ICANCERRESEARCH56.4922-4926.NovemberI,1996 ...cancerres.aacrjournals.org/content/56/21/4922.full.pdf · ICANCERRESEARCH56.4922-4926.NovemberI,1996] ABSTRACT...

ICANCER RESEARCH 56. 4922-4926. November I, 1996]

ABSTRACT

Phenolphthalein (a triphenylmethane derivative) has been commonlyused as a laxative for most of the twentieth century, but little Is knownabout its long-term carcinogenic potential in experimental studies. In ourstudies, phenolphthalein administered continuously In the feed for 2 yearsto F344 rats at doses of 0, 12,500, 25,000, and 50,000 ppm and to C57BIJ6 x CR3 F1(hereafter calledB6C3F1)miceat dosesofO,3,000,6,000,and12,000 ppm caused multiple carcinogenic effects. Treatment-related neoplasma occurred in the kidney and adrenal medulla in male rats, adrenalmedulla in female rats, hematopoletic system in male and female mice(histiocytic sarcomas and malignant lymphomas), and ovary of female

mice. Phenolphthalein has been shown to have estrogenic and clastogenicproperties. Previous studies of other estrogenic chemicals (e.g.,zearalenone) in the F344 rat and B6C3F1 mouse have not shown the samespectrum of carcinogenk activity as that found with phenolphthalein,suggesting that phenolphthaleln estrogenic activity alone is not responsi

ble for the spectrum oftwnors observed. It is more likely that the multiplebiological properties of phenolphthalein, including Its ability to form freeradicals, its clastogenic activity, and its estrogenic activity, contributed tothe carcinogenic effects observed. These studies show that phenolphthalem is a multisite/multispecies carcinogen. One of the sites for neoplasmthat is of particular concern is the ovary, and epidemiology studies areunder way to identify any potential effects of phenolphthalein exposure atthis site in humans.

INTRODUCTION

Epidemiological studies have indicated that many human cancersare influenced by environmental factors. Although recent studies haveidentified genes that are associated with cancer in certain populations,we have not been able to demonstrate that inherited mutations inspecific genes are solely responsible for human disease (1). As populations migrate from one area of the world to another, the incidencefor a cancer in immigrant populations may approach that of the hostcountry in one or two generations, suggesting that environmentalfactors may play an important role in the occurrence of some types ofcancer (2, 3). Phenolphthalein has been used as an over-the-counterlaxative during most of this century (4), and this chemical was studiedto determine its potential to cause cancer.

Phenolphthalein has multiple biological properties as demonstratedin various in vitro and in vivo studies. Phenolphthalein was negativefor genotoxicity in the Salmonella test both with and without metabolic activation, but was positive in an in vitro chromosomal aberration test conducted with 59 metabolic activation (5). In the B6C3F1mouse and the Swiss CD- I mouse, phenolphthalein increases thefrequency of micronucleated erythrocytes (6). The lowest dose level atwhich phenolphthalein causes micronuclei formation is 360 mg/m2body surface area (6), a dose that is approximately two times therecommended dose in humans (185 mg/m2 body surface area). Phenolphthalein has been shown to bind competitively to the estrogenreceptor in MCF-7 cells (7). Thus, phenolphthalein has the potential to

Received 6/14/96; accepted 8/30/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

@ To whom requestsfor reprintsshouldbe addressed,at National Instituteof Environmental Health Science, P. 0. Box 12233, Research Triangle Park, NC 27709.

cause adverse biological effects through its clastogenic activity or itsestrogenic activity. This article reports the toxic and carcinogeniceffects of phenolphthalein in the F344 rat and B6C3F1 mouse after a2-year exposure period.

MATERIALS AND METHODS

Animals. Male and female F3441Nrats and B6C3F1 mice were obtainedfrom Taconic Farms (Germantown, NY) and placed on study at 7—8 weeks ofage. Rats were housed by sex, five per cage. Mice were individually housed.Polycarbonate cages (LabProducts, Inc., Rochelle Park, NJ) provided with

Sani-chips bedding chips (P. J. Murphy Forest Products, Montville, NJ) wereused. Tap water and NIH 07 feed (Zeigler Bros., Gardners, PA) were providedad libitum. The animals were maintained in a room that was kept at 21—23°Cwith 10 air changes/h and a 12-h light cycle. All animals were checked dailyfor clinical signs and moribund animals were necropsied. Animal body weightswere taken once a week during the first 13 weeks of study and every 4 weeksthereafter. Feed consumption was recorded weekly.

Chemical. Phenolphthalein (P9189; Fig. I) was supplied by Pharmco Laboratory, Inc. (Titusville, FL; Fig. I). The chemical was examined with infared,

UV/visible, and nuclear magnetic resonance spectroscopy and found to be 99%pure by elemental analyses, Karl Fischer water analysis, titration of thefunctional groups thin-layer chromatography, and high-performance liquidchromatography (National Toxicology Program; Ref. 5). The lot met all USPrequirements. Formulated diets were prepared by mixing appropriate amountsof phenolphthalein, a yellow powder, with NIH 07 feed. Periodic analysis ofthe formulated diets of phenolphthalein were performed, and the diet wasfound to be within ±10% of the targeted concentrations of 0, 12,000, 25,000,or 50,000 ppm (rats) or 0, 3000, 6000, or 12,000 ppm (mice; Ref. 5).

Two-Year Study Design. Groups of 50 rats and 50 mice of each sexreceived control or phenolphthalein diets for 104 (males) or 105 weeks(females). A complete gross necropsy was performed on all animals from allgroups that died or were killed during and at the end of the experiment. Alltissues were preserved in 10% neutral buffered formalin. Major organs/tissuestrimmed, embedded in paraffin, sectioned, stained with H&E, and examinedmicroscopically included the adrenal gland, bone and marrow, brain, clitoral

gland, esophagus, gallbladder, heart, kidney, large intestine (cecum, colon,

rectum), liver. lung, mammary gland, mandibular and mesentenc lymph nodes,nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland,prostate, salivary gland, skin, small intestine (duodenum, jejunum, ileum),spleen, stomach (fore stomach and glandular), testis with epididymis andseminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus, Zym

bal's gland, and all gross lesions.Statistical Analysis. Differences in survival were analyzed using life table

methods (8). For the analysis of tumor incidence data, survival-adjustedprocedures were used to assess dose-response trends and to make pairwisecomparisons between dosed groups and controls (9, 10).Fisher exact tests andCochran-Armitage trend tests were also utilized to analyze tumor incidencedata. Results are considered as significant where the P < 0.05.

RESULTS

Survival, Body Weights, and Clinical Signs. Final survival ofexposed rats and mice was similar to the respective controls with theexception of high-dose female mice where survival was less than thatof controls. Mean body weights of treated rats were reduced by5—10%during most of the study, but toward the end of the study thebody weight reduction became more pronounced (Table 1).

Clinically, there were no treatment-related signs of toxicity. The

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Phenolphthalein Exposure Causes Multiple Carcinogenic Effects in ExperimentalModel Systems

June K. Dunnick' and James R. HaileyNational Institute of Environmental Health Sciences, Research Triangle Park@North Carolina 27709

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Table 1Survival and body weightDose

(ppm)012,000

25,00050,000Male

ratsSurvival21/5015/5025/5013/50Final

mean body weight (%)ag47979FemaleratsSurvival34/5041/5037/5040/50Final

mean body weight (%)86 8685Dose

(ppm)0300

6,00012,000Male

miceSurvival40/5033/5036/5036/50Final

mean body weight (%)979993FemalemiceSurvival42/5033/50

34/5031/50Finalmean body weight (%)929594a

Percentage relative to controls.

PHENOLPHTHALEIN EXPOSURE IN EXPERIMENTAL MODELS

0

@C@h@0H

Fig. 1. Phenolphthalein (CAS 77—09-8).

average feed consumption for exposed animals was similar to that ofcontrols. Male rats ate approximately 13—18g feed/day/animal; female rats ate 9—12g feed/day/animal; and male and female mice ate4—5g feed/day/animal.Basedon thefeedconsumption,dietarylevelsof 12,000, 25,000, and 50,000 ppm phenolphthalein resulted in average daily doses during the last year of the study of approximately 500,1000, or 2000 mg phenolphthaleinlkg body weight to male rats and500, 1000, or 2100 mg/kg to female rats. Based on the feed consumption, dietary levels of 3,000, 6,000, and 12,000 ppm phenolphthaleinresulted in average daily doses during the last year of the study ofapproximately 300, 600, or 1,200 mg phenotphthaleinlkg body weightto male and female mice (Table 2).

Treatment-related Lesions: Rats. There was an increased mcidence of proliferative lesions (hyperplasia and pheochromocytomas)in the adrenal medulla of male and female rats (Table 3). Also, thenumber of males that had pheochromocytomas in both adrenal medullas (bilateral) was increased in the treated groups. Focal hyperplasia was only marginally increased in the treated groups. However, thediagnosis offocal hyperplasia in a gland was only made in the absenceof a diagnosis of a neoplasm in that gland. Focal hyperplasia andpheochromocytoma are considered to constitute a morphological continuum in the adrenal medulla.

There was an increased incidence of proliferative lesions of therenal tubule epitheium of all treated groups of male rats. Although theincrease was predominantly of hyperplasia and adenoma, carcinomawas slightly increased. Both renal tubular adenoma and carcinoma arerelatively uncommon neoplasms in the male F344 rat (i.e. , occurringin <1% of untreated control male rats). In the kidney, hyperplasia,adenoma, and carcinoma are thought to represent a continuum in theprogression of proliferative lesions of the tubule epithelium.

The incidence of nephropathy of the kidney was increased in alltreated female groups, whereas the severity was increased in alltreated male groups. Nephropathy is a spontaneously occurring lesionin most aging F344 rats, particularly males, and involves a spectrumof degenerative and associated inflammatory and regenerativechanges. Marked tubule dilation with cyst formation and transitionalepithelial hyperplasia of the renal pelvis (components of nephropathy)occurred in kidneys from treated males with the most severe nephropathy.

Diffuse hyperplasia of the parathyroid gland, fibrous osteodystrophy of the bone, and mineralization of the glandular stomach (data notshown) were increased in treated groups of male rats. These lesionsare commonly observed in rats with severe nephropathy and areassociated with a calcium/phosphorous imbalance created by compromised functional capacity of the kidney.

Treatment-related Lesions: Mice. An increased incidence of histiocytic sarcoma was observed in male and female mice (Table 4).Spontaneous histiocytic sarcoma occurred in 0.5—1.0% of the untreated B6C3F1 mice. In this study, histiocytic sarcoma was consistently observed in the liver with several other sites (e.g. , spleen, lung,bone marrow, and various lymph nodes) less frequently involved.

Exposure to phenolphthalein was associated with an increasedincidence of malignant lymphoma (all sites) and of lymphoma ofthymic origin in all exposed groups of female mice. The incidences oflymphoma of thymic origin were increased in exposed groups of malemice, but were significantly increased only in the 6000 ppm group.Atypical hyperplasia of the thymus was also observed in treated malesand females. Lymphomas were considered of thymic origin if(a) theywere observed only in the thymus and other observed proliferativelymphocytic lesions were limited to the thoracic cavity or (b) thethymus clearly contained the largest proliferative lymphocytic lesionwhen systemic lymphocytic lesions occurred.

In the mouse, the thymus is a bibbed organ, with each lobe havinga distinct outer cortex composed of small hyperchromatic lymphocytes and an inner medulla composed of larger lymphocytes and fewerepithelial cells. In some treated mice, one or both of the lobes lackedthe normal corticomedullary arrangement. When these abnormal lobeswere less than the size of a normal thymic lobe and were comprisedof sheets of large lymphocytes admixed with variable numbers ofsmaller more normal-appearing lymphocytes, the change was diagnosed as atypical hyperplasia. The incidence of atypical hyperplasia ofthe thymus was also increased in treated males and females. In theearliest lesions diagnosed as malignant lymphoma, a fairly homogeneous population of lymphocytes extended beyond the confines ofnormal thymic tissue and mitoses were common. Larger thymiclymphomas occupied a large portion of the chest cavity.

There was an increased incidence of benign sex-cord stromal ovarian tumors and associated stromal cell hyperplasias in all treatedfemale groups. The neoplasms were discrete masses which variedfrom occupying most of the ovary to markedly expanding it (up toeight times) with compression of surrounding parenchyma. Neoplasms and hyperplasias were usually composed of sheets of round tooval stromal cells with abundant cytoplasm, which varied from finelygranular and eosinophilic to vesiculated (leuteinized). In most neoplasms the cells were rather homogeneous with a low mitotic index;however, in some of the larger ones there was a modest degree ofcellular pleomorphism with a higher mitotic rate.

Proliferative lesions (neoplasms and foci) of the liver were decreased in treated male and female mice. Furthermore, many of thehepatocellular adenomas in the control groups were multiple, but notin treated groups. Hepatocellular neoplasms occur with a high andvariable rate in the B6C3F1 mouse [with a range of 10—68% inuntreated male mice (mean 41%) and a range of 6—42%in untreatedfemale mice (mean 21%)]. Although there is a known association ofthe incidence of this neoplasm with body weight in the B6C3F1

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Table2 Summaryoffeed and compound consumption by rats and mice in thphenolphthalein studye

2-yearDose

(ppm)Males

Females012,000

25,000 50,000 0 12,00025,00050,000RatsMean

weeks1—13gfeed/day16.316.2 17.0 16.6 10.7 10.410.411.0mg/kg@/day

mg/m /day@'0 07891,714 3,375 0 787 1,655

4,102 8,912 17,550 0 4,092 8,6033,42317,799Meanweeks54—104g

feed/day14.415.3 15.9 15.811.4 11.311.411.8mg/kg/day0434953 1,880 0 4771,0132,125Dose

(ppm)Males

Females03,000

6,000 12,000 0 3,0006,00012.000MiceMean

weeks1—13gfeed/day4.34.3 4.4 4.2 5.1 4.95.35.1mg/k@day

mg/m /day―0 0453940 1.802 0 65 1 1,4O7

1,359 2,820 5,406 0 1,953 4,2212,6407,920Meanweeks54—104g

feed/day4.84.8 4.9 4.8 5.3 5.35.25.2mg/kg/day0291589 1,181 0 311 6171,255

Table3 Treatment-relatedlesionsin rats exposedto phenolphthalein for2yearsTumor

siteDose

(ppm)012,000

25,00050,000Male

ratAdrenalmedulla50―505050Hyperplasia

Pheochromocytoma, benignI3b322'

1819d 19―23c15―(bilateral)

Pheochromocytoma, all benignPheochromocytomas, benign and/or17 1834d

34d

35― 35d34d35dmalignantKidney

HephropathyCystHyperplasia, transitional epitheliumHyperplasia, renal tubule epithelium50

47 (l.8)eI4050

5049 (29)d@ (31)d

19d 21d

31― 34d6d 7d50

22d

29d

2Adenoma,renal tubule0426'Carcinoma,

renal tubule0112Adenomaand/or carcinoma, renal05'37dtubuleBone,

femurFibrous osteodystropy50 050

5017d 14d5012dParathyroid

glandHyperplasia41 048

4916d 14d4614dFemale

ratAdrenalmedulla50505049Hyperplasia1018

15IIPheochromocytoma,all benign31 1―92Pheochromocytoma,benign and/or312'10'2malignantKidney5050

5050Nephropathy34(1 .2)45C (1 .4) 43' ( I 5)C44( ( I.5fCyst02

01Hyperplasia,transitional epithelium2110Adenoma,

renal tubule0001a

Numberofanimalsineachgroup.b Number of animals withlesions.C

p <0.05.dp

< 0.01.

PHENOLPHTHALEIN EXPOSURE IN EXPERIMENTAL MODELS

studies (1 1). Thus, in our model systems, the mouse is more sensitiveto a chemically induced ovarian tumor response than the rat.

Cancer of the ovary is the fourth most common cancer in Americanwomen. There was an estimated 26,600 new cases diagnosed in 1995and 14,500 deaths resulted from this disease (13). In humans, approximately 80% of the ovarian tumors are of epithelial origin; otherovarian tumors may be of sex cord/stromal origin. Like many cancers,ovarian cancer is a disease of aging, with almost one half of new casesoccurring in women age 65 years or older.

Environmental factors that might contribute to ovarian cancer inhumans have not been identified. It is known that the rate for ovariancancer in American women is two to three times higher than that forOriental women (2). It is not known whether this difference in theovarian cancer rate is due to environmental or genetic factors. BRCAJand BRCA2 mutations have been associated with familial cancer butonly 5—10%of ovarian carcinomas are thought to result from hereditaly predispositions (14), and for the large majority of human ovarian

cancer cases examined, mutations in the BRCAJ or BRCA2 locationshave not been observed (14—16).

Like phenolphthalein, which has been shown to bind competitivelyto the estrogen receptor in MCF-7 cells (7), other chemicals tested inthe bioassay have also been shown to interfere with estrogen bindingat the estrogen receptor and to have mitogenic effects on breast cancercells (17). These include zearalenone, 2,4-dichlorophenol, and butylbenzyl phthalate (1 1, 17). However, of these chemicals, only phenolphthalein was shown to cause ovarian neoplasms in mice. Thus,demonstration of a chemical-induced estrogenic response does notnecessarily correlate with ovarian or mammary gland neoplasia inrodents. Furthermore, these chemicals with estrogenic activity showdifferent neoplasm patterns in other tissues. This suggests that phe

aCalculationforbodysurfaceareadosebasedonFreireichetaL(35);mg/m2 Km X (dose in mg/kg), where Km @537 for humans, 5.2 for rats, and 3.0 for mice.(Km 15a conversion based on average height:body weight ratio). Estimated human doseis 5 mg/kg body weight or 185 mg/m2 body surface area.

mouse, the slight decreases in body weights of treated groups couldnot account for the decreased incidences observed in this study.

In addition to the neoplastic effects, there were a number ofnonneoplastic effects considered related to administration of phenolphthalein in mice. These included mild to moderate testicular degeneration in males which involved germinal epithelium, increased mcidence of myelofibrosis of the bone marrow of males, and an increasedseverity of this lesion in treated females, a decreased incidence ofdegeneration of the teeth in treated males, and an increased incidenceof splenic hematopoietic cell proliferation in males.

DISCUSSION

At the end of the 2-year study period, phenolphthalein causedneoplasia in the ovary of female mice, the hematopoietic system ofmale and female mice, the adrenal of male and female rats, and thekidney of male rats.

The phenolphthalein-induced ovarian neoplasms were consideredto be of sex cord/stromal origin rather than epithelial or germ cell.However, they did not fit into one of the classical morphologicalcategories of sex cord/stromal tumors. In mice, there have been anumber of other chemicals [benzene, 1,3-butadiene, n-methylolacrylamide, 5-nitroacenaphthene, nitrofurantoin, nitrofurazone, 4-vinylcylcohexene, and 4-vinyl-l-cyclohexene diepoxide (1 1)] that causedovarian tumors. In almost all instances, these have been mixed tumorsor granulosa cell tumors. Early loss/destruction of oocytes and thesubsequent perturbation of the ovary-pituitary axis with a chronichormonally induced stimulation/proliferation is thought to lead totumor formation. The increased stimulation by gonadotropins isthought to stimulate stromal cell proliferation in the ovary and promote the eventual development of neoplasms (12).

Phenolphthalein is somewhat unique in that this killing of oocytesdoes not appear to be the mechanism for ovarian tumor formation,and, furthermore, the morphology of the ovarian tumors is not typicalof those seen as spontaneous or induced tumors in mice. No treatmentrelated ovarian tumors have been reported in rats in any of the NTP Severity of grade: I, minimal; 2, mild; 3, moderate; and 4, marked.

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Table 4 Treatment-related lesions in mice exposed to phenolphthalein for 2yearsTumor

siteDose

(ppm)03,000

6,00012,000Male

mouseHematopoieticsystem

Histiocytic sarcoma50― 1b50

50@ I lc4912'Malignant

lymphomas (all sites)68128Lymphoma(thymus)047C2Atypical

hyperplasia (thymus)037'@7CBonemarrow50505049Mylelofirosis

Pigmentation3 088

2 5dl9cl6cSpleen

Hematopoietic cell proliferation50 105050

22c 28c4921dTestis5050

5048Degeneration149C5@c47CFemale

mouseHematopoieticsystem50505050Histiocytic

sarcomaMalignant lymphoma (all sites)Lymphoma (thymus)0

1512

7C28― 33C

9' 10'7C25'

7dAtypical

hyperplasia (thymus)07'6'5COvary

Stromal cell hyperplasiaStromal cell tumor50

4049

5011d 107' 6d50

17'5dBone

marrow50505050Myelofibrosis34343836dPigmentation2

50133

1]C50 5014 20I

lc5021

PHENOLPHTHALEINEXPOSURE IN EXPERIMENTAL MODELS

lesion. Bone marrow myelofibrosis was characterized by focal tomultifocal replacement of bone marrow hematopoietic precursor cellsby clusters of loosely arranged spindle cells. Female sex hormones,especially estradiol, appear to play a role in the spontaneous development of myelofibrosis in mice. Estrogen has been reported to causeneutropenia, thromocytopenia, decreased bone marrow cellularity,and increased splenic hematopoiesis in mice (23, 24). Witt et a!. (6)have shown that phenolphthalein can induce micronuclei in bonemarrow erythrocytes of mice. Micronuclei are formed from acentricchromosomal fragments or whole chromosomes generated through avariety of mechanisms such as mitotic loss of acentric fragments,mechanical consequences of chromosomal breakage and exchangeevents resulting in abnormal anaphase separation, or through mitoticloss of whole chromosomes due to centromere or spindle failures.Induction of chromosomal breakage may be a mechanism for thephenolphthalein-induced bone marrow damage.

Other treatment-related effects in mice may have been related to theestrogenic activity of phenolphthalein, including the testicular degeneration and a decrease in tooth dysplasia in treated male mice. Estrogen has been shown to inhibit liver tumor promotion in mice (25),and, in NTP studies, spontaneous liver tumors occur in males at twicethe rate of females. In this study, the rates for liver tumors weremarkedly decreased in both sexes of mice.

The incidence of pheochromocytoma of the adrenal medulla wassignificantly increased in all groups of treated male rats and in lowand mid-dose female rats. The mechanism for the formation of thistumor is not known and has been previously observed with bothgenotoxic and nongenotoxic chemicals (1 1). Of approximately 250NTP studies in which chemicals were administered in the feed to rats,phenolphthalein is the only chemical that caused adrenal tumors inboth male and female rats. The carcinogenic effect at the same site inboth sexes reinforces the treatment-related carcinogenic response atthissite.

The kidney tumors observed in phenolphthalein-treated male ratsmay have been related to toxic effects of the chemical at this site.There was an increase in the severity of nephropathy in all treated ratgroups, and the severity was greater in males. There was an increasein the renal tubule proliferative lesions, including hyperplasia, adenoma, and carcinoma in treated male rat groups.

The findings in the male rat kidney are similar to those observedwith other cyclic lactone chemicals, including quercetin, coumarin,and dihydrocoumarin (26). With these chemicals, there was no morphological evidence of toxicity to the kidney early in the study, buttoward the end of the 2-year study the severity of nephropathy wasincreased in male rats (and to a lesser extent in female rats) and a fewkidney tumors occurred in the male rat. The greater sensitivity of themale rat to this kidney toxicity is apparently due to a greater susceptibility of male rats to spontaneous nephropathy during aging and theexacerbation of this disease by chemical administration.

There is no readily apparent common mechanism by which theserenal proliferative lesions developed. One possible mode of action isconsistent with the theory of increased cell replication providing afertile ground for increased mutation rates and neoplasm development. Renal cellular damage is thought to increase the amount of renaltubule epithelial regeneration via cell replication. In one study,[3H]thymidine labeling demonstrated increased levels of DNA synthesis to be directly proportional to the increased severity of nephropathy in aging female F344/NCr rats (27).

Phenolphthalein is absorbed in the small bowel and is conjugated inthe liver to form phenolphthalein glucuronide, which is eliminated inthe bile; as it passes through the small intestine it is partially deconjugated and reabsorbed (28, 29). The major metabolite of phenolphthalein is the glucuronide. The plasma level of free phenolphthalein

SpleenHematopoietic cell proliferation

a Number of animals in each group.

b Number of animals with lesions.‘P< 0.01.dp <@

nolphthalein-induced neoplasms may be due to multiple factors,which might include specific metabolism and distribution of thechemical as well as estrogenic or genotoxic properties of the chemical.

The hematopoietic system of the mouse was affected by phenolphthalein exposure. There was a clear increase in the incidence ofhistiocytic sarcomas and malignant lymphomas in all groups of maleand female treated mice. Chemical-associated increases in histiocyticsarcoma are not commonly observed in the bioassay in mice. Theincidence of spontaneous histiocytic sarcoma occurs two to threetimes more frequently in female than in male B6C3F1 mice; however,in this phenolphthalein study, incidences were greater in the treatedmale groups. Histiocytic sarcomas were observed most often in theliver and occasionally at other sites. Histiocytic sarcomas are generally considered to arise from a macrophage/histiocyte and possiblyfrom the specialized Kupifer cell of the liver, but definitive datarelative to the site of origin are lacking.

Many of the phenolphthalein-induced malignant lymphomas werethought to originate in the thymus. Spontaneously occurring lymphomas in mice in the NiP studies usually originate in the spleen orlymph nodes rather than in the thymus. With phenolphthalein, 1,3-butadiene, and DDC (18), the lymphomas originated primarily in thethymus.

Phenolphthalein, 1,3-butadiene, and DDC have in common positiveresults in in vivo genotoxic tests, suggesting that genotoxicity may bea contributing factor to the finding of thymic lymphomas. Althoughmutations that activate ras proto-oncogenes are common in thymiclymphomas induced by a number of environmental agents [includingN-methylnitrosurea (19, 20) and radiation (21)], ras mutations wereseen in only 2 of 11 thymic lymphomas from butadiene-treated mice(22). ras mutations were more common in butadiene-induced lung andliver tumors (6/7 lung tumors had K-ras mutations, 3/7 liver tumorshad K-ras, and 4/7 liver tumors had H-ras mutations; Ref. 22).

In female mice, there was an increase in the severity of bonemarrow myelofibrosis and in males an increase in the incidence of this

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PHENOLPHTHALEINEXPOSURE IN EXPERIMENTAL MODELS

is approximately 0.1—0.2 @tg/mlplasma over a dose range of 500-2000 mg/kg/day.2 The findings that the plasma level of free phenolphthalein were approximately the same for the three dose levels usedin the 2-year study may explain in part why the carcinogenic responsewas not proportional to dose in the kidney of male rats, ovary offemale mice, and hematopoietic system of mice. The low dose used inthe 2-year mouse study is within 10 times the human dose level whencompared on a mg/m2 body surface area basis (Table 2).

Other structurally related di- or triphenylmethane chemicals (e.g.,gentian violet) form free radicals (30, 31) and cause cancer in mice[hematological cancers and ovarian toxicity (32)]. Recent studies havealso confirmed that phenolphthalein fonns free radicals, suggesting thatphenolphthalein may be a significant source of oxidative stress in physiological systems. It is possible that one mechanism for the cancer seenin the phenolphthalein-treated animals is through the formation of oxygen

radicals and subsequent cellular damage (33). Phenolphthalein is capableof being converted to a quinoid structure. Quinoids are highly reactivechemicals capable of reacting with sulffiydryl groups and amino groupsand forming free oxygen radicals (34).

Epidemiology studies are now underway to identify any potentialovarian effects that phenolphthalein exposure may have on women inthe United States.

ACKNOWLEDGMENTS

We thank Dr. Joel Mahler and Dr. Thomas Burka, National Institute ofEnvironmental Health Sciences and Dr. Michael Elwell, Experimental Pathology Laboratories for their helpful comments and review of the manuscript.

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