Cancer Letters, 47 (1989) 99- 103

Elsevier Scientific Publishers Ireland Ltd

99

Clastogenic activity in urine of individuals with urinary bladder infections

M.P. Rosinazb, R.H. Seeb and R.H.C. Sane

“Laboratory of Cell Biology and Genetics, School of Kinesiology, Simon Fraser University, Burnaby, B.C., V5A 1S6,

bEnuironmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouuer,

B.C., V5Z lL3 (Canada) and ‘Microbiological Associates, Inc., 9900 Blackwell Rd., Rockoille, MD 20850, (U.S.A.)

(Received 20 April 1989)

(Accepted 24 April 1989)

Summary

The object of this study was to determine

whether an elevation in chromosome-damag- ing (clastogenic) activity occurred in the urine

of individuals with bladder infections. Urine

samples were collected from 18 patients with

chronic (long-term) bladder infections (CBI).

Organic material was extracted from urine by

preparative reversed-phase high-pressure

liquid chromatography and assayed for

chromosome-damaging activity in Chinese

Hamster Ovary (CHO) cell cultures.

Clastogenic activity was present in these urine

extracts at levels significantly above those

observed in control individuals (P < 0.001).

These levels were comparable to those

observed in smoker’s urine. In addition, 2 of 4 individuals with acute (short-term) bladder infection (ABI) showed a significant elevation

in clastogenic activity in their urine samples

(P = 0.025). This study indicates that clasto-

genie components can be produced during

bacterial infections in the urinary bladder and

supports a direct involvement of urinary tract

infections in the development of bladder can-

cer.

Correspondence to: M.P. Rosin, Occupational Health Unit, School of Kinesiology, Simon Fraser University, Burnaby, B.C.,

V5A lS6, Canada.

Keywords: genotoxicity of urine; bladder infections; chromosome-damaging; clasto- genie.

Introduction

Urinary tract infections may have an etiolog- ical role in the development of bladder cancer. Egyptians with Schistosoma haematobium

infections in the bladder [6,7], patients with long-term catheter bladder drainage [ 1,2,14,18-ZO] , and individuals with cystitis (reviewed in [17]) have all been reported to be at risk for bladder cancer. Whether this elevation in risk is directly associated with the presence of bladder infection is unknown. However, such a possibility is supported by animal studies in which bacterial infections have been shown to enhance the development of bladder tumors in carcinogen-exposed animals [5,11,15,16].

A possible approach to exploring the involvement of bacterial infections in human bladder cancer is to examine the urine of infected individuals for the presence of geno- toxic components. An elevation in such meta- bolites has been found in the urine of populations at risk for bladder cancer, such as cigarette smokers [24] and the examination of urine is widely used to monitor human

0304.3835/89/$03.50 is, 1989 Elsevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland

100

populations for exposure to occupational and environmental toxins (reviewed in 1231). Recently, we have developed HPLC procedures for the extraction of genotoxic components from the urine and have tested these components for chromosome-damaging (clastogenic) activity on CHO fibroblasts as an endpoint, an approach validated with smokers and pesticide workers [4,22].

The objective of this study is to determine whether bacterial infections of the urinary tract would result in the production of agents which cause chromosomal breakage. Paraplegics with long-term indwelling catheters were chosen for study since this group is character- ized by a continuous polymicrobial bladder infection and a 16- to 28-fold elevation in risk for squamous cell carcinoma of the bladder [ 1,14,18,20]. The demonstration of clasto- genie material in the urine of these individuals may provide further clues for the association between bacterial infections and cancer and provide a possible mechanism whereby such infections could lead to an elevation in cancer risk.

Materials and methods

Study group

Urine samples were obtained from 18 patients in the chronic care units of Shaugh- nessy and Pearson Hospitals, Vancouver, B.C. All patients were paraplegics with in- dwelling catheters and with chronic urinary bladder infections. Such infections were usually polymicrobial in nature involving Escherichia co/i and other Enterococci,

Proteus mirabilis, Psuedomonas aeruginosa,

Serratia marscescens and Klebsiella

pneumoniae. Bacterial typing and quantifica- tion of each urine sample were not performed in this initial study. However, significant bac- teriuria was confirmed in all specimens by light microscopy. Since all patients were asympto- matic they received no antibiotics during the sampling period. In addition to the aforementioned CBI patients, 4 patients who presented with ABI at a local family practice

were included in this study. Urine specimens were obtained from these individuals prior to antibiotic therapy. All patients chosen were non-smokers.

Control urine samples for the above study were obtained from 18 non-smoking Vancouver residents with no known bladder infection. For comparison purposes, 11 smokers were also included as a positive control population.

Collection of urine samples

Urine samples were collected over a 6-h period in sterile 500-ml polyethylene bottles (Nalgene, Rochester, NY) without any preser- vatives. Urines were refrigerated until transport to the laboratory where they were centrifuged and the supernatants stored at - 20°C until analysis.

Extraction and measurement of clastogenic

components from urine

Urine samples were thawed, filtered and a creatinine determination performed as described by Iosefsohn [ 131. Creatinine values were used to normalize urine samples for subject to subject differences in urine volumes and body weight. Organic material from urines was concentrated by reversed-phase high- pressure liquid chromatography, lyophilized and reconstituted in double-distilled water as previously described [22]. The urine extracts were diluted to yield a concentration range of 1.0-8.0 mg/ml creatinine equivalence. Clastogenic activity of these urine extracts was assayed in cultured CHO fibroblasts by established methods [4]. A minimum of 100 metaphase plates were analyzed for the presence of chromatid aberrations. Typically, distilled water passed through the HPLC columns and concentrated by the above procedures induced aberrant metaphases in less than 1% of treated cells.

Results

Clastogenic activity was assayed in urine samples collected from 18 controls (non-

101

smokers, no bladder infection) and from 18 patients with CBI. Mean values for urines from these subjects are shown in Table 1. All urine concentrates demonstrated chromosome- damaging activity above background frequencies obtained with medium alone. However, the mean level of breakage in cul- tures exposed to urine samples from CBI patients was significantly elevated (2.9-fold) above that of control subjects. This increase in clastogenic activity was comparable to that observed in a group of smokers (no bladder infection) whose urine was sampled concurrently. The latter group showed a 3.3- fold increase in clastogenic activity compared to controls. Table 1 also presents the data obtained with samples of urine from 4 patients with ABI. The mean level of chromosomal breakage produced by these samples was simi- lar to that observed in urine extracts from indi- viduals with CBI.

Figure 1 shows, for each subject, the urinary

Table 1. Presence of clastogenic activity in the urine of patients with urinary bladder infection.

Study group N % Metaphases P with chromatid

aberrations (mean + S.E.)a

Controls (non-smoking, 18 5.0 f 0.8 no bladder

infection) Smokers, llc 16.3 2 1.7

no bladder

infection) Acute bladder 4 14.2 & 6.6

infection

Chronic bladder 18 14.4 f 2.3

infection

< 0.001

0.025

< 0.001

“Average of the maximum value obtained for each individual over the dose range tested (1.0-8.0 mg/ml creatine equivalence). The frequency of chromatid aber- rations present in untreated CHO cultures was 0.9%.

bsignificance relative to control group (Mann-Whitney U- test)

cAverage of 15 cigarettes per day.

Fig. 1. Clastogenic activity of urine concentrates from control subjects, individuals with acute (ABI) and chronic (CBI) bladder infection. Each bar represents the maxi- mum percentage of aberrant metaphases over the tested dose range (1.0-8.0 mg/ml creatinine equivalence)

clastogenic activity observed among the control group and among patients with bladder infection. Values of aberrant metaphases in cultures exposed to urine samples from the control population ranged from 1 to 11.7%. Frequencies observed in cultures treated with urine samples from infected individuals ranged from 2.2 to 33.3% (4.4-33.3% for ABI and 2.2-33.3% for CBI). These data show that a considerable overlap occurs between the range of activities observed in these 3 populations. However, a greater percentage of individuals in the group with urinary bladder infection had urine extracts with high clastogenic activities (Fig. 2). Fifty-six percent of the individuals with CBI had urine samples which induced chromatid aberrations in more than 10% of metaphases. Only 6% of controls did so. Two of the 4 patients with ABI also had this level of clastogenic activity in their urine.

Discussion

The results of the present study show that, as a group, patients with bacterial infections in the urinary tract have a significant elevation of chromosome-damaging components in their urine. The level of activity observed in these

102

-0 c 40

8 20

0

% Metaphases With Chromatid Aberrations

:“~I 0 5 10 15 20 25 30 35

% Metaphases With Chromatid Aberrations

Fig. 2. Frequency distribution of clastogenic activity of

urine from controls (A) and patients with chronic bladder infection (B).

urine concentrates was similar to that found in smokers’ urine. To our knowledge, clastogenic activity has not been previously reported in the urines of individuals with urinary bladder infections. Everson and co-workers [9] tested urine from individuals with Schistosomal

infections using the Ames Salmonella assay and found no indication of an increase in mutation frequency above that produced by urine samples from controls. However, there was no indication of whether or not these individuals had concurrent bacterial infections. Furthermore, positive results in our study could be attributed to differences in assay systems (test organism and endpoint).

The identity of the clastogenic compo- nent(s) present in urine samples from infected individuals is unknown. By nature, however, they are non-polar and capable of producing damage in CHO cultures without external metabolic activation. There has been some speculation in the literature that nitrosamine production by urinary bladder infections could be involved in carcinogenesis at this site. Bac- terial isolates from such infections can produce nitrosamines in vitro [3]. Furthermore, the urine of both CBI and ABI patients has been shown to contain nitrosamines [6,8,10,12,21]. The hydrophobicity of the iso- lated clastogenic compounds and their direct- acting nature suggest that agents other than nitrosamines may also be present in the urine of infected individuals and that this group of compounds could contribute significantly to the genotoxic activity of such urine samples.

The relevance of the present study on cfas- togenic factors in urine from infected individuals to an assessment of risk for bladder cancer in the general population has yet to be determined. From the standpoint of the concentration of these components in the urine, most extracts are active at concentrations less than double that observed in urine voids. Hence, chromosome damage could be occurring in the bladder mucosa exposed to this urine. Such a possibility could be explored in the future by employing the micronucleus test on exfoliated cells collected from the urinary bladder of infected individuals [22]. However, it should be noted that this study primarily focused on CBI infections. The majority of infections in the general North American population would be acute in nature: short in duration and differing from chronic infections in both the number of bacterial species involved (single for ABI vs. multiple for CBI) and the total quantity of bacteria in the urine. Of the 4 urine samples obtained from outpatients with ABI, two demonstrated significant clastogenic activity. Further work is required to determine whether the active products in these urine extracts are similar to those present in urine voids of

individuals with CBI and to assess the propor- tion of ABI patients which would have such an elevation in activity in their urine.

Acknowledgements

This study was supported by a grant from National Sciences and Research Engineering Council of Canada. The authors would like to thank Ms. L. Hui for preparation of graphics.

References

1

2

3

4

5

6

7

8

9

10

Bors, E. and Comarr. A.E. (1971) Neurological Urology.

Physiology of Micturition, Its Neurological Disorders and

Sequelae, pp. 312-314. University Park Press, Balti- more.

Broecker. B.H., Klein, F.A. and Hackler, R.H. (1981)

Cancer of the bladder in spinal cord injury patients. J. Ural., 125, 196- 197.

Calmels, S., Ohshima, H., Vincent, P.. Gounot, A.-M.

and Bartsch, H. (1985) Screening of microorganisms for nitrosation catalysis at pH 7 and kinetic studies on

nitrosamine formation from secondary amines by E. coli

strains. Carcinogenesis, 6 (6). 911-915.

Curtis, J.R. and Dunn, B.P. (1985) High-pressure liquid

chromatography for isolation of clastogenic agents from

urine. Mutat. Res., 146, 171-177.

Davis, C.P., Cohen, S.M., Groeber, M.B., Anderson,

M.D. and Warren, M.M. (1984) Urothelial hyperplasia and

neoplasia: response to chronic urinary tract infection in

rats. J. Ural.. 132, 1025-1031.

El-Aaser, A., El-Merzabani. M., Higgy, N. and El-Habet,

A. (1982) A study on the etiological factors of bilharzial

bladder cancer in Egypt, 6. The possible role of urinary

bacteria. Tumori, 68, 23-28.

El Boulkany, M.N.. Ghoneium M.A. and Mansour, M.A.

(1972) Carcinoma of the bilharzial bladder in Egypt.

Clinical and pathological features. Br. J. Ural., 44, 561-

570.

Evans, C.M. and Bowen, J.G. (1977) Demonstration of nitrosamines in human urine: preliminary observations on

a possible etiology for bladder cancer in associati,m with

chronic urinary tract infections. Proc. R. Sot. Med., 70, 413-417.

Everson, R.B., Gad-El-Mawia, N.M., Attia, M.A., Chev-

len, E.M., Thorgeirsson, S.S., Alexander, L.A., Flack,

P.M., Staiano, N. and Ziegler, J.L. (1983) Analysis of

human urine for mutagens associated with carcinoma of

the bilharzial bladder by the Ames Salmonella plate assay.

Cancer, 51,371-377.

Hicks, R.M.. Walters, C.L., Elseboi, I., El-Aaser, A.B., El-

Merzanbani, M. and Gouch, T.A. (1977) Demonstration

of nitrosamines in human urine: preliminary observation with chronic urinary tract infections. Proc. R. Sot. Med.,

70,413-416.

11

12

13

14

15

16

17

18

19

20

21

22

23

24

103

Higgy, N.A., Verma, A.K., Erturk, E., Oberly, T.D., El-

Aaser, A.A., El-Merzabani, M.M. and Bryan, G.T. (1987)

Escherichio co/i infection of the urinary bladder: induction

of tumours in rats receiving nitrosamine precursors and

augmentation of bladder carcinogenesis by n-nitrosobutyl

(4.hydroxybutyl)amine. IARC Sci. Pub. (France) 84,

380-383.

Hill, M.G., Hawksworth, G. and Tattersall, G. (1973) Bac-

teria, nitrosamines, and stomach cancer. Br. J. Cancer,

28,562-567.

losefsohn, M. (1982) Creatinine in serum and urine with

Fuller’s earth. In: Selected Methods for the Small Clinical

Laboratory, Vol. 9, pp. 201-205. Editors: W.R. Faulkner and S. Meites, American Association for Clinical

Chemistry, Wash., D.C.,

Jewett, H.J. (1970) Tumors of the bladder. In: Urology,

3rd ed., pp.1003-1142. Editors: M.F. Campbell and

J.H. Harrison. W.B. Saunders C., Philadelphia.

Johansson, S.L. (1987) The influence of urinary tract

infection on the incidence of urinary tract tumors in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide induced

carcinogenesis in male Sprague-Dawley rats. Cancer

Lett., 37,87-98.

Johansson, S.L., Anderstrom, C., von Schultz, L. and

Larsson, P. (1967) Enhancement of N-[4-(S-nitro-2-furyI)-

2-thiazolyl]formamide induced carcinogenesis by urinary

tract infection. Cancer Res., 47,559-562.

Kantor. A.F., Hartege, PI, Hoover, R.N., Narayana, A.S..

Sullivan, J. W. and Fraumeni Jr., J.F. (1984) Urinary tract

infection and risk of bladder cancer. Am. J. Epidemiol., 119,510-515.

Kaufman, J.M., Fam. B, Jacobs, S.C.. Gabilondo, F.,

Yalla, S., Kane, J.P. and Rossier, A.B. (1977) Bladder

cancer and squamous metaplasia in spinal cord injury patients. J. Ural., 118, 967-971.

Locke, J.R., Hill, D.E. and Walzer, Y. (1985) Incidence of

squamous cell carcinoma in patients with long-term

catheter drainage. J. Ural., 133, 1034-1035.

Melzak, J. (1966) The incidence of bladder cancer in

paraplegia. Paraplegia, 4, 85.

Radomski, J.L., Greenwald, D., Hearn, W.L., Block,

N.L. and Woods, F.M. (1978) Nitrosamine formation in bladder infections and its role in the etiology of bladder

cancer. J. Ural., 120.48-50.

San, R.H.C., Rosin, M.P.. See, R.H., Dunn, B.P and Stich, H.F. (1989) Use of urine for monitoring human exposure to genotoxic agents. In: Biological Monitoring for

Pesticide Exposure: Measurement, Estimation, and Risk

Reduction, pp. 98-116. Editors: R.G.M. Wang, C.A. Franklin, R.C. Honeycutt and J.C. Reinert. American

Chemical Society Symposium Series, 382, Wash., D.C.

Venitt, S. (1988) The use of short-term tests for the

detection of genotoxic activity in body fluids and excreta.

Mutat. Res., 205, 331-353. Yamasaki. E. and Ames, B.N. (1977) Concentration of

mutagens from urine by adsorption with the nonpolar resin XAD2: Cigarette smokers have mutagenic urine. Proc.

Natl. Acad. Sci. USA, 74,3555-3559.