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Effect of contrast material on transitional cell carcinoma viability
BASIC SCIENCE
EFFECT OF CONTRAST MATERIAL ON TRANSITIONAL CELL CARCINOMA VIABILITY KELLY A. LINDERT
AND
MARTHA K. TERRIS
ABSTRACT Objectives. To assess the effects of contrast material on the viability of transitional cell carcinoma (TCC) cells and the ability of such cells to attach to a recipient bed, because seeding of TCC into the upper urinary tract is a possibility during retrograde pyelography or percutaneous procedures. Methods. Primary cultures of TCC cells were established and placed in either quarter, half, or full-strength contrast for 10 minutes or one-quarter strength contrast for 10, 30, and 60 minutes. Cells were then removed from the contrast agent, resuspended in urothelium-specific media, and incubated for 5 days, after which the cells were counted. Results. A pronounced decrease in cell viability was observed with increasing exposure time and contrast material concentration. Cells incubated for 10, 30, and 60 minutes with contrast yielded an average of 79%, 60%, and 12% of the control group growth, respectively (P ⬍0.001). Likewise, plates incubated with quarter, half, and full-strength contrast yielded 79%, 27%, and 10% of the control group growth, respectively (P ⬍0.001). The difference in the response of low-grade superficial and high-grade invasive bladder tumors was not statistically significant. Conclusions. TCC cells that have been exposed to dilute contrast material for a short period are able to attach and grow on an adequate recipient bed. However, increasing the contrast concentration and/or the exposure time appears to decrease the viability and adherence of the TCC cells. UROLOGY 56: 876–879, 2000. © 2000, Elsevier Science Inc.
T
he possibility of implantation of transitional cell carcinoma (TCC) cells onto adjacent areas of the urinary epithelium is a concern during endoscopic procedures to treat such tumors.1–3 This concern is supported both by clinical studies that demonstrated a high rate of upper tract tumors in patients with bladder TCC and vesicoureteral reflux4,5 and by laboratory research demonstrating that many synchronous and metachronous tumors are monoclonal.6,7 Some urologists, therefore, advocate avoiding any percutaneous approaches to TCC treatment or concomitant procedures at the time of transurethral bladder tumor resection; Presented at the 75th Annual Meeting of the Western Section of the American Urological Association, Monterey, California, October 3, 1999. From the Department of Urology, Stanford University Medical Center, Stanford and Section of Urology, Veterans Affairs Palo Alto Health Care System, Palo Alto, California Reprint requests: Martha K. Terris, M.D., Section of Urology (112C), Veterans Affairs Palo Alto Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304 Submitted: January 10, 2000, accepted (with revisions): May 26, 2000
876
© 2000, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
some investigators recommend administration of intravesical chemotherapy at the end of the endoscopic procedure to minimize the risk of tumor seeding.2– 4 Arguments against this concern include the potential toxicity of the contrast materials used, which is supported by the well-documented induction of urinary cytologic abnormalities by contrast material.8 –10 Cytologically, urothelial cells show cellular shrinkage, nuclear pyknosis, fragmentation, and occasionally cytoplasmic vacuolization with exposure to contrast material. McClennan et al.8 found that these changes were similar whether this exposure was in vitro or in vivo. No prior studies have investigated the toxic effects of contrast material on TCC cells. This study was designed to evaluate the in vitro effect of contact with contrast material on primary human TCC cells harvested from a variety of tumor grades and stages. MATERIAL AND METHODS Primary cultures of human TCC were established from transurethral resection specimens from three low-grade, su0090-4295/00/$20.00 PII S0090-4295(00)00717-2
perficial bladder tumors and two high-grade invasive bladder tumors. The specific characteristics of the patients and tumors from which these specimens were acquired are shown in Table I. In the operating suite, specimens were transferred immediately into hepes-buffered saline (HBS) composed of HEPES 30 mM, glucose 4 mM, KCl 3 mM, NaCl 130 mM, Na2HPO7 䡠 7H2O, phenol red 䡠 Na salt 0.0033 mM, and gentamicin 100 mg/mL and transported to the tissue culture laboratory. Each specimen was washed with red blood cell lysis buffer (ammonium chloride 8.3 g/L, potassium bicarbonate 1.0 g/L, ethylenediaminetetraacetic acid [EDTA] 1.0 g/L)11 and finely minced and incubated in 0.02% trypsin/0.002% EDTA in HBS for 30 seconds. The trypsin digestion was terminated by addition of HBS containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, Utah). Specimens were then vigorously pipetted to yield a single-cell suspension, centrifuged for 5 minutes at 5000g, and resuspended in urothelium-specific media consisting of Ham’s F-12 medium (Gibco, Grand Island, NY) supplemented with L-aspartate 2.66 g/L, L-glutamate 2.94 g/L, L-alanine 1.78 g/L, glycine 150 g/L, L-proline 1.15 g/L, L-serine 1.05 g/L, L-asparagine 䡠 H2O 1.50 g/L, Lglutamine 2.92 g/L, D-glucose 6.48 g/L, phosphoethanolamine 0.1 mM, hydrocortisone 1 mg/mL, selenium 5 ⫻ 10⫺8 M, transferrin 5 mg/mL, insulin 10 mg/mL, calcium 22 mg/mL, gentamicin 100 mg/mL, and 1% FBS.12 Cells were incubated on 60-mm dishes previously coated with 0.2 mL of a solution containing 1 mL Vitrogen 100 purified type 1 collagen (Collagen, Menlo Park, Calif) suspended in 4 mL 0.013N HCl at 37°C in 95% air/5% carbon dioxide. Initially, the cells were plated in 2 mL of modified Ham’s F-12, 1% FBS medium to facilitate cell attachment to the collagen coating. An additional 3 mL was added after 2 days. The cultures were allowed to proliferate for 7 to 10 days with media changes every 2 days until near-confluent growth was attained. The cells were detached from the dishes by incubation with 0.02% trypsin/0.002% EDTA in HBS for 1 minute. The trypsin digestion was terminated by addition of HBS containing 10% FBS. Cells were centrifuged for 5 minutes at 5000g, counted, and then divided into aliquots and placed in either full, half, or quarter-strength of a 43% solution of meglumine iothalamate (Conray, Mallinckrodt Medical, St. Louis, Mo) for 10 minutes (to reflect our estimate of the usual amount of time cells are exposed to contrast during retrograde pyelography at our institution) at 6 ⫻ 105 cells/mL. A control group was incubated in HBS alone at 6 ⫻ 105 cells/mL. Cells were incubated in quarter-strength meglumine iothalamate (to reflect the usual concentration of contrast used at our institution for retrograde pyelography) for 10, 30, and 60 minutes. After incubation for the allotted period, cells were centrifuged for 5 minutes at 5000g to remove the contrast agent and resuspended in modified Ham’s F-12 media with 1% FBS. The cells were plated on 60-mm collagen-coated culture dishes at 6 ⫻ 105 cells/dish and incubated at 37°C in 5% carbon dioxide/95% air. Culture media was changed every 48 hours. During the initial media change, the nonadherent cells floating in the media being removed were collected and tested for viability using the trypan blue exclusion test.13 Nonadherent cells from each dish were suspended separately in serumfree medium at a concentration of 1 ⫻ 105 cells/mL. To a 5-mL aliquot of this suspension, 0.1 mL of 0.5% trypan blue stain was added and mixed thoroughly. The solution was allowed to stand for 5 minutes at room temperature, then the total number of cells and the number of stained (nonviable) cells were counted using a hemocytometer, and the percentage of nonviable cells was calculated. The mean number of unattached cells in each experimental group was expressed as a percentage of the mean number of unattached cells observed in the control dishes. After 5 days, the cells were detached from the plates by UROLOGY 56 (5), 2000
digestion with 0.02% trypsin/0.002% EDTA and counted. All experiments were repeated in quadruplicate. The Student t test was used to test for statistical significance using the raw data of cell counts from all four dishes for each experiment. The mean cell counts (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean cell counts observed in the control dishes.
RESULTS A pronounced decrease in cell growth occurred after incubation with contrast material with both increasing exposure time and increasing concentration (Table I). The difference between the response of the cells from three grade 2, superficial TCCs and the high-grade, invasive TCCs was not statistically significant. The TCC cells incubated for 10, 30, or 60 minutes with contrast material (at one-quarter strength) yielded an average of 79%, 60%, and 12% of the control group growth, respectively (Table I). The decrease in cell counts with increasing exposure time was statistically significant (P ⬍0.001). Cells incubated for 10 minutes with quarter, half, and full-strength media yielded 79%, 27%, and 10% of the control group growth, respectively (Table I). The decrease in cell counts with increasing concentration was also statistically significant (P ⬍0.001). The number of nonadherent cells increased significantly with increasing contrast concentrations and exposure times (Table I). Of these nonadherent cells, 87% of those in control media were nonviable, and the percentage of nonviable cells increased with increasing contrast concentrations and exposure times, although this difference did not reach statistical significance. The TCC cells incubated for 10, 30, or 60 minutes with contrast material yielded an average of 108%, 131%, and 162% of the control group nonadherent cells, respectively (P ⬍0.001). Of these nonadherent cells, 91%, 94%, and 97% were nonviable in the 10, 30, and 60-minute exposure groups, respectively. Cells incubated with quarter, half, and fullstrength media yielded 108%, 121%, and 153% of the control group nonadherent cells, respectively (P ⬍0.001). Of these nonadherent cells, 91%, 93%, and 95% were nonviable in the quarter, half, and full-strength media, respectively. COMMENT In this study, increasing the contrast concentration and/or exposure time decreased the growth and adherence of TCC cells using the high osmolar ionic contrast medium, meglumine iothalamate. Similarly, several investigators have shown that the toxicity of contrast material to vascular endothelial cells in culture is related to the concentration and time of exposure; this toxicity is minimized if non877
153 121 108 * Mean cell counts (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean cell count observed in the control dishes. † Mean numbers of unattached cells (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean number of unattached cells observed in the control dishes.
10 27 79 162 131 108 60 79
61 5
67.6
72 4
Mean
62 3
Male
75 2
Male
68 1
Male
Patient Age (yr) Tissue Culture Number
Male
12
164 124 117 18 26 72 169 142 117 20 58 72 T3 4
180 116 98 0 20 78 139 118 98 15 62 78 T2 3
146 129 105 5 33 87 146 122 105 8 55 87 T1 2
146 123 112 10 27 81 170 138 112 10 63 81 T1 2
131 112 110 15 30 75 187 134 110 0 61 75 T1
Transurethral resection Transurethral resection Transurethral resection Transurethral resection Radical cystectomy Male
2
Full Strength 0.5 Strength 0.25 Strength Full Strength 0.5 Strength 0.25 Strength 60 Min 30 Min 10 Min 60 Min 30 Min Harvest Procedure Patient Sex
Tumor Grade
Tumor Stage
10 Min
Control Nonadherent Cells† (%) Source of Tissue Culture
Response to Contrast Concentration
Control Cell Growth* (%) Control Nonadherent Cells† (%) Control Cell Growth* (%)
Response to Contrast Exposure Time
TABLE I. Specimen characteristics and response to contrast concentration and exposure time 878
ionic, low osmolar contrast is used.14,15 Identical trends of increasing toxicity with increasing osmolality have been documented with kidney cell lines,14 –17 vascular smooth muscle cells,18 myocardial cells,19,20 neural cells,21,22 and human fibroblasts.23 The toxicity of contrast material in malignant cells (human cervical carcinoma cell lines and prostate cancer cell lines) has also been documented.24 –26 Factors other than direct cellular toxicity may also affect the ability of malignant transitional cells that have been transported with contrast material to implant on other sites in the collecting system. Zhan et al.27 have evaluated neutrophil adhesion to contrast-exposed vascular endothelial cells. They observed increasing cellular adhesion with decreasing contrast concentration. This increased adhesion of the cells was thought not to be related to osmolality, since the adhesion did not increase with decreasing concentrations of noncontrast solutions with similar osmolality. The investigators believed that the contrast material, therefore, caused some form of modulation of the cell-adhesion molecules. This theory is supported by the research of Owens et al.15 and Rasmussen et al.,28 who found decreased adherence of blood components to the endothelium with contrast exposure. In our study, TCC cultures were placed on a collagen substrate to encourage adherence. The number of nonadherent cells increased with increasing contrast concentrations and exposure times, suggesting an effect of the contrast on cellular adhesion as well (Table I). Potier et al.23 found that rat mesangial cells were more sensitive to the cytotoxic effects of contrast agents than the less-differentiated human fibroblasts. This might suggest that the more poorly differentiated TCC cells would not be as sensitive to contrast material as the low-grade tumors. We did not observe any differences in the response of the high versus low-grade TCC tumors to exposure to contrast material; however, the number of tumors tested was too small to confirm this finding statistically. Future studies evaluating larger numbers of tumors, a variety of other contrast agents, and tumor cell implantation in vivo after contrast exposure may further improve our understanding of the risk of tumor seeding during contrast studies. CONCLUSIONS Intravenous contrast administration can lead to multiple adverse reactions, such as cardiotoxicity, nephrotoxicity, and hemodynamic toxicity, making nonionic, low-osmolality agents preferable to ionic, high-osmolality agents.29 However, the direct introduction of material into the urinary colUROLOGY 56 (5), 2000
lecting system for the evaluation of TCC may be less prone to seeding of viable malignant cells if ionic, high-osmolar contrast agents, such as meglumine iothalamate, are used. Since cytologic abnormalities are worsened by exposure to ionic, high-osmolar contrast materials, any specimens for cytologic analysis should be collected before the use of any contrast agents.30 REFERENCES 1. Pode D, Horowitz AT, Vlodavsky I, et al: Prevention of human bladder tumor cell implantation in an in vitro assay. J Urol 137: 777–781, 1987. 2. Soloway MS, and Masters S: Urothelial susceptibility to tumor cell implantation: influence of cauterization. Cancer 46: 1158 –1163, 1980. 3. Solomon LZ, Jennings AM, Foley SJ, et al: Bladder cancer recurrence by implantation of exfoliated cells: is gammalinolenic acid an effective tumoricidal agent? Br J Urol 82: 122–126, 1998. 4. De Torres Mateos JA, Banus Gassol JM, Palou Redorta J, et al: Vesicorenal reflux and upper urinary tract transitional cell carcinoma after transurethral resection of recurrent superficial bladder carcinoma. J Urol 138: 49 –51, 1987. 5. Amar AD, and Das S: Upper urinary tract transitional cell carcinoma in patients with bladder carcinoma and associated vesicoureteral reflux. J Urol 133: 468 – 471, 1985. 6. Li M, and Cannizzaro LA: Identical clonal origin of synchronous and metachronous low-grade, noninvasive papillary transitional cell carcinomas of the urinary tract. Hum Pathol 30: 1197–1200, 1999. 7. Fadl-Elmula I, Gorunova L, Mandahl N, et al: Cytogenetic monoclonality in multifocal uroepithelial carcinomas: evidence of intraluminal tumour seeding. Br J Cancer 81: 6 –12, 1999. 8. McClennan BL, Oertel YC, Malmgren RA, et al: The effect of water soluble contrast material on urine cytology. Acta Cytol 22: 230 –233, 1978. 9. Andriole GL, McClennan BL, Becich M, et al: Effect of low osmolar, ionic and nonionic, contrast media on the cytologic features of exfoliated urothelial cells. Urol Radiol 11: 133–135, 1989. 10. Fisher S, Nielsen ML, Clausen S, et al: Increased abnormal urothelial cells in voided urine following excretory urography. Acta Cytol 26: 153–158, 1982. 11. Liedl T: Flow cytometric DNA/cytokeratin analysis of bladder lavage: methodical aspects and clinical implications. Urol Int 54: 22– 47, 1995. 12. Loretz LJ, and Reznikoff CA: Clonal growth of normal human uroepithelial cells. In Vitro Cell Dev Biol 24: 333–342, 1988.
UROLOGY 56 (5), 2000
13. Freshney R: Culture of Animal Cells: A Manual of Basic Technique. New York, Alan R. Liss, 1987, p 117. 14. Dascalu A, and Peer A: Effects of radiologic contrast media on human endothelial and kidney cell lines: intracellular pH and cytotoxicity. Acad Radiol 1: 145–150, 1994. 15. Owens MR, Ribes JA, Marder VJ, et al: Effects of ionic and nonionic radiographic contrast agents on endothelial cells in vitro. J Lab Clin Med 119: 315–319, 1992. 16. Andersen KJ, Christensen EI, and Vik H: Effects of iodinated x-ray contrast media on renal epithelial cells in culture. Invest Radiol 29: 955–962, 1994. 17. Schick CS, and Haller C: Comparative cytotoxicity of ionic and non-ionic radiocontrast agents on MDCK cell monolayers in vitro. Nephrol Dial Transplant 14: 342–347, 1999. 18. Wang YX, Chan P, and Morcos SK: The effect of radiographic contrast media on human vascular smooth muscle cells. Br J Radiol 71: 376 –380, 1998. 19. Davis WL, Nelson JA, and Barry WH: Effects of contrast media on cultured myocardial cells. Invest Radiol 21: 626 – 630, 1986. 20. Chuck LH, Refsum H, Rouleau JL, et al: Direct pharmacologic and osmolal effects of contrast media on the mechanics of heart muscle isolated from cats. Am Heart J 108: 97–104, 1984. 21. Kormano M, and Frey H: Toxicity of X-ray contrast media in cell cultures. Invest Radiol 15: 68 –71, 1980. 22. Kormano M, and Hervonen H: Use of tissue culture to examine neurotoxicity of contrast media. Radiology 120: 727– 729, 1976. 23. Potier M, Lagroye I, Lakhdar B, et al: Comparative cytotoxicity of low- and high-osmolar contrast media to human fibroblasts and rat mesangial cells in culture. Invest Radiol 32: 621– 626, 1997. 24. Nordby A, Tvedt KE, Halgunset J, et al: Incorporation of contrast media in cultured cells. Invest Radiol 24: 703–710, 1989. 25. Nordby A, Halgunset J, Thorstensen K, et al: Short-term effects of radiographic contrast media on monolayer cell cultures and hepatocytes. Invest Radiol 22: 603– 607, 1987. 26. Nordby A, Halgunset J, and Haugen OA: Effects of radiographic contrast media on monolayer cell cultures. Invest Radiol 21: 234 –239, 1986. 27. Zhan X, Agrawal DK, and Thorpe PE: Effect of iodinated contrast media on neutrophil adhesion to cultured endothelial cells. J Vasc Interv Radiol 9: 808 – 816, 1998. 28. Rasmussen F, Antonsen S, and Georgsen J: Granulocyte adherence is inhibited by radiographic contrast media in vitro. Acta Radiol 33: 379 –383, 1992. 29. Dawson P: Chemotoxicity of contrast media and clinical adverse effects: a review. Invest Radiol 20: 84 –91, 1985. 30. Carter HB: Instrumentation and endoscopy, in Walsh PC, Retik AB, Vaughan ED, et al (Eds): Campbell’s Urology. Philadelphia, WB Saunders, 1998, pp 167–168.
879
EFFECT OF CONTRAST MATERIAL ON TRANSITIONAL CELL CARCINOMA VIABILITY KELLY A. LINDERT
AND
MARTHA K. TERRIS
ABSTRACT Objectives. To assess the effects of contrast material on the viability of transitional cell carcinoma (TCC) cells and the ability of such cells to attach to a recipient bed, because seeding of TCC into the upper urinary tract is a possibility during retrograde pyelography or percutaneous procedures. Methods. Primary cultures of TCC cells were established and placed in either quarter, half, or full-strength contrast for 10 minutes or one-quarter strength contrast for 10, 30, and 60 minutes. Cells were then removed from the contrast agent, resuspended in urothelium-specific media, and incubated for 5 days, after which the cells were counted. Results. A pronounced decrease in cell viability was observed with increasing exposure time and contrast material concentration. Cells incubated for 10, 30, and 60 minutes with contrast yielded an average of 79%, 60%, and 12% of the control group growth, respectively (P ⬍0.001). Likewise, plates incubated with quarter, half, and full-strength contrast yielded 79%, 27%, and 10% of the control group growth, respectively (P ⬍0.001). The difference in the response of low-grade superficial and high-grade invasive bladder tumors was not statistically significant. Conclusions. TCC cells that have been exposed to dilute contrast material for a short period are able to attach and grow on an adequate recipient bed. However, increasing the contrast concentration and/or the exposure time appears to decrease the viability and adherence of the TCC cells. UROLOGY 56: 876–879, 2000. © 2000, Elsevier Science Inc.
T
he possibility of implantation of transitional cell carcinoma (TCC) cells onto adjacent areas of the urinary epithelium is a concern during endoscopic procedures to treat such tumors.1–3 This concern is supported both by clinical studies that demonstrated a high rate of upper tract tumors in patients with bladder TCC and vesicoureteral reflux4,5 and by laboratory research demonstrating that many synchronous and metachronous tumors are monoclonal.6,7 Some urologists, therefore, advocate avoiding any percutaneous approaches to TCC treatment or concomitant procedures at the time of transurethral bladder tumor resection; Presented at the 75th Annual Meeting of the Western Section of the American Urological Association, Monterey, California, October 3, 1999. From the Department of Urology, Stanford University Medical Center, Stanford and Section of Urology, Veterans Affairs Palo Alto Health Care System, Palo Alto, California Reprint requests: Martha K. Terris, M.D., Section of Urology (112C), Veterans Affairs Palo Alto Medical Center, 3801 Miranda Avenue, Palo Alto, CA 94304 Submitted: January 10, 2000, accepted (with revisions): May 26, 2000
876
© 2000, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
some investigators recommend administration of intravesical chemotherapy at the end of the endoscopic procedure to minimize the risk of tumor seeding.2– 4 Arguments against this concern include the potential toxicity of the contrast materials used, which is supported by the well-documented induction of urinary cytologic abnormalities by contrast material.8 –10 Cytologically, urothelial cells show cellular shrinkage, nuclear pyknosis, fragmentation, and occasionally cytoplasmic vacuolization with exposure to contrast material. McClennan et al.8 found that these changes were similar whether this exposure was in vitro or in vivo. No prior studies have investigated the toxic effects of contrast material on TCC cells. This study was designed to evaluate the in vitro effect of contact with contrast material on primary human TCC cells harvested from a variety of tumor grades and stages. MATERIAL AND METHODS Primary cultures of human TCC were established from transurethral resection specimens from three low-grade, su0090-4295/00/$20.00 PII S0090-4295(00)00717-2
perficial bladder tumors and two high-grade invasive bladder tumors. The specific characteristics of the patients and tumors from which these specimens were acquired are shown in Table I. In the operating suite, specimens were transferred immediately into hepes-buffered saline (HBS) composed of HEPES 30 mM, glucose 4 mM, KCl 3 mM, NaCl 130 mM, Na2HPO7 䡠 7H2O, phenol red 䡠 Na salt 0.0033 mM, and gentamicin 100 mg/mL and transported to the tissue culture laboratory. Each specimen was washed with red blood cell lysis buffer (ammonium chloride 8.3 g/L, potassium bicarbonate 1.0 g/L, ethylenediaminetetraacetic acid [EDTA] 1.0 g/L)11 and finely minced and incubated in 0.02% trypsin/0.002% EDTA in HBS for 30 seconds. The trypsin digestion was terminated by addition of HBS containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, Utah). Specimens were then vigorously pipetted to yield a single-cell suspension, centrifuged for 5 minutes at 5000g, and resuspended in urothelium-specific media consisting of Ham’s F-12 medium (Gibco, Grand Island, NY) supplemented with L-aspartate 2.66 g/L, L-glutamate 2.94 g/L, L-alanine 1.78 g/L, glycine 150 g/L, L-proline 1.15 g/L, L-serine 1.05 g/L, L-asparagine 䡠 H2O 1.50 g/L, Lglutamine 2.92 g/L, D-glucose 6.48 g/L, phosphoethanolamine 0.1 mM, hydrocortisone 1 mg/mL, selenium 5 ⫻ 10⫺8 M, transferrin 5 mg/mL, insulin 10 mg/mL, calcium 22 mg/mL, gentamicin 100 mg/mL, and 1% FBS.12 Cells were incubated on 60-mm dishes previously coated with 0.2 mL of a solution containing 1 mL Vitrogen 100 purified type 1 collagen (Collagen, Menlo Park, Calif) suspended in 4 mL 0.013N HCl at 37°C in 95% air/5% carbon dioxide. Initially, the cells were plated in 2 mL of modified Ham’s F-12, 1% FBS medium to facilitate cell attachment to the collagen coating. An additional 3 mL was added after 2 days. The cultures were allowed to proliferate for 7 to 10 days with media changes every 2 days until near-confluent growth was attained. The cells were detached from the dishes by incubation with 0.02% trypsin/0.002% EDTA in HBS for 1 minute. The trypsin digestion was terminated by addition of HBS containing 10% FBS. Cells were centrifuged for 5 minutes at 5000g, counted, and then divided into aliquots and placed in either full, half, or quarter-strength of a 43% solution of meglumine iothalamate (Conray, Mallinckrodt Medical, St. Louis, Mo) for 10 minutes (to reflect our estimate of the usual amount of time cells are exposed to contrast during retrograde pyelography at our institution) at 6 ⫻ 105 cells/mL. A control group was incubated in HBS alone at 6 ⫻ 105 cells/mL. Cells were incubated in quarter-strength meglumine iothalamate (to reflect the usual concentration of contrast used at our institution for retrograde pyelography) for 10, 30, and 60 minutes. After incubation for the allotted period, cells were centrifuged for 5 minutes at 5000g to remove the contrast agent and resuspended in modified Ham’s F-12 media with 1% FBS. The cells were plated on 60-mm collagen-coated culture dishes at 6 ⫻ 105 cells/dish and incubated at 37°C in 5% carbon dioxide/95% air. Culture media was changed every 48 hours. During the initial media change, the nonadherent cells floating in the media being removed were collected and tested for viability using the trypan blue exclusion test.13 Nonadherent cells from each dish were suspended separately in serumfree medium at a concentration of 1 ⫻ 105 cells/mL. To a 5-mL aliquot of this suspension, 0.1 mL of 0.5% trypan blue stain was added and mixed thoroughly. The solution was allowed to stand for 5 minutes at room temperature, then the total number of cells and the number of stained (nonviable) cells were counted using a hemocytometer, and the percentage of nonviable cells was calculated. The mean number of unattached cells in each experimental group was expressed as a percentage of the mean number of unattached cells observed in the control dishes. After 5 days, the cells were detached from the plates by UROLOGY 56 (5), 2000
digestion with 0.02% trypsin/0.002% EDTA and counted. All experiments were repeated in quadruplicate. The Student t test was used to test for statistical significance using the raw data of cell counts from all four dishes for each experiment. The mean cell counts (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean cell counts observed in the control dishes.
RESULTS A pronounced decrease in cell growth occurred after incubation with contrast material with both increasing exposure time and increasing concentration (Table I). The difference between the response of the cells from three grade 2, superficial TCCs and the high-grade, invasive TCCs was not statistically significant. The TCC cells incubated for 10, 30, or 60 minutes with contrast material (at one-quarter strength) yielded an average of 79%, 60%, and 12% of the control group growth, respectively (Table I). The decrease in cell counts with increasing exposure time was statistically significant (P ⬍0.001). Cells incubated for 10 minutes with quarter, half, and full-strength media yielded 79%, 27%, and 10% of the control group growth, respectively (Table I). The decrease in cell counts with increasing concentration was also statistically significant (P ⬍0.001). The number of nonadherent cells increased significantly with increasing contrast concentrations and exposure times (Table I). Of these nonadherent cells, 87% of those in control media were nonviable, and the percentage of nonviable cells increased with increasing contrast concentrations and exposure times, although this difference did not reach statistical significance. The TCC cells incubated for 10, 30, or 60 minutes with contrast material yielded an average of 108%, 131%, and 162% of the control group nonadherent cells, respectively (P ⬍0.001). Of these nonadherent cells, 91%, 94%, and 97% were nonviable in the 10, 30, and 60-minute exposure groups, respectively. Cells incubated with quarter, half, and fullstrength media yielded 108%, 121%, and 153% of the control group nonadherent cells, respectively (P ⬍0.001). Of these nonadherent cells, 91%, 93%, and 95% were nonviable in the quarter, half, and full-strength media, respectively. COMMENT In this study, increasing the contrast concentration and/or exposure time decreased the growth and adherence of TCC cells using the high osmolar ionic contrast medium, meglumine iothalamate. Similarly, several investigators have shown that the toxicity of contrast material to vascular endothelial cells in culture is related to the concentration and time of exposure; this toxicity is minimized if non877
153 121 108 * Mean cell counts (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean cell count observed in the control dishes. † Mean numbers of unattached cells (of the four dishes) for each experiment were calculated and expressed as a percentage of the mean number of unattached cells observed in the control dishes.
10 27 79 162 131 108 60 79
61 5
67.6
72 4
Mean
62 3
Male
75 2
Male
68 1
Male
Patient Age (yr) Tissue Culture Number
Male
12
164 124 117 18 26 72 169 142 117 20 58 72 T3 4
180 116 98 0 20 78 139 118 98 15 62 78 T2 3
146 129 105 5 33 87 146 122 105 8 55 87 T1 2
146 123 112 10 27 81 170 138 112 10 63 81 T1 2
131 112 110 15 30 75 187 134 110 0 61 75 T1
Transurethral resection Transurethral resection Transurethral resection Transurethral resection Radical cystectomy Male
2
Full Strength 0.5 Strength 0.25 Strength Full Strength 0.5 Strength 0.25 Strength 60 Min 30 Min 10 Min 60 Min 30 Min Harvest Procedure Patient Sex
Tumor Grade
Tumor Stage
10 Min
Control Nonadherent Cells† (%) Source of Tissue Culture
Response to Contrast Concentration
Control Cell Growth* (%) Control Nonadherent Cells† (%) Control Cell Growth* (%)
Response to Contrast Exposure Time
TABLE I. Specimen characteristics and response to contrast concentration and exposure time 878
ionic, low osmolar contrast is used.14,15 Identical trends of increasing toxicity with increasing osmolality have been documented with kidney cell lines,14 –17 vascular smooth muscle cells,18 myocardial cells,19,20 neural cells,21,22 and human fibroblasts.23 The toxicity of contrast material in malignant cells (human cervical carcinoma cell lines and prostate cancer cell lines) has also been documented.24 –26 Factors other than direct cellular toxicity may also affect the ability of malignant transitional cells that have been transported with contrast material to implant on other sites in the collecting system. Zhan et al.27 have evaluated neutrophil adhesion to contrast-exposed vascular endothelial cells. They observed increasing cellular adhesion with decreasing contrast concentration. This increased adhesion of the cells was thought not to be related to osmolality, since the adhesion did not increase with decreasing concentrations of noncontrast solutions with similar osmolality. The investigators believed that the contrast material, therefore, caused some form of modulation of the cell-adhesion molecules. This theory is supported by the research of Owens et al.15 and Rasmussen et al.,28 who found decreased adherence of blood components to the endothelium with contrast exposure. In our study, TCC cultures were placed on a collagen substrate to encourage adherence. The number of nonadherent cells increased with increasing contrast concentrations and exposure times, suggesting an effect of the contrast on cellular adhesion as well (Table I). Potier et al.23 found that rat mesangial cells were more sensitive to the cytotoxic effects of contrast agents than the less-differentiated human fibroblasts. This might suggest that the more poorly differentiated TCC cells would not be as sensitive to contrast material as the low-grade tumors. We did not observe any differences in the response of the high versus low-grade TCC tumors to exposure to contrast material; however, the number of tumors tested was too small to confirm this finding statistically. Future studies evaluating larger numbers of tumors, a variety of other contrast agents, and tumor cell implantation in vivo after contrast exposure may further improve our understanding of the risk of tumor seeding during contrast studies. CONCLUSIONS Intravenous contrast administration can lead to multiple adverse reactions, such as cardiotoxicity, nephrotoxicity, and hemodynamic toxicity, making nonionic, low-osmolality agents preferable to ionic, high-osmolality agents.29 However, the direct introduction of material into the urinary colUROLOGY 56 (5), 2000
lecting system for the evaluation of TCC may be less prone to seeding of viable malignant cells if ionic, high-osmolar contrast agents, such as meglumine iothalamate, are used. Since cytologic abnormalities are worsened by exposure to ionic, high-osmolar contrast materials, any specimens for cytologic analysis should be collected before the use of any contrast agents.30 REFERENCES 1. Pode D, Horowitz AT, Vlodavsky I, et al: Prevention of human bladder tumor cell implantation in an in vitro assay. J Urol 137: 777–781, 1987. 2. Soloway MS, and Masters S: Urothelial susceptibility to tumor cell implantation: influence of cauterization. Cancer 46: 1158 –1163, 1980. 3. Solomon LZ, Jennings AM, Foley SJ, et al: Bladder cancer recurrence by implantation of exfoliated cells: is gammalinolenic acid an effective tumoricidal agent? Br J Urol 82: 122–126, 1998. 4. De Torres Mateos JA, Banus Gassol JM, Palou Redorta J, et al: Vesicorenal reflux and upper urinary tract transitional cell carcinoma after transurethral resection of recurrent superficial bladder carcinoma. J Urol 138: 49 –51, 1987. 5. Amar AD, and Das S: Upper urinary tract transitional cell carcinoma in patients with bladder carcinoma and associated vesicoureteral reflux. J Urol 133: 468 – 471, 1985. 6. Li M, and Cannizzaro LA: Identical clonal origin of synchronous and metachronous low-grade, noninvasive papillary transitional cell carcinomas of the urinary tract. Hum Pathol 30: 1197–1200, 1999. 7. Fadl-Elmula I, Gorunova L, Mandahl N, et al: Cytogenetic monoclonality in multifocal uroepithelial carcinomas: evidence of intraluminal tumour seeding. Br J Cancer 81: 6 –12, 1999. 8. McClennan BL, Oertel YC, Malmgren RA, et al: The effect of water soluble contrast material on urine cytology. Acta Cytol 22: 230 –233, 1978. 9. Andriole GL, McClennan BL, Becich M, et al: Effect of low osmolar, ionic and nonionic, contrast media on the cytologic features of exfoliated urothelial cells. Urol Radiol 11: 133–135, 1989. 10. Fisher S, Nielsen ML, Clausen S, et al: Increased abnormal urothelial cells in voided urine following excretory urography. Acta Cytol 26: 153–158, 1982. 11. Liedl T: Flow cytometric DNA/cytokeratin analysis of bladder lavage: methodical aspects and clinical implications. Urol Int 54: 22– 47, 1995. 12. Loretz LJ, and Reznikoff CA: Clonal growth of normal human uroepithelial cells. In Vitro Cell Dev Biol 24: 333–342, 1988.
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