Dietary Effects of ortho-Phenylphenol and Sodium ortho-Phenylphenate on Rat Urothelium

Margaret K. St. John, Lora L. Arnold, Traci Anderson, Martin Cano, Sonny L. Johansson and Samuel M. Cohen1

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are pesticides used commercially in the food industry that have been shown to be carcinogenic to rat urothelium. Dietary administration of 1.25% OPP or 2.0% NaOPP caused increased incidences of urothelial hyperplasia and eventually caused tumors in male F344 rats, with NaOPP apparently having a more potent effect. In other studies, various sodium salts such as saccharin and ascorbate enhanced bladder carcinogenesis, although the acid forms of these salts did not. In studies with high dietary doses of these sodium salts, an amorphous precipitate was produced in the urine; precipitate formation was pH dependent. In previous experiments in which high doses of OPP were fed for up to 17 weeks, severe hyperplasia of the urothelium was produced, but without the formation of an urinary amorphous precipitate, calculi, or abnormal microcrystalluria. In addition, we found no evidence of OPP-DNA adduct formation in the urothelium. The present study was conducted to determine if feeding NaOPP • 4 H20 to male F344 rats as 2.0% of the diet resulted in the formation of an amorphous precipitate in the urine, and if NaOPP caused an increased mineral concentration in the urine and/or kidneys. NaOPP administration produced a higher urinary pH than did OPP fed as 1.25% of the diet. Neither amorphous precipitate nor other solids were observed in the urine of the OPP or NaOPP-treated rats, and urinary calcium concentrations in the treated groups were similar to control. OPP and NaOPP had similar proliferative effects on rat urothelium after 10 weeks of treatment by light microscopy, scanning electron microscopy (SEM), and bromodeoxyuridine (BrdU) labeling indices. The results of this study indicate that formation of abnormal urinary solids is not part of the mechanism by which OPP or NaOPP exert their effects on the rat bladder epithelium.

Key Words: ortho-phenylphenol; sodium ortho-phenylphenate; urothelial effects; rats..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ortho-phenylphenol (OPP) and sodium ortho-phenylphenate (NaOPP) are widely used fungicides and antibacterial agents (IARC, 1983). Dietary studies with high doses of OPP (1.25% and 2.5%; Hiraga and Fugii, 1984) and NaOPP (0.5, 1.0, 2.0, and 4.0%; Hiraga and Fugii, 1981) resulted in hyperplastic lesions in male rats and/or tumors of the urinary bladder and renal pelvis. Tumors were found in female rats, but only at the highest dose of NaOPP (4%). In a subsequent study performed in the United States, there was an increased incidence of proliferative lesions and bladder tumors in male rats fed 0.8% OPP in a chronic 2-year bioassay (Wahle et al., 1997Go). Few hyperplastic lesions and no tumors were found in OPP-treated female rats in this bioassay, despite a higher consumption of OPP in the females (647 mg OPP/kg body weight/day compared to 402 mg OPP/kg body weight/day in males).

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., 1983Go). 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., 1985Go; Hagiwara et al., 1984Go; Hasegawa et al., 1990Go). 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., 1995aGo; Cohen, 1998Go, 1999Go). 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, 1999Go; Cohen et al., 2000Go). No toxic or proliferative effects were observed with the acid forms of those sodium salts that have been tested (Cohen, 1999Go; Cohen et al., 1991Go, 1995bGo). 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., 1983Go; Hiraga and Fujii, 1981Go, 1984Go). 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., 1998Go; Wahle et al., 1997Go). 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., 1998Go).

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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test articles.
Technical grade OPP was purchased from Aldrich Chemical Co. (Milwaukee, WI), and NaOPP•4 H20 was purchased from Fluka Chemical Corp. (Milwaukee, WI). The purity of the chemicals was determined by the respective companies to be 99.8% for OPP and 101.6% for NaOPP. The chemicals were mixed by Dyets, Inc. (Bethlehem, PA.) into Purina® Rodent Chow 5002 (Ralston Purina Co., St. Louis, MO), at doses of 1.25% for OPP and 2.0% for NaOPP•4 H20.

Animals/husbandry.
Thirty male Fischer F344 rats from Charles River Breeding Laboratories (Raleigh, NC), approximately 4–5 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., 1984Go) 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, 1983Go, 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, 1955Go). 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, 1966Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body weights for both the OPP- and NaOPP-treated groups were significantly lower than the controls, but the percent weight gain over the course of the study was similar in all groups (Table 1Go). Food consumption was slightly increased during week 3 and significantly increased during week 8 in the treated groups. Water consumption was increased in the treatment groups at both time points measured, but the increase was only significant during week 8 in the NaOPP-treated group.


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TABLE 1 Body Weight, Food and Water Consumption
 
The urinary pH in the OPP-treated group was similar to the control group at week 1, but it was decreased compared to control throughout the rest of the study (Table 2Go). The urinary pH in the NaOPP group was significantly higher compared to the control and OPP-treated rats at each time point tested. Urinary calcium levels at week 9 were 13.2 ± 0.2 mg/dl for the OPP-treated rats, 13.3 ± 0.3 mg/dl for NaOPP-treated rats, and 15.3 ± 2.0 mg/dl for controls. A small amount of calcium phosphate–containing amorphous precipitate was observed in all groups including the control group after 1 week of treatment (data not shown). This is not uncommon at the start of an experiment in young rats and may be due to stress from handling or acclimation to experimental conditions rather than to dietary treatment (Cohen et al., 1996Go). In subsequent urine collections there were light deposits of amorphous precipitate at similar levels in all groups observed at week 5, but no deposits were observed by week 9. The amorphous precipitate was composed primarily of calcium and phosphate, but contained varying amounts of potassium, silicon, chloride, and sulfur as detected by X-ray energy dispersive spectroscopy (Cohen et al., 1995aGo, 1995bGo). No abnormal crystals, increased number of crystals, or calculi were detected in the urine of any rat in any group.


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TABLE 2 Fresh Void Urine pH
 
The incidence of simple hyperplasia was significantly higher in both the OPP- and NaOPP-treated rats, whereas the occurrence of papillary/nodular hyperplasia was significantly increased only in the OPP-treated rats (Table 3Go). The labeling indices were significantly increased in the OPP- and NaOPP-treated rats, with a greater increase in the OPP group (Table 3Go). No differences between OPP and NaOPP treatments were observed by SEM (Figs. 1 and 2GoGo). The bladder from one control rat was classified as Class 3, but the remaining control bladders were all Class 1 and Class 2 (Fig. 3Go). Class 1 and 2 are considered normal whereas Class 4 and 5 are considered abnormal, based on previous studies. Class 3 is equivocal (Cohen et al., 1990Go).


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TABLE 3 Effects of OPP/NaOPP on the Urinary Bladder after 10 Weeks of Treatment
 


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FIG. 1. Bladder from rat fed 1.25% OPP, with areas of piling up of round cells and exfoliation, Class 5. Original magnification x203.

 


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FIG. 2. Bladder from rat fed 2.0% NaOPP, with areas of piling up of round cells, Class 5. Original magnification x170.

 


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FIG. 3. Control bladder, Class 1, with normal flat polygonal cells. Original magnification x137.

 
Examination by light microscopy of the kidneys of all animals except two control rats showed calcification. The calcification was not localized to any particular area; it was observed in the collecting ducts of the papilla, in the medulla and corticomedullary junction, and to a lesser extent in the pelvis. The light microscopic evaluation of the kidneys was otherwise unremarkable. All kidneys examined by TEM also showed extensive areas of calcification.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, a comparison of the urinary and urothelial effects of dietary treatment with OPP or NaOPP was conducted to compare the proliferative effects observed in male rat bladders following treatment with OPP and NaOPP and to determine if there was formation of urinary solids in the bladder or kidney following treatment with either chemical. We previously showed that urinary solids did not form following OPP administration (Smith et al., 1998Go). In the present experiment, in spite of increased urinary pH to well above 7.0, there was no precipitate formation or other urinary solids observed following NaOPP treatment. This is in contrast to what has been observed with several other sodium salts (Cohen et al., 1995aGo, 1995bGo, 2000Go), such as saccharin or ascorbate, where precipitate occurred when the urinary pH was >= 6.5. Additionally, unlike the acid form of these and other sodium salts, OPP itself produced a cytotoxic and regenerative proliferative effect on the bladder urothelium even at a pH that was significantly lower than the pH found in the NaOPP-treated group. These results suggest that a mechanism other than formation of urinary solids and increased urinary pH is causing or enhancing the toxic effects observed following administration of high doses of either OPP or NaOPP. Thus, NaOPP is not acting through a mechanism containing precipitate as has been found with other sodium salts. A direct, chemical-induced mechanism must be operative in producing the toxicity following administration of either NaOPP or OPP.

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., 1998Go; Freyberger and Degen, 1998Go; Kwok and Eastmond, 1997Go; Kwok et al., 1999Go; Kolachana et al., 1991Go; Reitz et al., 1983Go, 1984Go). PBQ is suspected of being the ultimate metabolite in the induction of bladder epithelial injury and hyperplasia (Hasegawa et al., 1991Go; Kolachana et al., 1991Go; Kwok et al., 1999Go). 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, 1997Go; Kwok et al., 1999Go). 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., 1987Go; Hasegawa et al., 1991Go). 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., 1987Go; Fukushima et al., 1989Go; Hasegawa et al., 1991Go).

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, 1997Go; Kwok et al., 1999Go; Murata et al., 1999Go; Nagai et al., 1990Go; Smith et al., 1998Go). 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, 1992Go, 1993Go; Ushiyama et al., 1992Go). 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, 1990Go).

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, 1997Go; Kwok et al., 1999Go; Murata et al., 1999Go).


    ACKNOWLEDGMENTS
 
We gratefully acknowledge the assistance of Dr. Mary Fidler and Rick Vaughn for assistance with the TEM processing and analysis, and to Kai Jones and Denise Miller for their assistance in the preparation of this manuscript. The constructive critiques of this manuscript by Dr. T. Lawson are greatly appreciated. This research and publication served as partial fulfillment of the degree requirements for the Master of Science for Margaret K. St. John. S. M. Cohen is the Havlik Wall Professor of Oncology.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (402) 559-9297. E-mail: scohen{at}unmc.edu. Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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