Research ArticleExperimental Studies

192R Allele of Paraoxanase 1 (PON1) Gene as a New Marker for Susceptibility to Bladder Cancer

OĞUZ ÖZTÜRK, ÖMER F. KAĞNICI, TÜLIN ÖZTÜRK, HAYDAR DURAK, BORA M. TÜZÜNER, HALIL I. KISAKESEN, CANSER ÇAKALIR and TURGAY İSBIR
Anticancer Research October 2009, 29 (10) 4041-4046;

Abstract

Background: The human body has a number of endogenous free-radical scavenging systems. Paraoxonase1 (PON1) is one of the enzymes involved in such systems. The purpose of this study was to investigate the relationship between bladder cancer and the polymorphism of PON1 which results in Q/R substitution at codon 192. This is the first report of this polymorphism in bladder cancer. Patients and Methods: Seventy-six bladder cancer patients and 135 healthy controls took part in the study. DNA from paraffin-embedded tissues for patients and from blood cells for controls was extracted. The distribution of PON1192 polymorphism was determined by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) techniques. Results: There was significant difference in PON1 genotype frequencies (patients/controls QQ: 8/37, QR: 53/84, RR: 15/14, p=0.007) between the control and bladder cancer patients. Moreover, in patients there was significant association of the RR genotype of PON1 with invasive growth pattern (p=0.027), perineural invasion (p=0.016), distant metastasis (p<0.001), radical cystectomy (p=0.016) and death (p=0.005). Conclusion: In patients, the QQ genotype was statistically less frequent, while the RR genotype frequency was somewhat less than in controls. QQ type PON1 enzyme activity might be protective for bladder carcinogenesis. These findings support that genotypical differences of PON1 might contribute to prognosis and pathogenesis of bladder cancer. Arylamines are well-known bladder carcinogens, especially after a sulfate or an acetate esterification and PON1 has arylesterase activity. We hypothesize that arylesterase activity of PON1 would help the formation of free-radical type arylamine derivates on the bladder epithelial surface, so that secondary metabolites of paraoxon or related chemicals and biotransformed intermediates of arylamines might be involved in formation of bladder carcinoma.

According to the American National Cancer Institute, bladder cancer is the fourth most common cancer in men and the eight most common cancer in women, and the second most common malignancy of the genitourinary tract (1). Males are more prone to the disease than females: 70% of patients are males. Tobacco, aromatic amines and amides (e.g. 4-aminobiphenyl, benzidine and 2-naphthylamine) and the combustion of fossil fuels and some anticancer drugs, such as cyclophosphamide, are found to be associated with the risk of bladder cancer (2). Most bladder carcinogens exert their action by direct contact with the bladder epithelium. Inhaled directly carcinogenic compounds, or metabolized carcinogenic byproducts, reach the urinary bladder via urinary excretion. Although the human body has detoxification systems, polymorphic differentiation among individuals results in varying tolerence and susceptibility to cancer.

In this study, the association of bladder cancer and the detoxifying enzyme paraoxanase 1 (PON1), named after paroxon, first identified substrate of this enzyme family was studied. The PON genes are clustered on human chromosome 7 (q21.22). Besides its hydrolytic activity on paroxon and metabolites of organophosphorous insecticides (Figure 1), it has broader physiological role and substrate range. Mackness et al. first showed PON1 mechanism in metabolism of oxidized low- and high-density lipoproteins (HDL) in 1991 (3); following this, in 2003, Costa et al. revealed that PON1 also protects phospholipids in high-density lipoprotein (HDL) from oxidation (4). Moreover in 2000, Biggadike et al. discovered the lactonase activity of PON1 (5).

Previous studies show modular activity of PON1, hence it has different kinetics and distinct activities in different physiological conditions. Moreover, polymorphic diversity boosts the modularity of the enzyme activity. There are more than 160 defined polymorphisms, both in introns, exons and regulatory regions of the gene. Earlier studies indicate that some polymorphisms may cause high, intermediate or low paraoxonase activity (6). Frequencies of these high or low metabolizers vary among groups of different ethnic or geographical origins. PON1 and disease-related studies examine mostly 3 single nucleotide polymorphisms which cause missense mutation resulting in amino acid substitutions that are Q192R, L55M and C108T.

Figure 1.
Figure 1.

A sample reaction of aryl-ester hydrolase. Because of the toxicity of paraoxon, phenyl acetate is widely preferred for measuring PON1 activity (21).

In the light of findings from Mackness et al. and Costa et al., previous studies investigated the role of PON1 in atherosclerosis and cardiovascular diseases. However, recent studies suggest various roles in distinct diseases including lung, breast, prostate and ovarian cancer, and even age- and diabetes-related cataract formation (7-11). Here in this study for the first time, Q192R substitution of PON1, which determines low or high paroxonase activity by Q and R alleles respectively, and its relation with bladder cancer was investigated.

Patients and Methods

Patient selection and clinical investigation. A total of 211 unrelated individuals were included in this study: 76 bladder cancer patients, 64 male (81.25%) (median age: 64, range 20-91 years) and 135 controls, 84 male (62.20%) (median age: 59, range 20-91 years). Patients were selected from the Department of Pathology, Istanbul University Cerrahpasa Medicine School, Istanbul in 2002-2007. During assessment, the 2004 WHO definitions and criteria for cancer were used (12). The control group was selected from patients attending the General Surgery, Urology and Orthopedic Clinics of the same hospital and who were treated for non-neoplastic diseases such as inguinal hernia or trauma. Medical history and physical, pathological features were recorded for all patients (Table I).

DNA extraction. Patients' DNA was extracted from paraffin-embedded tissue with the used of a method taken from Wright and Manos (13). Blood specimens of control the group were collected in tubes containing EDTA, and DNA was prepared from leukocyte pellets by SDS lysis, ammonium acetate extraction and ethanol precipitation (14).

View this table:
Table I.

Pathological features of patients with bladder cancer.

View this table:
Table II.

Distributions of PON1 genotypes in patient and control groups.

View this table:
Table III.

Effect of PON1 RR genotype on pathological variables in multivariate model (by forward logistic regression analysis). PON1 192 gene polymorphism was compared as a dependent variable with the other group of variables. Only survival as a related variable reached the statistical signifigance with the RR genotype of PON1.

Genotyping method for paraoxonase Q192R polymorphism. For genotyping, DNA extracted from blood of controls and from paraffin-embedded non-tumoral neighboring tissues of cystectomized patients and also from paraffin-embedded samples from non-tumoral neighboring bladder mucosa after transurethial resection (TUR) operation was used. PON1 genotypes were determined following PCR according to the method of Adkins et al. (15).

Statistical analysis. Statistical analyses were performed using SPSS version 7.5 including the Chi-square (χ2) test, Fisher's exact test and the Pearson correlation test. Multivariate analysis was performed with logistic regression (forward: LR). Odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated. In multivariate analysis, comparison of PON1 192 gene polymorphism as a dependent variable with the other groups of variables (survival, superficial lamina propria infiltration, tumors grade, p53 immune reactivity) was planned. Kaplan-Meier survival analysis was performed with the log-rank (Mantel-Cox) test of equality of survival distributions for the genotypes.

Results

Genotype frequencies of patients and the control group are shown in Table II. These results showed a significantly lower QQ genotype frequency in the patient group compared to controls (p=0.004, OR: 3.21, 95% CI: 1.407-7.317). The frequency of the PON1 192 RR genotype of bladder cancer patients was higher than in the control group. We observed strong interactions between the presence of the RR allele of PON1 and bladder cancer (p=0.058, OR: 2.125, 95% CI: 0.963-4.686) (Table II).

The mean age for patients with QQ, QR and RR genotypes was 68.87, 63.28 and 58.86 years, respectively. In the light of these results, the patients with RR genotype had cancer at an earlier age than did patients with QQ or QR genotypes but difference did not reach statistical importance.

Genotypes were analyzed with some dependent/independent pathological features. Significant association of the RR genotype with: radical cystectomy, invasive growth pattern, perineural invasion, distant metastasis and death were found (Table II). Additionally, 0-25 month survival surveys of the patients reveal that those with the RR genotype have shorter life expectancy around 10.70±3.154 (4.521-6.884) months (remaining genotypes: 17.10±1.867) which was of borderline significance (κ2: 3.75, p=0.079). In model 1 multivariate analysis, significant association between death and the RR genotype of PON1 (Table III) was found. In the light of these findings, bladder cancer patients with PON1 RR genotype would seem to have poor clinical outcomes.

Discussion

Oxidative stress and free radicals have been associated with the increased risks of various types of cancer. The human body has a number of endogenous free-radical scavenging systems, including PON1. PON1 binds to HDL and contributes to the detoxification of organophosphorus compounds, such as paraoxon, and carcinogenic lipid-soluble radicals from lipid peroxidation (16). Parathion is an organophousphorus chemical which is used widely as an insecticide for fruits, corn and olive. Parathion is converted to paraoxon in vivo in mammals by microsomal oxidation reaction. PON1 is capable of hydrolyzing paraoxon to produce p-nitrophenol which causes DNA and cell damage. The RR genotype of PON1 results in higher activity of paraoxon, which increases p-nitrophenol formation rate, but lowers activity for lipid-soluble free radicals and some organophosphorus compounds. Due to the lower metabolization rate by PON1, lipid-soluble free radicals accumulate, and along with many organophosphorus compounds and as yet unidentified chemical substrates of PON1 may also be involved in bladder carcinoma formation.

In this study, a statistically lower frequency of the QQ genotype in bladder cancer patients (p<0.01) was found and also in patients a non-significant but increased association of the RR genotype with bladder cancer (p=0.058, OR 2.13, 95% CI: 0.96-4.68) was observed. In addition, early age of diagnosis of patients with RR genotype (highly active type) may suggest that secondary metabolites of paraoxon and paraoxon-related chemicals or the esterase activity of PON1 on N-sulfonyloxy-, N-acetoxy- and N-prolyoxy- derivatives of arylamines or unidentified substrates detoxified by the RR type may be involved in bladder carcinoma. Thus, the activity of the QQ type PON1 enzyme might be protective for bladder carcinoma. In comparison, patients with the PON1 RR genotype also had a more deadly course of disease than the others (p<0.01): risk for death in the RR genotype group was fivefold higher than that for the others in logistic regression (p<0.01, OR 5.89, 95% CI: 1.58-21.9). Multivariate analysis also showed that increased death risk of the patients with the RR genotype is independent of p53 immune reactivity or other pathological features. PON1 Q192R polymorphism may predict poor prognosis in bladder carcinoma. These findings suggest that different genotypes of PON1 may be significant contributors to pathogenesis of bladder cancer. Moreover, we also studied PON1 192 polymorphism in a small case-control group and found that patients who carried the PON1 192 RR genotype had more than a fourfold increase in breast cancer development risk (8).

Figure 2.
Figure 2.

Arylamine biotransformation pathway scheme and possible role of PON1. Hypothesized reaction is shown as dashed arrows.

We hypothesize a greatly possible reaction, catalyzed by PON1 in bladder epithelium, might have a role in formation of bladder cancer, depending on the aryl esterase activity of PONI. In the bladder epithelium, a variety of reactive arylamine ester derivatives produced in the liver can be hydrolyzed to form hydroxylarylamine by aryl esterase activity and excreted via urine (Figure 2). Under physiological conditions in liver tissue, hydroxylamine is known to be a less potent carcinogen than arylamine derivatives, however, in acidic environments such as bladder epithelium, these compounds can form nitrenium ion, which is a potent bladder carcinogen (17). Thus, by leading to nitrenium ion formation, the arylesterase activity of PON1 may play a key role in bladder carcinogenesis.

In the 1960s, paraoxon was not thought to be a mutagen, but Gichner et al. demonstrated a mutagenic synergy between paraoxon and 2-acetoxyacetylaminofluorene (2AAAF). This synergy was dependent on the acetylated form of the arylamine (18). Miller et al. showed that N-hydroxy-acetylaminofluorine (AAF) is converted in rat liver to a sulfate, N-sulfonoxy-AAF, by means of a cytosol sulfotransferase activity, or an acetate, N-acetoxy-AAF, by means of acetyltransferase activity. The acid pH of urine can convert these esterified compounds to nitrenium ion or this can happen spontaneously. Nitrenium ion reacts with nucleic acids and proteins and appears to be the ultimate carcinogen in the urinary bladder epithelium (19).

Although, the exact carcinogenic metabolite(s) involved in the initiation of bladder carcinogenesis remains uncertain, it seems reasonable to expect that any enzyme system involved in the further metabolic activation of N-hydroxyarylamines might serve as a potential determinant of bladder cancer susceptibility (Figure 2). In addition, electrophilic forms of the aromatic amines result from their metabolic activation, and the positively charged nitrenium ion formed from naphthylamine and aminobiphenyl compounds has been implicated as the ultimate urinary bladder carcinogen in dogs and humans (20-21).

PON1 may be a candidate enzyme in activation of hydroxylamine formation from acetoxy esters. While acetoxy esters have not been to be shown known substrates under physiological conditions, structure-activity relation (SAR) studies suggest their possible role. Khersonsky et al. had studied lactonase and esterase activity of PON1 both in silico and in vivo. For esterase activity of PON1, they determined that PON1 has plateau-like kinetic parameters for most aryl esters independent of the leaving group pKa. They suggest that the catalysis rate may be limited by physical steps other than substrate binding, such as product release, or a conformational change, rather than a chemical step such as bond-breaking (22). Therefore, PON1 may be a universal aryl esterase and a conformational change in structure may increase or gain activity in cleavage of acetoxy esters of carcinogenic aryl ester, which are also detoxified by phase I enzymes such as N-acetyl-transferase 1 (NAT1), leading to bladder carcinogenesis.

In summary, previous studies suggest that cancer related to PON activity depended on lipid peroxidates and its byproducts. Arylamines are well-known bladder carcinogens especially after a sulfate or an acetate esterification. We hypothesize that arylesterase activity of PON1 would help the formation of free-radical type arylamine derivatives on the bladder epithelial surface. Hence secondary metabolites of paraoxon or related chemicals, or some biotransformed intermediates of arylamines or unidentified chemicals might be involved in bladder carcinoma. In future studies, additional PON1 192 genotyping and multivariate analysis should be performed in other populations with a larger number of patients to confirm the association of the PON1 192 polymorphism with the risk of bladder cancer.

Acknowledgements

This study was supported by a grant from the Istanbul University, Research Foundation (project T-464/250604), Turkey. We also appreciate the cancer epidemiologist Dr. Hakan Camlica for support and correction of statistical analysis and Professor Dr. Makbule Aydin for evaluating the suggested chemical reactions.

  • Received April 30, 2009.
  • Revision received July 15, 2009.
  • Accepted August 17, 2009.

References

  1. ↵
    1. Natioanal Cancer Institute
    . What You Need to Know About Bladder Cancer. http://www.cancer.gov/cancertopics/wyntk/bladder (Accessed May 8, 2009).
  2. ↵
    1. Cohen SM,
    2. Shirai T,
    3. Steineck G
    : Epidemiology and etiology of premalignant and malignant urothelial changes. Scand J Urol Nephrol Suppl 205: 105-115, 2000.
    OpenUrlPubMed
  3. ↵
    1. Mackness MI,
    2. Arrol S,
    3. Durrington PN
    : Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 286: 152-154, 1991.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Costa LG,
    2. Cole TB,
    3. Jarvik GP,
    4. Furlong CE
    : Functional genomics of the paraoxonase (PON1) polymorphisms: effects on pesticide sensitivity, cardiovascular disease, and drug metabolism. Annu Rev Med 54: 371-392, 2003.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Biggadike K,
    2. Angell MR,
    3. Burgess CM,
    4. Farrell RM,
    5. Hancock AP,
    6. Harker AJ,
    7. Irving WR,
    8. Ioannou C,
    9. Procopiou PA,
    10. Shaw RE,
    11. Solanke YE,
    12. Singh OMP,
    13. Snowden MA,
    14. Stubbs RJ,
    15. Walton S,
    16. Weston HE
    : Selective plasma hydrolysis of glucocorticoid γ-lactones and cyclic carbonates by the enzyme paraoxonase: An ideal plasma inactivation mechanism. J Med Chem 43: 19-21, 2000.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Furlong CE,
    2. Holland N,
    3. Richter RJ,
    4. Bradman A,
    5. Ho A,
    6. Eskenazi B
    : PON1 status of farmworker mothers and children as a predictor of organophosphate sensitivity. Pharmocogenet Genomics 16: 183-190, 2006.
    OpenUrl
  7. ↵
    1. Lee CH,
    2. Lee KY,
    3. Choe KH,
    4. Hong YC,
    5. Kim YD,
    6. Kang JW,
    7. Kim H
    : Effects of oxidative DNA damage induced by polycyclic aromatic hydrocarbons and genetic polymorphism of the paraoxonase-1 (PON1) gene on lung cancer. J Prev Med Public Health 38: 345-350, 2005.
    OpenUrlPubMed
  8. ↵
    1. Bedia Ağaçhan,
    2. İlhan Yaylim,
    3. Arzu Ergen H.,
    4. Soykan Arikan,
    5. Seden Küçücük,
    6. Hülya Yilmaz,
    7. Ümit Zeybek S.,
    8. Savaş Bayrak,
    9. Nilüfer Bozkurt,
    10. Uzay Görmüş,
    11. Turgay İsbir
    : Is paraoxonase 1 192 BB genotype risk factor for breast cancer? Adv Mol Med 2(1): 37-40, 2006.
    OpenUrl
    1. Stevens VL,
    2. Rodriguez C,
    3. Talbot JT,
    4. Pavluck AL,
    5. Thun MJ,
    6. Calle EE
    : Paraoxonase 1 (PON1) polymorphisms and prostate cancer in the CPS-II Nutrition Cohort. Prostate 68: 1336-1340, 2008.
    OpenUrlPubMed
    1. Lurie G,
    2. Wilkens LR,
    3. Thompson PJ,
    4. McDuffie KE,
    5. Carney ME,
    6. Terada KY,
    7. Goodman MT
    : Genetic polymorphisms in the paraoxonase 1 gene and risk of ovarian epithelial carcinoma. Cancer Epidemiol Biomarkers Prev 17: 2070-2077, 2008.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Hashim Z,
    2. Zarina S
    : Assessment of paraoxonase activity and lipid peroxidation levels in diabetic and senile subjects suffering from cataract. Clin Biochem 40: 705-709, 2007.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Eble NJ,
    2. Sauter G,
    3. Epstein JI,
    4. Sesterhann IA
    : Pathology and Genetics: Tumours of the Urinary System and Male Genital Organs. WHO Classification of Tumours. Lyon, IARC Pres, pp. 89-154, 2004.
  13. ↵
    1. Inis MA,
    2. Gelfand DH,
    3. Sninsky JJ,
    4. White TJ
    1. Wright DK,
    2. Manos MM
    : Sample preparation from paraffin-embedded tissues. In: PCR Protocols, A Guide to Methods and Applications. Inis MA, Gelfand DH, Sninsky JJ, White TJ, (eds.). UK Academic Press, pp. 153-158, 1990.
  14. ↵
    1. Miller SA,
    2. Dykes DD,
    3. Polesky HS
    : Simple salting-out procedure for extracting DNA from human nucleated cells. Nucleic Acid Res 16: 1215, 1988.
    OpenUrlFREE Full Text
  15. ↵
    1. Adkins S,
    2. Gan KN,
    3. Mody M,
    4. La Du BN
    : Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes. Am J Hum Genet 52: 598-608, 1993.
    OpenUrlPubMed
  16. ↵
    1. Shih DM,
    2. Gu L,
    3. Xia YR,
    4. Navab M,
    5. Li WF,
    6. Hama S,
    7. et al.
    : Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394: 284-287, 1998.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Lower GM,
    2. Nilsson T,
    3. Nelson CE,
    4. Wolf H,
    5. Gamsky TE,
    6. Bryan GT
    : N-Acetyltransferase phenotype and risk in urinary bladder cancer: approaches in molecular epidemiology: Preliminary results in Sweden and Denmark. Int J Epidemiol 36(1): 11-18, 2007.
    OpenUrlFREE Full Text
  18. ↵
    1. Gichner T,
    2. Veleminský J,
    3. Wagner ED,
    4. Plewa MJ
    : Mutagenic synergy between paraoxon and plant-activated m-phenylenediamine or 2-acetoxyacetylaminofluorene. Environ Mol Mutagen 27: 59-66, 1996.
    OpenUrlPubMed
  19. ↵
    1. Miller EC
    : Some current perspectives on chemical carcinogenesis in humans and experimental animals. Cancer Res 38: 1479-1496, 1978.
    OpenUrlFREE Full Text
  20. ↵
    1. Young JF,
    2. Kadlubar FF
    : A pharmacokinetic model to predict exposure of the bladder epithelium to urinary N-hydroxyarylamine carcinogens as a function of urine pH, voiding interval, and resorption. Drug Metab Dispos 10(6): 641-644, 1982.
    OpenUrlAbstract
  21. ↵
    1. Ruddon RW
    : Cancer Biology: New York: Oxford University Press, pp. 22-23, 2007.
  22. ↵
    1. Khersonsky O,
    2. Tawfik DS
    : Structure reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 44: 6371-6382, 2005.
    OpenUrlCrossRefPubMed
    1. Jung I,
    2. Messing E
    : Molecular mechanisms and pathways in bladder cancer development and progression. Cancer Control 7: 325-334, 2000.
    OpenUrlPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
192R Allele of Paraoxanase 1 (PON1) Gene as a New Marker for Susceptibility to Bladder Cancer
OĞUZ ÖZTÜRK, ÖMER F. KAĞNICI, TÜLIN ÖZTÜRK, HAYDAR DURAK, BORA M. TÜZÜNER, HALIL I. KISAKESEN, CANSER ÇAKALIR, TURGAY İSBIR
Anticancer Research Oct 2009, 29 (10) 4041-4046;
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section