Department of Pathology and Microbiology and the Eppley Institute, University of Nebraska Medical Center, 983135 Nebraska Medical Center, Omaha, Nebraska 68198-3135
Received July 31, 2000; accepted October 30, 2000
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ABSTRACT |
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Key Words: ortho-phenylphenol; sodium ortho-phenylphenate; urothelial effects; rats..
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INTRODUCTION |
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NaOPP enhanced bladder carcinogenesis when administered to male rats after a brief exposure to the bladder-specific carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in the drinking water, but OPP did not (Fukushima et al., 1983). The reason for a lack of effect of OPP following BBN treatment is unclear, especially considering the positive carcinogenicity of OPP and NaOPP in standard bioassays without prior BBN dosing. Comparing effects in different species, increased urothelial tumors were found in male rats fed 2.0% NaOPP, but no tumors were found in guinea pigs, hamsters, or mice (Fukushima et al., 1985
; Hagiwara et al., 1984
; Hasegawa et al., 1990
). This evidence suggests that the urothelial effects of OPP and NaOPP are a high-dose, species-specific phenomenon, with NaOPP having an apparently greater effect than OPP, and the effect was much greater in male than in female rats. However, as the mechanism for the urothelial effects of OPP and NaOPP in the rat remains unclear, the potential risk to humans is uncertain.
High dietary doses of the sodium salts of ascorbate, aspartate, glutamate, citrate, erythorbate, bicarbonate, and saccharin produce urothelial toxicity, regenerative hyperplasia, and enhanced tumorigenic effects in male rats (Cohen et al., 1995a; Cohen, 1998
, 1999
). A cytotoxic, calcium phosphate-containing amorphous precipitate forms in the urine of rats treated with these sodium salts and is associated with an urinary pH
6.5 (Cohen, 1999
; Cohen et al., 2000
). No toxic or proliferative effects were observed with the acid forms of those sodium salts that have been tested (Cohen, 1999
; Cohen et al., 1991
, 1995b
). Coadministration of these sodium salts with ammonium chloride produces an acidic (pH < 6.0) urine and prevents formation of the precipitate and prevents induction of the urothelial toxicity and proliferative effect.
Like these other sodium salts, NaOPP produces urothelial hyperplasia and tumors, and its effects on the rat urothelium appear to be greater than those seen following treatment with equimolar doses of OPP, although they were tested at different times in separate experiments (Fukushima et al., 1983; Hiraga and Fujii, 1981
, 1984
). Feeding NaOPP produces a significantly higher urinary pH than that produced by OPP, and urinary acidification by coadministration of ammonium chloride decreases the tumorigenic effect of NaOPP, as it does with other sodium salts. In contrast to these other sodium salts, however, the parent acid, OPP, is also carcinogenic in rats and produces toxic and proliferative urothelial effects at doses of 0.4% and higher of the diet (Smith et al., 1998
; Wahle et al., 1997
). Examination of the urine from OPP-treated rats in previous experiments has shown no evidence of the formation of an amorphous precipitate, calculi, or abnormal microcrystalluria (Smith et al., 1998
).
The current study was performed to compare the cytotoxic and proliferative effects produced by NaOPP and OPP in male F344 rats and to determine if formation of an amorphous precipitate in the urine occurs following NaOPP feeding, which could accentuate the effects produced by OPP. This study included evaluation of urinary and urothelial parameters.
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MATERIALS AND METHODS |
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Animals/husbandry.
Thirty male Fischer F344 rats from Charles River Breeding Laboratories (Raleigh, NC), approximately 45 weeks of age, were quarantined for 1 week prior to study initiation. Animals were maintained in a level 4 barrier facility accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). The level of care provided to the animals met or exceeded the basic requirements outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23, revised 1996). Five rats were housed per polycarbonate cage (Lab Products, Maywood, NJ) with corncob bedding (Anderson, Inc., Maumee, OH). The room was maintained at a targeted temperature of 21 ± 2°C, a relative humidity of 50 ± 20% and with a 12-h. light/dark (0600 h/ 1800 h) cycle. Food and tap water were available ad libitum throughout the experiment.
Parameters/treatment.
Animals were randomized into three groups of 10 rats each using a weight stratification method (Martin et al., 1984) and were identified with a Monel self-piercing ear tag. Food and water consumption was determined over a 7-day interval for each cage of five rats beginning on days 14 and 49 of the experiment by weighing the food and water present at the beginning and end of the 7-day evaluation period. Body weights were determined on study days 0, 21, 56, and at termination on day 71.
Urine collection and analyses.
Fresh-void urine was collected from individual rats between 0700 and 0900 h during weeks 1, 5, and 9 directly into 1.5-mlplastic conical tubes and analyzed for pH using a microelectrode (Microelectrodes, Inc., Londonderry, NH; Fisher et al., 1989). A 100-µl aliquot of each specimen was centrifuged at 7000 rpm for 10 min. The supernatant was removed and analyzed for calcium (Sigma Diagnostics, St. Louis, MO) using a colorimetric micro method (Moorehead and Briggs, 1974). Samples were read at a dual wavelength of 570/650. The pellet was vacuum dried and processed for analysis of urinary solids using a Phillips 515 SEM (Phillips, Inc., Eindhoven, The Netherlands) with an attached Kevex Micro-X 7000 X-ray energy dispersive spectrometer (Kevex Inc., Haywood, CA) at 20 kev.
Necropsy and tissue processing.
Animals were sacrificed after 10 weeks of treatment, with an overdose ip injection of 50 mg/kg Nembutal® (Abbott Laboratories, North Chicago, IL). One hour prior to sacrifice, all animals received an ip injection of 100 mg/kg of bromodeoxyuridine (BrdU) (Sigma Chemical, St. Louis, MO). At necropsy the bladders and stomachs were inflated in situ with Bouin's fixative, removed, and placed in the same fixative. After fixation the bladders were rinsed in 70% ethanol and bisected sagittally. One-half of the bladder was processed for SEM and classified according to Cohen et al. (1990). Briefly, Class 1 bladders show flat, polygonal, superficial urothelial cells; Class 2 bladders show occasional small foci of urothelial necrosis; Class 3 bladders show numerous small foci of superficial urothelial necrosis; Class 4 bladders show extensive superficial urothelial necrosis, especially in the dome of bladder; and Class 5 bladders show necrosis and also piling up of rounded urothelial cells. Normal bladders are usually Class 1 or 2, but occasionally Class 3. The other half of the bladder was cut into four longitudinal strips, embedded in paraffin, and processed for examination by light microscopy and immunohistochemistry. A slice of stomach containing the limiting ridge was included with the bladder tissue to serve as a positive control for BrdU immunohistochemistry. Histopathological diagnosis of hematoxylin and eosin-stained slides was based on previously published criteria (Cohen, 1983, 1990). In brief, the diagnosis of simple hyperplasia was made when the number of cell layers in the bladder epithelium was increased above three. Bladder epithelium with endophytic or exophytic growths in hyperplastic areas was diagnosed as papillary/nodular hyperplasia. Unstained slides of the bladder and stomach tissue were used for immunohistochemical detection of incorporation of BrdU into the urothelial cells. Anti-BrdU (Chemicon International, Temecula, CA) was used at a dilution of 1:50. The number of BrdU-labeled cells in at least 3000 urothelial cells was counted to determine a labeling index.
The right kidney from the first five animals in each group was placed in PAPG (picric acid, paraformaldehyde, and glutaraldehyde) fixative for examination by transmission electron microscopy (TEM). After fixation the kidneys were cut into 1-mm3 pieces, with the renal medulla, papilla, and pelvis processed separately for each animal. The tissue was rinsed in Sorenson's phosphate buffer, postfixed in 1.0% OsO4 in 0.1 M phosphate buffer for 1 h, rinsed with buffer, dehydrated in a graded ethanol series and propylene oxide, infiltrated overnight in 1:1 propylene oxide:Araldite®, and embedded in Araldite®. The blocks were polymerized at 60°C overnight. Thick and thin sections were cut on a Sorvall MT7 ultramicrotome. Thin sections were stained with uranyl acetate and lead citrate and viewed in a Phillips 410 transmission electron microscope. Both kidneys from the last five animals in each group and the left kidney from the first five animals in each group were placed in phosphate-buffered formalin, and sections though the papilla were processed for light microscopic evaluation.
Analyses and statistics.
Statistical comparisons between groups for continuous data were made using Duncan's multiple range test (Duncan, 1955). Histopathology results were analyzed using Fisher's exact test, 2-tail method. SEM data were analyzed using nonparametric one-way analysis of variance with chi square. All analyses were done using software from SAS Institute, Inc. (Cary, NC). A p value of < 0.05 was considered significant. The Dixon Criterion for rejection of an observation in only one direction was used to identify outliers in the labeling index data (Natrella, 1966
).
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RESULTS |
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DISCUSSION |
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OPP and its sodium salt are metabolized by oxidation to o-phenylhydroquinone (PHQ) and o-phenylbenzoquinone (PBQ) and then conjugated by glucuronidation or sulfation (Bartels et al., 1998; Freyberger and Degen, 1998
; Kwok and Eastmond, 1997
; Kwok et al., 1999
; Kolachana et al., 1991
; Reitz et al., 1983
, 1984
). PBQ is suspected of being the ultimate metabolite in the induction of bladder epithelial injury and hyperplasia (Hasegawa et al., 1991
; Kolachana et al., 1991
; Kwok et al., 1999
). It has been demonstrated that PHQ is present in small amounts at low urinary pH (< 6.3) when there was no occurrence of bladder lesions (Kwok and Eastmond, 1997
; Kwok et al., 1999
). However, at higher pH (> 7.0) there was an increased presence of free PHQ and production of the reactive species PBQ from auto-oxidation, with a subsequent increased occurrence of bladder lesions. This observation is consistent with previous results in which urinary acidification with NaOPP administration was produced by concurrent feeding of high doses of ammonium chloride and resulted in decreased carcinogenicity levels similar to OPP (Fujii et al., 1987
; Hasegawa et al., 1991
). Similarly, urinary alkalinization during OPP administration was produced by concurrent administration of high doses of sodium bicarbonate and resulted in increased carcinogenicity (Fujii et al., 1987
; Fukushima et al., 1989
; Hasegawa et al., 1991
).
The effect of oxidation is one possible mechanism for the carcinogenicity of OPP, which would be in keeping with the pH effects on OPP oxidation. We (R. A. Smith and S. M. Cohen, unpublished observations) and others have found some evidence for increased oxidation in the urothelium, including formation of 8-oxoguanine (Kwok and Eastmond, 1997; Kwok et al., 1999
; Murata et al., 1999
; Nagai et al., 1990
; Smith et al., 1998
). Oxidative damage could be the mechanism for the cytotoxicity of OPP and NaOPP, with regenerative proliferation leading to the tumorigenic effects in the urothelium.
Recently, Kwok et al. (1999) reported binding of OPP to bladder proteins but not to DNA in male F344 rats. Others have reported OPP-DNA adduct formation following OPP administration. However this was observed only in mouse skin following direct application or in rats orally administered OPP, and using assays involving the whole bladder and not restricted to the urothelium (Pathak and Roy, 1992, 1993
; Ushiyama et al., 1992
). However, Smith et al. (1998) reported a lack of detection of OPP-DNA adducts from bladder scrapings in which only the urothelium of OPP-treated rats was analyzed.
After 10 weeks of dietary treatment with OPP or NaOPP, the proliferative effects on the urothelium were similar. An amorphous precipitate did not form, nor were there any abnormal microcrystalluria or calculus formation in the bladder or kidneys. Calcium deposits in the kidney were seen in all rats and did not appear to be treatment related. Renal calcium deposits are frequently detected in male rat kidneys, particularly in the F344 strain (Lord and Newberne, 1990).
In summary, both OPP and NaOPP administered at high doses in the diet for 10 weeks produced urothelial cytotoxicity and regenerative hyperplasia without formation of urinary solids in either treatment group, despite the higher urinary pH in rats fed NaOPP compared to those fed OPP. The results of our experiments strongly support chemical toxicity as the basis for both OPP and NaOPP rat bladder carcinogenesis, and this is compatible with the hypothesized mechanism based on oxidative damage (Kwok and Eastmond, 1997; Kwok et al., 1999
; Murata et al., 1999
).
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Cohen, S. M. 1983. Pathology of experimental bladder cancer in rodents. In The Pathology of Bladder Cancer, (S. M. Cohen and G. T. Bryan, Eds.), Vol. II, pp. 140. CRC Press, Boca Raton, FL.
Cohen, S. M. (1998). Urinary bladder carcinogenesis. Toxicol. Pathol. 26, 121127.[ISI][Medline]
Cohen, S. M. (1999) Calcium phosphate-containing urinary precipitate in rat urinary bladder carcinogenesis. International Agency for Research on Cancer, IARC Scientific Publications 147, 175189.
Cohen, S. M., Arnold, L. L., Cano, M., Ito, M., Garland, E. M., and Shaw, R. A. (2000). Calcium phosphate-containing precipitate and the carcinogenicity of sodium salts in rats. Carcinogenesis 21, 783792.
Cohen, S. M., Cano, M., Anderson, T., and Garland, E. M. (1996). Extensive handling of rats leads to mild urinary bladder hyperplasia. Toxicol. Pathol. 24, 251257.[ISI][Medline]
Cohen, S. M., Cano, M., Garland, E. M., St. John, M., and Arnold, L. L. (1995a). Urinary and urothelial effects of sodium salts in male rats. Carcinogenesis 16, 343348.[ISI][Medline]
Cohen, S. M., Ellwein, L. B., Okamura, T., Masui, T., Johansson, S. L., Smith, R. A., Wehner, J. M., Khachab, M., Chappel, C. I., Schoenig, G. P., Emerson, J. L., and Garland, E. M. (1991). Comparative bladder tumor promoting activity of sodium saccharin, sodium ascorbate, related acids, and calcium salts in rats. Cancer Res. 51, 17661777.[Abstract]
Cohen, S. M., Fisher, M. J., Sakata, T., Cano, M., Schoenig, G. P., Chappel, C. I., and Garland, E. M. (1990). Comparative analysis of the proliferative response of the rat urinary bladder to sodium saccharin by light and scanning electron microscopy and autoradiography. Scanning Microscopy 4, 135142.[ISI][Medline]
Cohen, S. M., Garland, E. M., Cano, M., St. John, M. K., Khachab, M., Wehner, J. M., and Arnold, L. L. (1995b). Effects of sodium ascorbate, sodium saccharin and ammonium chloride on the male rat urinary bladder. Carcinogenesis 16, 27432750.[Abstract]
Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, 142.[ISI]
Fisher, M. J., Sakata, T., Tibbels, T. S., Smith, R. A., Patil, K., Khachab, M., Johansson, S. L., and Cohen, S. M. (1989). Effect of sodium saccharin and calcium saccharin on urinary parameters in rats fed Prolab 3200 or AIN-76 diet. Food Chem. Toxicol. 27, 19.[ISI][Medline]
Freyberger, A., and Degen, G. H. (1998). Inhibition of prostaglandin-H-synthase by o-phenylphenol and its metabolites. Arch. Toxicol. 72, 637644.[ISI][Medline]
Fujii, T., Nakamura, K., and Hiraga, K. (1987). Effects of pH on the carcinogenicity of o-phenylphenol and sodium o-phenylphenate in the rat urinary bladder. Food Chem. Toxicol. 25, 359362.[ISI][Medline]
Fukushima, S., Inoue, T., Uwagawa, S., Shibata, M-A., and Ito, N. (1989). Co-carcinogenic effects of NaHCO3 on o-phenylphenol-induced rat bladder carcinogenesis. Carcinogenesis 10, 16351640.[Abstract]
Fukushima, S., Kurata, Y., Ogiso, T., Okuda, M., Miyata, Y., and Ito, N. (1985). Pathological analysis of the carcinogenicity of sodium o-phenylphenate and o-phenylphenol. Oncology 42, 304311.[ISI][Medline]
Fukushima, S., Kurata, Y., Shibata, M-A., Ikawa, E., and Ito, N. (1983). Promoting effect of sodium o-phenylphenate and o-phenylphenol on two-stage urinary bladder carcinogenesis in rats. Gann 74, 625632.[ISI][Medline]
Hagiwara, A., Shibata, M., Hirose, M., Fukushima, S., and Ito, N. (1984). Long-term toxicity and carcinogenicity study of sodium o-phenylphenate in B6C3F1 mice. Food Chem. Toxicol. 22, 809814.[ISI][Medline]
Hasegawa, R., Fukuoka, M., Takahashi, T., Yamamoto, A., Yamaguchi, S., Shibata, M-A., Tanaka, A., and Fukushima, S. (1991). Sex differences in o-phenylphenol and sodium o-phenylphenate rat urinary bladder carcinogenesis: Urinary metabolites and electrolytes under conditions of aciduria and alkalinuria. Jpn. J. Cancer Res. 82, 657664.[ISI][Medline]
Hasegawa, R., Takahashi, S., Asamoto, M., Shirai, T., and Fukushima, S. (1990). Species differences in sodium o-phenylphenate induction of urinary bladder lesions. Cancer Lett. 50, 8791.[ISI][Medline]
Hiraga, K., and Fujii, T. (1981). Induction of tumours of the urinary system in F344 rats by dietary administration of sodium ophenylphenate. Food Chem. Toxicol. 19, 303310.
Hiraga, K., and Fujii, T. (1984). Induction of tumours of the urinary bladder in F344 rats by dietary administration of o-phenylphenol. Food Chem. Toxicol. 22, 865870.[ISI][Medline]
International Agency for Research on Cancer. (1983). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans 13, 329344.
Kolachana, F., Subrahmanyam, V. V., Eastmond, D. A., and Smith, M. T. (1991). Metabolism of phenylhydroquinone by prostaglandin (H) synthase: Possible implications in o-phenylphenol carcinogenesis. Carcinogenesis 12, 145149.[Abstract]
Kwok, E. S. C., and Eastmond, D. A. (1997). Effects of pH on nonenzymatic oxidation of phenylhydroquinone: Potential role in urinary bladder carcinogenesis induced by o-phenylphenol in Fischer F344 rats. Chem. Res. Toxicol. 10, 742749.[ISI][Medline]
Kwok, E. S. C., Buchholz, B. A., Vogel, J. S., Turteltaub, K. W., and Eastmond, D. A. (1999). Dose-dependent binding of ortho-phenylphenol to protein but not DNA in the urinary bladder of male F344 rats. Toxicol. Appl. Pharmacol. 159, 1824.[ISI][Medline]
Lord, G. H., and Newberne, P. M. (1990). Renal mineralization a ubiquitous lesion in chronic rat studies. Food Chem. Toxicol. 28, 449455.[ISI][Medline]
Martin, R. A., Daly, A. M., DiFonzo, C. J., and de la Iglesia, F. A. (1984). Randomization of animals by computer program for toxicity studies. J. Am. Coll. Toxicol. 3, 111.
Moorehead, W. R., and Biggs, H. G. (1974). 2-Amino-2-methyl-1-propanol as the alkalizing agent in an improved continuous-flow cresophthalein complexone procedure for calcium in serum. Clin. Chem. 20, 14581460.
Murata, M., Moriya, K., Inoue, S., and Kawanishi, S. (1999). Oxidative damage to cellular and isolated DNA by metabolites of fungicide ortho-phenylphenol. Carcinogenesis 20, 851857.
Nagai, F., Ushiyama, K., Satoh, K., and Kano, I. (1990). DNA cleavage by phenylhydroquinone: The major metabolite of a fungicide o-phenylphenol. Chem. Biol. Interactions 76, 163179.[ISI][Medline]
Natrella, M. G. (1966). Experimental Statistics: National Bureau of Standards Handbook, pp. 17.217.6. U.S. Government Printing Office, Washington, DC.
Pathak, D. N., and Roy, D. (1992). Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique. Carcinogenesis 13, 15931597.[Abstract]
Pathak, D. N., and Roy, D. (1993). In vivo genotoxicity of sodium ortho-phenylphenol: Phenylbenzoquinone is one of the DNA-binding metabolite(s) of sodium ortho-phenylphenol. Mutat. Res. 286, 309319.[ISI][Medline]
Reitz, R. H., Fox, T. R., Quast, J. F., Hermann, E. A., and Watanabe, P. G. (1983). Molecular mechanisms involved in the toxicity of ortho-phenylphenol and its sodium salt. Chem. Biol. Interact. 43, 99119.[ISI][Medline]
Reitz, R. H., Fox, T. R., Quast, J. F., Hermann, E. A., and Watanabe, P. G. (1984). Biochemical factors involved in the effects of orthophenylphenol (OPP) and sodium orthophenylphenate (SOPP) on the urinary tract of male F344 rats. Toxicol. Appl. Pharmacol. 73, 345349.[ISI][Medline]
SAS/STAT User's Guide. (1989). Statistics. Vol. II, Ver. 6, 4th ed. SAS Institute, Cary, NC.
Smith, R. A., Christenson, W. R., Bartels, M. J., Arnold, L. L., St. John, M. K., Cano, M., Garland, E. M., Lake, S. G., Wahle, B. S., McNett, D. A., and Cohen, S. M. (1998). Urinary physiologic and chemical metabolic effects on the urothelial cytotoxicity and potential DNA adducts of o-phenylphenol in male rats. Toxicol. Appl. Pharmacol. 150, 402413.[ISI][Medline]
Ushiyama, K., Nagai, F., Nakagawa, A., and Kano, I. (1992). DNA adduct formation by o-phenylphenol metabolite in vivo and in vitro. Carcinogenesis 13, 14691473.[Abstract]
Wahle, B. S., Christenson, W. R., Lakel, S. G., Elcock, L. E., Moore, K. D., Sangha, G. K., and Thyssen, J. H. (1997). Technical grade ortho-phenylphenol: A combined chronic toxicity/ oncogenicity testing study in the rat. Toxicologist 36, 341.