Evaluation of Mutagenic Effects of Chronic Renal Disease and Hemodialysis with Micronucleus Test

The present study aimed to evaluate the mutagenic effects of chronic renal disease (CRD) and haemodialysis (HD) using the mutagenic test known as the micronucleus assay The study involved 97 individuals, including 32 CRD patients on HD, 34 CRD patients not on HD and 31 controls. Buccal smears were examined with a light microscope at 400, and cells showing micronuclei, binucleated nuclei, “broken egg” nuclei, pyknosis, karyorrhexis and karyolysis were counted in 2000 cells for each participant. Our main results demonstrate that the clastogenicity indicators micronuclei, binucleated cells, broken egg cells, sum of binucleated plus broken egg cells and sum of pyknosis plus karyorrhexis were statistically significant higher in the CRD and HD group compared to the CRD and non-HD groups and the control group (p=0.0001). Males had more binucleated cells (p=0.002) and sum of binucleated plus broken egg cells (p=0.0001). Those with a family history of genetic disease had more binucleated cells (p=0.002). Those on HD who did not consume wine showed more micronuclei (p=0.042). Consumers of distilled alcoholic beverages had more karyolysis (p=0.038).Those not on HD but exposed to chemical agents had more karyolysis (p=0.041). Controls who drank wine had a lower sum of binucleated plus broken egg ells (p=0.050). Ex-smokers had more broken egg cells (p=0.006). We conclude that the CRD and HD combined with occupational and genetic factors and certain lifestyle habits promote the occurrence and increase of micronuclei and other nuclear changes, possibly able to contribute to the emergence of renal cancer.


Introduction
Chronic renal disease (CRD) causes damage to the kidney tubules and can cause premature cardiovascular death. In the late phase, patients need dialysis or a kidney transplant. The main risk factors in Brazil, the United States (US) and Canada are systemic arterial hypertension (SAH), diabetes mellitus (DM) and family history. CRD in the US is the leading cause of death and affects about 26 million people [1][2][3][4].
In Brazil, CRD affects about 1 million people, causing about 15,000 deaths/year. Currently, 100,000 people are estimated to be on HD, and the mortality rate is 17%. The most affected are the elderly, women and ethnic groups such as Asians. The prevalence in different countries is 7.2% in people over 30 years and 28 to 46% in individuals over 64 years [5,6].
The inflammatory process in uremic patients with CRD causes tissue damage, endothelial dysfunction, atherogenesis and cardiovascular disease. Hemodialysis (HD) increases the risk of oxidative stress by the interaction between blood and biocompatible membranes, which may cause DNA damage, genomic instability and inhibition of the repair mechanism [7][8][9][10][11]. In an in vitro study, Ha and colleagues (2000) demonstrated that the solutions used in the process of DP with low pH and or with high glucose levels can promote damage in the peritoneum in addition to the death of the mesothelial cells in the DNA.
Genotoxic agents induce cell damage. The formation of micronuclei (MN) is due to clastogenic agents that promote chromosome breaks in single-and double-stranded DNA. MNs can be used as a biomarker of genotoxic damage and an increase their frequency may represent the action of genotoxic/clastogenic agents. Successive exposure may show cumulative degenerative damage and malignant transformation [10,[12][13][14][15].
It was estimated that in 2013, there were about 273,518 new cases of renal cancer in the world [16]. Incidence varied by geographic region [17], where it was higher in Europe and North America. [18] The etiology has not yet been identified, but there is evidence of a strong association with genetic and environmental factors such as lifestyle, tobacco use, obesity and occupation [19].
MN analysis can be used as a biomarker for chromosomal DNA damage in a variety of cell types. [13][14][15]19]. Guven et al. investigated the presence of nuclear damage in peripheral lymphocytes by associating the frequency of micronuclei with hemodialysis (HD) and peritoneal dialysis (DP), concluding that patients in the final stage of renal disease exhibit advanced nuclear damage [20][21][22].
Oral mucosa is used for the quantitative analysis of nuclear alterations in patients who are smokers of many kinds of tobacco, alcoholics, exposed to chemical agents, pesticides, antineoplastic drugs and other kinds of genotoxic agents [19].
The aim of this study was to evaluate the mutagenic effects of CRD combined with HD, using the micronucleus test, analyzing cells from the oral mucosa of patients with CRD on HD, with CRD not on HD and controls.

Materials and Methods
Samples were randomly obtained from 120 volunteers, and of these 97 participants were analyzed. This was carried out at the hospital care complex, Rio Grande do Sul, Brazil, between January and December 2014. The first group consisted of 32 individuals with CRD undergoing conventional HD for over 3 months with duration of 3 to 4 hr per session, three times a week, with 17 L from Gambro Polyflux dialyzer and peracetic acid sterilant. The second group consisted of 34 individuals with CRD on HD. The control group was formed by 31 individuals without CRD and who never underwent HD. Twenty-three participants was excluded: 12 smokers and 11 over 76 years old or under 21 or those with inflammatory or infectious lesions in the oral mucosa (Figures 1 and 2).  The collection and testing of cytogenetic materials followed biosafety guidelines. We used the Cytobrush Plus (Kolplast Commercial Industrial Ltda, São Paulo, SP, Brazil). To obtain samples from the oral mucosa smears the slides were stained by the May-Grünwald/Giemsa (MGG) technique with 10% Giemsa (Merck, Darmstadt, Germany). A total of 2000 cells of each participant were analyzed with a Leica CME® binocular light microscope at 400X, and we counted cells with micronuclei, broken egg cells, binucleated cells, and cells showing apoptosis (karyorrhexis, pyknosis) and karyolysis [20][21].
Statistical analysis was performed with the Statistical Package for Social Sciences (SPSS), version 19.0, and Kolmogorov-Smirnov test for normality of data. Descriptive statistics used frequencies, means and standard deviations (±). Comparisons between groups used the Student t-test and one-way ANOVA followed by the Tukey post-test. p<0.05 was considered statistically significant.

Sample description
We studied 97 subjects aged 21 Table 1: Characteristics of the CRD on HD, CRD not on HD and control groups.

Evaluation of nuclear alterations
By morphological analysis, cell quantification and statistical analysis of constant nuclear abnormalities in Table 3, we found a higher prevalence of MN, BNC, BEC, sum of BNC+BEC, karyorrhexis, karyolysis and sum of pyknosis+karyorrhexis in the CRD on HD group, followed by the CRD not on HD and control groups. The prevalence of pyknosis was lowest in the control group. All these differences were statistically significant (p=0.0001).  Comparing the number of abnormal cells and the sex of the participants, we found that the CRD not on HD group showed a higher number of BNC in men versus women (p=0.002), and also for the sum of BNC plus BEC in men (p=0.011) versus women, statistically significant differences.
The comparison between the ethnicity of the three groups studied and the number of abnormal cells showed that Caucasians in the CRD on HD group had higher rate of karyorrhexis (p=0.031). In the CRD not on HD group, the number of BNC was greater (p=0.001). However, non-Caucasians showed a significantly greater number of BEC (p=0.007) and BNC plus BEC (p=0.0001), all statistically significant differences.  Regarding the associated exposure factors (Table 2), in the CRD on HD group, there was a lower prevalence of: beer and wine consumption (28.1%), exposure to chemical (34.4%) and physical (3.1%) agents and chimarrão drinking (31.3%) compared with those in the on HD (p=0.007) and control (p=0.004) groups. Those who drank wine had a greater number of MN (p=0.042). Those who drank distilled alcoholic beverages showed a higher rate of karyolysis (p=0.038).

Variable/abnormal cells CRD on HD group CRD not on HD group
In the CRD not on HD group, there was greater frequency of exposure to chemical agents (70.6%) and greater rate of karyolysis in those exposed versus not exposed (p=0.041) ( Table 2).

Discussion
We observed a higher mean number of MN in the CRD on HD group compared to the CRD not on HD and control groups, indicating that CRD and HD combined have a mutagenic effect on cells causing genetic damage. This increase in MN may also be related to the uremic state caused by CRD, occupational exposures, genetics and lifestyle habits. These results corroborate other studies, where there was a higher frequency of MN in HD patients compared to control groups [22][23][24][25].
Long treatments with HD are associated with damage to the genome, due to the inflammatory state and the oxidative "stress." In this study, treatment time with HD averaged 4.15 years (± 4.15). In a previous study, patients on HD for a period ≥ 7 years had a mean MN count of 8.89 (± 5.96), and those on HD for a period ≤ 6 years had a lower mean MN of 2.91 (± 2.74). A similar situation occurred in with BEC with respective values of 3.78 (± 5.09) and 1.00 (± 3.00) (p<0.05). We noted that the greater the time of exposure to HD, the higher the genotoxic damage as evidenced by the increase in the number of MN [7][8][9]23].
BNC indicate delayed cell division and cytotoxicity. We observed a higher mean BNC frequency in the CRD on HD group and CRD not on HD groups, which might have been related to the genotoxic effects of CRD, since the controls did not have CRD. In another study, BNC frequency was also higher in the HD group than in controls, with 46.2% (± 4.3%) versus 24.4% (± 9.5%) [20,25].
The high frequency of BEC can indicate normal adaptation of the epithelium, but we observed a greater incidence in patients with CRD on HD and with CRD not on HD, compared to controls. The sum of BNC plus BEC showed a mean of 37.28 (± 15.78) in the CRD on HD group versus 32.47 (± 22.10) in the CRD not on HD group versus 14.25 (± 6.92) in controls (p=0.0001), reinforcing the notion of this increase being related to the mutagenic effects of CRD and HD [23,26].
The increase in pyknosis in the CRD on HD group can be related to shear stress caused by prolonged HD and by CRD, which results in a state of uremia. The mean frequency of karyorrhexis and karyolysis with the sum of the variables pyknosis plus karyorrhexis between the groups was statistically significant in relation to controls (p=0.0001), demonstrating that CRD and HD determine the increase in these cellular changes [7][8][9]20,24].
Mean age was 53.62 (± 14.20) years in the CRD on HD group, 53.26 (± 13.95) in the CRD not on HD group, and 47.00 (± 15.24) in the control group, a similar age profile as in other studies [23][24][25].
There was no statistically significant relation with exposure to chemical agents. In another study, workers exposed to farm pesticides for 16.3 (± 10.0) years had a mean MN of 3.55 (± 2.13), significantly higher than in the non-exposed group with 1.78 (± 1.23) (p=0.001) [27].
We observed in comparing the number of cells with alterations and weekly consumption of chimarrão a statistically significant increase in BEC only in the control group, although it might have been related to the diet restrictions and consumption of chimarrão of those on HD. Another study found that among daily chimarrão drinkers, 28% had BNC, 14% karyorrhexis, 2% karyolysis, 6% BEC, 7% pyknosis and 43% MN [28].
Comparing the cellular changes and the consumption of alcohol, in the CRD on HD group, wine drinkers had a lower mean MN of 5.66 (± 3.60) (p=0.095), while the controls had a lower sum of BNC plus BEC with a mean of 11:50 (± 4:41) (p=0.050). This decrease may further indicate a more protective property of wine related to the reduced  [29].
In the CRD on HD group who drank distilled spirits had a higher mean rate of karyolysis at 93.00 (± 52.32) (p=0.038). The effects of alcohol together with a HD may result in potentiated cell damage, since ethanol acetaldehyde may promote carcinogenesis and since alcohol consumption is a risk factor for cancers of the head and neck, intestine, liver and breast. In another study, workers exposed to pesticides with a habit of alcohol consumption had higher mean MN levels compared to controls. In this study, we found that CRD, by affecting renal filtration, and HD, by causing oxidative stress, together influence this process [27,30,31].
This study differs from similar ones by analyzing CRD and HD separately in three groups which made it clear that patients undergoing HD had a significant increase in genetic changes, evidenced by a higher frequency of MN and BNC, compared to patients with CRD not subjected to HD [22][23][24][25].
New studies are warranted to evaluate genetic polymorphisms and susceptibility to the development of renal cancer by genotyping patients with CRD undergoing HD.

Conclusion
We demonstrated using the micronucleus test that there were statistically significant differences in mean number of cells with MN, BNC, BEC, pyknosis, karyorrhexis and karyolysis between groups, with higher numbers in the CRD on HD group in relation to the CRD not on HD and control groups.
The increase in cellular changes might have been related to CRD, the HD and also other occupational exposure factors, eating habits, underlying diseases, genetic and ethnic factors denoting greater susceptibility to mutagenic effects.
We noted that those on HD, Caucasian males had higher rates of cellular changes that may be associated with the occurrence of mutagenic damage. We showed that CRD and HD contribute to the emergence and increase of metanuclear changes such as pyknosis, karyorrhexis and karyolysis.
Finally, we believe that CRD and HD are mutagenic factors alone, together or in combination with other occupational exposure factors, genetics and lifestyle habits, and that they significantly contribute to the emergence and increase of micronuclei and other nuclear changes. Therefore, CRD and HD may be factors that are related to the emergence of renal cancer. In addition, our results show that the MN test and the frequency test of other nuclear alterations using the oral mucosa can be an extremely sensitive and non-invasive biomarker to evaluate the deleterious effects of various xenobiotic substances, including procedures such as HD.