Phenolphthalein metabolite inhibits catechol-O-methyltransferase-mediated metabolism of catechol estrogens: a possible mechanism for carcinogenicity.

Phenolphthalein (PT), used in over-the-counter laxatives, has recently been identified as a multisite carcinogen in rodents, but the molecular species responsible for the carcinogenicity is not known. A catechol metabolite of PT, hydroxyphenolphthalein (PT-CAT), was recently identified and may be the molecular species responsible for at least part of the toxicity/carcinogenicity of PT. We hypothesize that PT-CAT inhibits the enzyme catechol-O-methyltransferase (COMT) and therefore potentiates genotoxicity by either PT-CAT itself or the endogenous catechol estrogens (CEs) in susceptible tissues. The present studies were conducted to determine the effects of PT treatment and PT-CAT itself on the COMT-mediated metabolism of 4- and 2-hydroxyestradiol both in vitro and in vivo. Female mice were treated with PT (50 mg/kg/d) for 21 days and then euthanized. PT-CAT concentration in urine reached plateau levels by 7 days of exposure. An O-methylated metabolite of PT-CAT was detected in feces. In vitro experiments demonstrated that PT treatment resulted in an increase in free CEs, which are normally cleared by COMT and a concurrent decrease in the capacity of hepatic catechol clearance by COMT. In vitro, PT-CAT was a substrate of COMT, with kinetic properties within the range measured with endogenous substrates. PT-CAT was an extremely potent mixed-type inhibitor of the O-methylation of the catechol estrogens, with 90-300 nM IC50s. The above data, when taken together, suggest that chronic administration of PT may enhance metabolic redox cycling of both PT-CAT and the catechol estrogens and this, in turn, may contribute to PT-induced tumorigenesis.

Cell-transforming activity and genotoxicity of phenolphthalein in cultured Syrian hamster embryo cells.

Phenolphthalein is a cathartic agent widely used in non-prescription laxatives. For the simultaneous assessment of in vitro carcinogenicity and mutagenicity of phenolphthalein, the ability of this chemical to induce cell transformation and genetic effects was examined using the Syrian hamster embryo (SHE) cell model. Cell growth was reduced by treatment with phenolphthalein at 10-40 microM in a dose-related manner. Treatment with phenolphthalein for 48 hr induced a dose-dependent increase in morphological transformation of SHE cells. Over the dose range that resulted in cell transformation ( 10-40 microM), treatment of SHE cells with phenolphthalein induced gene mutations at the hprt locus but not at the Na+/K+ ATPase locus. A statistically significant level of chromosomal aberrations was elicited in SHE cells treated with phenolphthalein at the highest dose (40 microM). Meanwhile, neither numerical chromosomal changes nor DNA adduct formation, analyzed by the nuclease P1 enhancement version of 32P-post-labeling, were induced by treatment with phenolphthalein at any concentrations examined. We thus report cell-transforming activity and mutagenicity of phenolphthalein assessed with the same mammalian cells in culture. Our results provide evidence that phenolphthalein has cell-transforming and genotoxic activity in cultured mammalian cells. The mutagenic and clastogenic activities of phenolphthalein could be a causal mechanism for carcinogenicity in rodents.

Phenolphthalein induces thymic lymphomas accompanied by loss of the p53 wild type allele in heterozygous p53-deficient (+/-) mice.

Epidemiology studies have indicated that many human cancers are influenced by environmental factors. Genetically altered mouse model systems offer us the opportunity to study the interaction of chemicals with genetic predisposition to cancer. Using the heterozygous p53-deficient (+/-) mouse, an animal model carrying one wild type p53 gene and one p53 null allele, we studied the effects of phenolphthalein on tumor induction and p53 gene alterations. Earlier studies showed that phenolphthalein caused carcinogenic effects in Fisher 344 rats and B6C3F1 mice after a 2-yr dosing period (Dunnick and Hailey, Cancer Res. 56: 4922-4926, 1996). The p53 (+/-) mice received phenolphthalein in the feed at concentrations of 200, 375, 750, 3,000, or 12,000 ppm (approximately 43, 84, 174, 689, or 2,375 mg/kg body weight/day or 129, 252, 522, 2,867, or 7,128 mg/m2 body surface area/day) for up to 6 mo. A target organ cancer site that accumulated p53 protein in the B6C3F1 mouse (i.e., thymic lymphoma) was also a target site for cancer in the p53 (+/-) mouse. In the p53 (+/-) mouse, treatment-related atypical hyperplasia and malignant lymphoma of thymic origin were seen in the control and dosed groups at a combined incidence of 0, 5, 5, 25, 100, and 95%, respectively. Twenty-one of the thymic lymphomas were examined for p53 gene changes, and all showed loss of the p53 wild type allele. Chemical-induced ovarian tumors in the B6C3F1 mouse showed no evidence for p53 protein accumulation and did not occur in the p53 (+/-) mouse. The p53-deficient (+/-) mouse model responded to phenolphthalein treatment with a carcinogenic response in the thymus after only 4 mo of dosing. This carcinogenic response took 2 yr to develop in the conventional B6C3F1 mouse bioassay. The p53-deficient (+/-) mouse is an important model for identifying a carcinogenic response after short-term (< 6 mo) exposures. Our studies show that exposure to phenolphthalein combined with a genetic predisposition to cancer can potentiate the carcinogenic process and cause p53 gene alterations, a gene alteration found in many human cancers.

Phenolphthalein exposure causes multiple carcinogenic effects in experimental model systems.

Phenolphthalein (a triphenylmethane derivative) has been commonly used as a laxative for most of the twentieth century, but little is known about its long-term carcinogenic potential in experimental studies. In our studies, phenolphthalein administered continuously in the feed for 2 years to F344 rats at doses of 0, 12,500, 25,000, and 50,000 ppm and to C57BL/6 x CH3 F1 (hereafter called B6C3F1) mice at doses of 0, 3,000, 6,000, and 12,000 ppm caused multiple carcinogenic effects. Treatment-related neoplasms occurred in the kidney and adrenal medulla in male rats, adrenal medulla in female rats, hematopoietic system in male and female mice (histiocytic sarcomas and malignant lymphomas), and ovary of female mice. Phenolphthalein has been shown to have estrogenic and clastogenic properties. Previous studies of other estrogenic chemicals (e.g., zearalenone) in the F344 rat and B6C3F1 mouse have not shown the same spectrum of carcinogenic activity as that found with phenolphthalein, suggesting that phenolphthalein estrogenic activity alone is not responsible for the spectrum of tumors observed. It is more likely that the multiple biological properties of phenolphthalein, including its ability to form free radicals, its clastogenic activity, and its estrogenic activity, contributed to the carcinogenic effects observed. These studies show that phenolphthalein is a multisite/multispecies carcinogen. One of the sites for neoplasm that is of particular concern is the ovary, and epidemiology studies are under way to identify any potential effects of phenolphthalein exposure at this site in humans.

Phenolphthalein: induction of micronucleated erythrocytes in mice.

Phenolphthalein was tested for the induction of micronucleated erythrocytes in mice. Results of an initial investigation revealed significant, dose-related increases in micronucleated polychromatic erythrocytes (MN-PCE) and normochromatic erythrocytes (MN-NCE) in peripheral blood samples of male and female mice exposed to 0.6% to 5% phenolphthalein (approximately 1100 to 10,000 mg/kg/day) in feed for 90 days (Dietz et al., 1992). Results from a second long-term feed study with Swiss CD-1 mice confirmed this effect. However, administration of comparable doses of phenolphthalein by corn oil gavage on two consecutive days gave negative results in a mouse bone marrow micronucleus test. Subsequent tests were performed to clarify the conflicting results seen in the chronic exposure, dosed-feed, peripheral blood studies and the acute, corn oil gavage, bone marrow studies. Phenolphthalein was administered to male B6C3F1 mice in feed (3%) for 14 days. Peripheral blood samples taken at 4, 7, and 14 days all showed significant increases in micronucleated PCE; bone marrow samples taken on days 7 and 14 also were clearly positive for micronucleus induction. Therefore, comparable results were obtainable from both bone marrow and peripheral blood analyses. Because of the negative results in the two-exposure gavage test, additional tests were then designed to investigate the effects of bolus vs continuous dosing, feeding vs gavage administration, and corn oil vs feed as a carrier for phenolphthalein. Results of these tests indicated that the rate of exposure to phenolphthalein affects the frequency of induced MN-PCE and that micronucleated erythrocytes can be induced by phenolphthalein either by feeding or by corn oil gavage administration. In all the acute exposure studies, relatively high doses of phenolphthalein (2000-6000 mg/kg/day for at least 2 days) were required to induce micronuclei. The positive results obtained with phenolphthalein in vivo were consistent with the results of an in vitro chromosomal aberration test in Chinese hamster ovary cells, where dose-related increases in aberrations were noted only in cells treated in the presence of induced rat liver S9.

Subchronic (13-week) toxicity studies of oral phenolphthalein in Fischer 344 rats and B6C3F1 mice.

Phenolphthalein is a cathartic agent that is widely used in over-the-counter laxatives. Thirteen-week toxicity studies of phenolphthalein were performed using F344/N rats and B6C3F1 mice. Rats and mice were fed ad libitum with a NIH 07 diet containing 0; 3000; 6000; 12,000; 25,000; or 50,000 ppm phenolphthalein. On a milligram per kilogram body weight basis, rats and mice fed 50,000 ppm phenolphthalein ingested more drug than would be expected during human laxative abuse. Phenolphthalein produced little evidence of toxicity in rats. There was slightly lower weight gain among the 25,000 and 50,000 ppm groups. Treated rats showed elevated relative kidney weights (males only) and elevated absolute and relative liver weights at 12,000-50,000 ppm phenolphthalein. Rat serum bile acids were depressed early (Days 5 and 6) by phenolphthalein treatment. Several treatment-related toxic effects, however, were identified in mice who received more phenolphthalein per unit body weight than rats. Although there were no effects on body weight gain, elevated liver weights were noted in female mice receiving 6000-50,000 ppm phenolphthalein. The primary treatment-related findings that occurred during the mouse studies involved the reproductive and hematopoietic systems. Reproductive changes including depressed testis and right epididymal weights and sperm density, an elevated production of abnormal sperm, and morphologic alterations in seminiferous tubules occurred at all levels of exposure (3000-50,000 ppm). Hematopoietic changes included bone marrow hypoplasia (12,000-50,000 ppm), increased splenic hematopoiesis (males only; 25,000 and 50,000 ppm), and an elevated incidence of micronucleated erythrocytes (6000-50,000 ppm).