done to collect the following data: socio-demographic data (name, age, sex, occupation, residence) and clinical data of cases (Karimianpour et al. 2008).

Surgical specimens were collected and stored at − 80 °C; the patients were selected from those admitted in Assiut University Hospital.

Sample collection and testing

Surgically resected specimens were collected, and part of it were sent to the pathology laboratory in order to know the type of the mass; the samples were used to determine the mutation in k-ras gene.

Five milliliters of blood was collected from patients and controls on EDTA in vacutainer tubes to measure the level of cholinesterase enzyme and P53.

Cholinesterase enzyme level was measured from blood samples which were collected using plain vacutainers tubes and proceeded quickly in the lab to prevent damage associated with storage according to the method of Knedel and Kin (1967).

P53 were measured using the human p53 ELISA kit from the blood of the patient according to the kit method (www.glorybioscience.com).

DNA extraction

The DNA was extracted from the primary tumor tissue and adjacent noncancerous tissues using the DNA extraction kit (proteinase K and phenol extraction) and then stored at 4 °C, for examining the mutations in the K-ras gene according to Nanda et al. (2010). In polymerase chain reaction (PCR), primer was designed from previous studies for amplifying sequences around codons 12 and 13 of KRAS; the primer sequences used were as follows: 5′-ACTGAATATAAACTTGTGGTAGTTGGACCT-3′ and 5′-TTCTCCATCAATTACTACTTGCTTCCTGTA-3′ (Przybojewska et al. 2000).

Statistical analysis

The statistics were done using SPSS program version 15, to explore the risk factors for those patients especially the exposure to organophosphates and explore the relation between organophosphate, level of p53, the k-ras gene mutation, and the occurrence of bladder cancer.

Ethical aspects

An informed consent was taken from all the participants involved in the study before participation. The consent was taken in written form after giving the patient’s full information about the research and after clarifying that there will be no hazards for him if he refused to share in the research as regards the services offered for him in the hospital. All ethical statements of the institute were followed to maintain the confidentiality of the results.

Results

This study was done to prove or reject the hypothesis of that exposure to organophosphates in pesticides which can lead to mutation in certain genes that lead to bladder cancer in patients who had been proved to have cancer by pathology and also to prove or reject the hypothesis that the P53 level correlates with the occurrence of cancer. The study was done on 100 patients of bladder cancer in Assiut University Hospital.

As shown in Table 1 summary of the data related to the patients and the control group, it seems that in our era, the occurrence of bladder cancer is more in males than in females. The median age group of the patient group was 57 years old (from 49 to 66 years old). There are many risk factors that related to the appearance of bladder cancer among the patients as shown in Tables 1 and 2: the presence of bladder stones (p < 0.001), positive family history of bladder cancer (p < 0.001), exposure to organophosphates (0.001), smoking (0.005), occurrence of recurrent cystitis (0.001), and bilharziasis (0.013). Table 2 shows the logistic regression of the significant risk factors that contribute in the development of the bladder cancer.

Table 1 Basic characteristic of bladder cancer patients and controls at presentation (T test)
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Table 2 Logistic regression for the significant risk factor in patients with bladder cancer
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Table 3 shows the relation between level of acetylcholinesterase enzyme, the pesticide exposure, types of tumors, and occurrence of mutation in the K-ras gene in patients and in the control group. The table shows that there was a highly significant relation between these multiple factors. The most common type of cancer bladder which is the UC type is highly correlated with the pesticide exposure and occurrence of the K-ras mutation.

Table 3 Relation between exposure to pesticide, the acetylcholinesterase enzyme level, type of bladder cancer, and occurrence of the mutation in K-ras (multivariate analysis)
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Table 4 shows the relation between the acetylcholinesterase level (AChE) and the P53 level in the serum of bladder cancer patients and control group. It shows that there was a significant inhibition of AChE in patient group in comparison with that of the control group (p < 0.001). Inhibition of AChE level is related to high level of organophosphates in blood. In the same table, there is highly significant elevation of P53 level in those with bladder cancer in comparison with the control group (p < 0.001).

Table 4 Relation between presence of bladder cancer and abnormal AChE and serum P53 levels (T test)
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Table 5 shows the relation between inhibition of the AChE level and the k-ras mutation. Most of the patients with AChE inhibition had a k-ras mutation in 65 patients (65%) with p value < 0.001.

Table 5 Relation between inhibition of AChE and K-ras mutation (T test)
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Discussion

Identification of the risk factors that lead to bladder cancer may decrease the incidence of the cases and give our era an alarm to decrease the unsafely excessive use of the organophosphates. Early screening by using an ideal marker (P53) may improve the diagnosis and prognosis. In this study, we found that inhibition of AChE level was associated with the occurrence of mutation in the K-ras gene. Also, high level of P53 was associated with the occurrence of bladder cancer.

Many studies were done to evaluate the risk factors that lead to bladder cancer among people, but few try to know the mechanism by which this factors lead to bladder cancer. The present study tries to find a correlation between the K-ras mutation and exposure to organophosphates. Lee et al. (2004) and Koutros et al. (2015) in their study found that there was a highly significant correlation between the pesticide exposure and occurrence of bladder cancer especially in those who never smoke to exclude the smoking factor. Many other studies were done to find the relation between cancers and some types of pesticides like atrazine; these studies were done in different era with different types of users and did not find a good relation between atrazine exposure and bladder cancer (Rusiecki et al. 2004; Freeman et al. 2011). This may give an alarm about the method and amount of organophosphorous used by Egyptian farmers.

Few studies were done to know how pesticides can cause different types of cancers; in the present study, the authors searched for K-ras gene mutation and found that, on the one hand, there was a significant correlation between pesticide exposure and mutation in K-ras gene and, on the other hand, there is a correlation between the k-ras mutation and the occurrence of the bladder cancer. This was indicated by the increase in the level of P53 in the blood of the bladder cancer patients exposed to the organophosphorus compounds and the significant decrease in the cholinesterase enzyme level. Barbacid (1990) agreed with the present study results that the ras gene have a role in different types of neoplasia. Noaishi et al. (2011) agreed with the present results and found mutation in the K-ras gene in the peripheral blood of Egyptian workers occupationally exposed to pesticides. Also, Ahrendt et al. (2001) found a correlation between cigarette smoking and mutation of the k-ras gene in patients with lung cancer; Riely et al. (2009), Roberts et al. (2010), Karachaliou et al. (2013), Rau et al. (2016), and Li et al. (2016) found an association between Kars mutation and non-small cell lung cancer. Alguacil et al. (2002) found that occupational exposure to organic solvents can also lead to k-ras mutation in pancreatic cancer, and Xiong et al. (2016) found that k-ras can be mutated in endometrial carcinoma. So, K-ras is an important indicator gene which can be affected easily by toxin exposure; few studies were done to find the relation between exposure to pesticides and k-ras mutation. Many studies were done to explain the mechanisms by which pesticide exposure can develop cancer. These include immunotoxicity (Galloway and Handy 2003), gene mutations (Menozzi et al. 2004), oxidative stress (Abdollahi et al. 2004; and John et al. 2001), and proliferation in cells (Cabello et al. 2001). In this study, authors found a relation between the k-ras gene and exposure to pesticides as a correlated factor to produce cancer in the bladder.

P53 level and its relation with the appearance of cancer were studied in previous studies, but few of them reported its relation with the bladder cancer; in this study, the authors found a significant correlation between high level of P53 and appearance of bladder cancer. P53 as a marker is a noninvasive method if it is found in high level, giving an alarm to physicians that there is something wrong with his patient. Shim et al. (1998) in their study suggested that mutation in P53 protein can be used as a biomarker in the management of patients with colorectal cancer. Also, Calaf et al. (2009) suggested that pesticide exposure can alter P53 and c-Ha ras and induce malignancy of breast cells through genetic factors. Nigro et al. (1989), Levine et al. (1991), and Callahan (1992) all agree with the results of the present study that P53 play a role in the development of many types of cancers. Also, Du et al. (2016) found that in their study, there is a correlation between P53 over estimation with progression of T1 non-muscle invasive bladder cancer (NMIBC) patients due to the heterogeneity and other limitations.

Conclusion

In conclusion, the present study supports the hypothesis of direct relationships between organophosphorus exposure and the activation of the K-ras gene and P53 level. However, future studies are needed on larger number of patients with follow-up, P53 level, and acetylcholinesterase level to confirm that excision of the cancer and correction of the exposure to organophosphates can improve the level of P53 and help in prognosis.