PBDE-47 and PBDE mixture (DE-71) toxicities and liver transcriptomic changes at PND 22 after in utero/postnatal exposure in the rat.

Pentabromodiphenyl ethers (PBDE) are found in human tissue, in household dust, and in the environment, and a particular concern is the potential for the induction of cancer pathways from these fat-soluble persistent organic pollutants. Only one PBDE cancer study has been conducted and that was for a PBDE mixture (DE-71). Because it is not feasible to test all PBDE congeners in the environment for cancer potential, it is important to develop a set of biological endpoints that can be used in short-term toxicity studies to predict disease outcome after long-term exposures. In this study, PBDE-47 was selected as the test PBDE congener to evaluate and compare toxicity to that of the carcinogenic PBDE mixture. The toxicities of PBDE-47 and the PBDE mixture were evaluated at PND 22 in Wistar Han rat (Crl: WI (Han)) pups after in utero/postnatal exposure (0, 0.1, 15, or 50 mg/kg; dams, GD6-21; pups, PND 12-PND 21; oral gavage daily dosing). By PND 22, PBDE-47 caused centrilobular hypertrophy and fatty change in liver, and reduced serum thyroxin (T4) levels; similar effects were also observed after PBDE mixture exposure. Transcriptomic changes in the liver included induction of cytochrome p450 transcripts and up-regulation of Nrf2 antioxidant pathway transcripts and ABC membrane transport transcripts. Decreases in other transport transcripts (ABCG5 & 8) provided a plausible mechanism for lipid accumulation, characterized by a treatment-related liver fatty change after PBDE-47 and PBDE mixture exposure. The benchmark dose calculation based on liver transcriptomic data was generally lower for PBDE-47 than for the PBDE mixture. The up-regulation of the Nrf2 antioxidant pathway and changes in metabolic transcripts after PBDE-47 and PBDE mixture exposure suggest that PBDE-47, like the PBDE mixture (NTP 2016, TR 589), could be a liver toxin/carcinogen after long-term exposure.

Carcinogenic activity of pentabrominated diphenyl ether mixture (DE-71) in rats and mice.

Pentabrominated diphenyl ether (PBDE) flame retardants have been phased out in Europe and in the United States, but these lipid soluble chemicals persist in the environment and are found human and animal tissues. PBDEs have limited genotoxic activity. However, in a 2-year cancer study of a PBDE mixture (DE-71) (0, 3, 15, or 50 mg/kg (rats); 0, 3, 30, or 100 mg/kg (mice)) there were treatment-related liver tumors in male and female Wistar Han rats [Crl:WI(Han) after in utero/postnatal/adult exposure, and in male and female B6C3F1 mice, after adult exposure. In addition, there was evidence for a treatment-related carcinogenic effect in the thyroid and pituitary gland tumor in male rats, and in the uterus (stromal polyps/stromal sarcomas) in female rats. The treatment-related liver tumors in female rats were unrelated to the AhR genotype status, and occurred in animals with wild, mutant, or heterozygous Ah receptor. The liver tumors in rats and mice had treatment-related Hras and Ctnnb mutations, respectively. The PBDE carcinogenic activity could be related to oxidative damage, disruption of hormone homeostasis, and molecular and epigenetic changes in target tissue. Further work is needed to compare the PBDE toxic effects in rodents and humans.

Tetrabromobisphenol A activates the hepatic interferon pathway in rats.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant in printed circuit boards, paper, and textiles. In a two-year study, TBBPA showed evidence of uterine tumors in female Wistar-Han rats and liver and colon tumors in B6C3F1 mice. In order to gain further insight into early gene and pathway changes leading to cancer, we exposed female Wistar Han rats to TBBPA at 0, 25, 250, or 1000mg/kg (oral gavage in corn oil, 5×/week) for 13 weeks. Because at the end of the TBBPA exposure period, there were no treatment-related effects on body weights, liver or uterus lesions, and liver and uterine organ weights were within 10% of controls, only the high dose animals were analyzed. Analysis of the hepatic and uterine transcriptomes showed TBBPA-induced changes primarily in the liver (1000mg/kg), with 159 transcripts corresponding to 132 genes differentially expressed compared to controls (FDR=0.05). Pathway analysis showed activation of interferon (IFN) and metabolic networks. TBBPA induced few molecular changes in the uterus. Activation of the interferon pathway in the liver occurred after 13-weeks of TBBPA exposure, and with longer term TBBPA exposure this may lead to immunomodulatory changes that contribute to carcinogenic processes.

Environmental chemical exposure may contribute to uterine cancer development: studies with tetrabromobisphenol A.

Tetrabromobisphenol A (TBBPA), a widely used flame retardant, caused uterine tumors in rats. In this study, TBBPA was administered to male and female Wistar Han rats and B6C3F1/N mice by oral gavage in corn oil for 2 years at doses up to 1,000 mg/kg. TBBPA induced uterine epithelial tumors including adenomas, adenocarcinomas, and malignant mixed Müllerian tumors (MMMTs). In addition, endometrial epithelial atypical hyperplasia occurred in TBBPA-treated rats. Also found to be related to TBBPA treatment, but at lower incidence and at a lower statistical significance, were testicular tumors in rats, and hepatic tumors, hemangiosarcomas (all organs), and intestinal tumors in male mice. It is hypothesized that the TBBPA uterine tumor carcinogenic mechanisms involve altered estrogen levels and/or oxidative damage. TBBPA treatment may affect hydroxysteroid-dehydrogenase-17ß (HSD17ß) and/or sulfotransferases, enzymes involved in estrogen homeostasis. Metabolism of TBBPA may also result in the formation of free radicals. The finding of TBBPA-mediated uterine cancer in rats is of concern because TBBPA exposure is widespread and endometrial tumors are a common malignancy in women. Further work is needed to understand TBBPA cancer mechanisms.

Toxicokinetics of bis(2-chloroethoxy)methane following intravenous administration and dermal application in male and female F344/N rats and B6C3F1 mice.

In the National Toxicology Program's toxicity studies, rats were more sensitive than mice to Bis(2-chloroethoxy)methane (CEM) - induced cardiac toxicity following dermal application to male and female F344/N rats and B6C3F1 mice. Thiodiglycolic acid (TDGA) is a major metabolite of CEM in rats. It has been implicated that chemicals metabolized to TDGA cause cardiac toxicity in humans. Therefore, the toxicokinetics of CEM and TDGA were investigated in male and female F344/N rats and B6C3F1 mice following a single intravenous administration or dermal application of CEM to aid in the interpretation of the toxicity data. Absorption of CEM following dermal application was rapid in both species and genders. Bioavailability following dermal application was low but was higher in rats than in mice with females of both species showing higher bioavailability than males. CEM was rapidly distributed to the heart, thymus, and liver following both routes of administration. Plasma CEM C(max) and AUC(∞) increased proportionally with dose, although at the dermal dose of 400mg/kg in rats and 600mg/kg in mice non-linear kinetics were apparent. Following dermal application, dose-normalized plasma CEM C(max) and AUC(∞) was significantly higher in rats than in mice (p-value<0.0001 for all comparisons except for C(max) in the highest dose groups where p-value=0.053). In rats, dose-normalized plasma CEM C(max) and AUC(∞) was higher in females than in males: however, the difference was significant only at the lowest dose (p-value=0.009 for C(max) and 0.056 for AUC(∞)). Similar to rats, female mice also showed higher C(max) and AUC(∞) in females than in male: the difference was significant only for C(max) at the lowest dose (p-value=0.002). Dose-normalized heart CEM C(max) was higher in rats than in mice and in females than their male counterparts. The liver CEM C(max) was lower compared to that of heart and thymus in both rats and mice following intravenous administration and in rats following dermal application. This is likely due to the rapid metabolism of CEM in the liver as evidenced by the high concentration of TDGA measured in the liver. Dose-normalized plasma and heart TDGA C(max) values were higher in rats compared to mice. In rats, females had higher plasma and heart TDGA C(max) than males; however, there was no gender difference in plasma or heart TDGA C(max) in mice. These findings support the increased sensitivity of rats compared to mice to CEM-induced cardiac toxicity. Data also suggest that, either CEM C(max) or AUC can be used to predict the CEM-induced cardiac toxicity. Although, both plasma and heart TDGA C(max) was consistent with the observed species difference and the gender difference in rats, the gender difference in mice to cardiac toxicity could not be explained based on the TDGA data. This animal study suggests that toxicologically significant concentrations of CEM and TDGA could possibly be achieved in the systemic circulation and/or target tissues in humans as a result of dermal exposure to CEM.

Somatic mutation databases as tools for molecular epidemiology and molecular pathology of cancer: proposed guidelines for improving data collection, distribution, and integration.

There are currently less than 40 locus-specific databases (LSDBs) and one large general database that curate data on somatic mutations in human cancer genes. These databases have different scope and use different annotation standards and database systems, resulting in duplicated efforts in data curation, and making it difficult for users to find clear and consistent information. As data related to somatic mutations are generated at an increasing pace it is urgent to create a framework for improving the collecting of this information and making it more accessible to clinicians, scientists, and epidemiologists to facilitate research on biomarkers. Here we propose a data flow for improving the connectivity between existing databases and we provide practical guidelines for data reporting, database contents, and annotation standards. These proposals are based on common standards recommended by the Human Genome Variation Society (HGVS) with additions related to specific requirements of somatic mutations in cancer. Indeed, somatic mutations may be used in molecular pathology and clinical studies to characterize tumor types, help treatment choice, predict response to treatment and patient outcome, or in epidemiological studies as markers for tumor etiology or exposure assessment. Thus, specific annotations are required to cover these diverse research topics. This initiative is meant to promote collaboration and discussion on these issues and the development of adequate resources that would avoid the loss of extremely valuable information generated by years of basic and clinical research.

Cardiotoxicity of Ma Huang/caffeine or ephedrine/caffeine in a rodent model system.

Ma Huang (equivalent to 0, 12.5, 25, or 50 mg/kg ephedrine) or ephedrine (0, 6.25, 12.5, 25 mg/kg) were administered as one bolus oral dose to male F344 rats with and without caffeine. The herbal medicine Ma Huang (ephedra) in combination with caffeine caused rapid clinical signs of toxicity including salivation, hyperactivity, ataxia, and eventually lethargy, and failure to respond to stimuli. When this syndrome of clinical signs emerged, animals were moribund sacrificed, and a histological analysis for heart lesions performed. Cardiotoxicity included hemorrhage, necrosis, and degeneration in the ventricles or interventricular septum within 2-4 hours after treatment with Ma Huang (ephedra)/caffeine or ephedrine (the principal active component in Ma Huang)/caffeine. There was a steep dose response curve for cardiotoxicity with minimal toxicity seen at levels of Ma Huang (equivalent to 12.5 mg/kg ephedrine) with caffeine. However, cardiotoxic lesions occurred in 28% of animals with Ma Huang dosages equivalent to 25 mg/kg ephedrine with 15 or 30 mg/kg caffeine, and in 90% of animals at Ma Huang exposures equivalent to 50 mg/kg ephedrine with 15 or 30 mg/kg caffeine. Cardiotoxic lesions occurred in 47% of animals in the 25 mg/kg ephedrine groups with caffeine at 7.25, 15, or 30 mg/kg. There was no statistical difference in the occurrence of cardiotoxic lesions when 15 or 30 mg/kg caffeine was combined with Ma Huang equivalent to 25 or 50 mg/kg ephedrine; likewise there was no statistical difference in the occurrence of cardiotoxic lesions when 7.25, 15, or 30 mg/kg caffeine was combined with 25 mg/kg ephedrine. These results show that the cardiotoxic effects of the herbal medicine, Ma Huang, are similar to that of ephedrine, the principal active ingredient in the herbal medicine. The combination of Ma Huang or ephedrine with caffeine enhanced the cardiotoxicity over that with the herbal medicine or the active ingredient alone.

Genetic pathways and mutation profiles of human cancers: site- and exposure-specific patterns.

Cancer is a complex disease that involves the accumulation of both genetic and epigenetic alterations of numerous genes. Data in the Genetic Alterations in Cancer database for gene mutations and allelic loss [loss of heterozygosity (LOH)] in human tumors (e.g. lung, oral, esophagus, stomach and colon/rectum) were reviewed. Results for the genes and pathways implicated in tumor development at these sites are presented. Mutation incidence, spectra and codon specificity are described for lung, larynx and oral tumors. LOH occurred more frequently than gene mutations in tumors from all sites examined. The cell cycle gene, TP53 (all sites), and cell signaling gene, APC (colorectal and gastric cancers), were the only genes with similar incidences of LOH and mutation. Alterations of one or more cell cycle and cell signaling genes were reported for tumors from each site. Site-specific activation was apparent in the cell signaling mitogen-activated protein kinase oncogenes (KRAS in lung, HRAS in oral cancers and BRAF in esophageal and colorectal cancers). Analysis of genetic changes in lung tumors showed that the incidence of mutations in the TP53 and KRAS genes and the incidence of LOH in the FHIT gene were significantly greater in smokers versus non-smokers (P < 0.01). In lung and oral cancers, the TP53 GC --> TA transversion frequency increased with tobacco smoke exposure (P < 0.05). Furthermore, the TP53 mutational hot spots for lung and laryngeal cancers from smokers included codons 157, 245 and 273, whereas for oral tumors included codons 280 and 281.

Critical pathways in heart function: bis(2-chloroethoxy)methane-induced heart gene transcript change in F344 rats.

Gene transcript changes after exposure to the heart toxin, bis(2-chloroethoxy)methane (CEM), were analyzed to elucidate mechanisms in cardiotoxicity and recovery. CEM was administered to 5-week-old male F344/N rats at 0, 200, 400, or 600 mg/kg by dermal exposure, 5 days per week, for a total of 12 doses by study day 16. Heart toxicity occurred after 2 days of dosing in all 3 regions of the heart (atrium, ventricle, interventricular septum) and was characterized by myofiber vacuolation, necrosis, mononuclear-cell infiltration, and atrial thrombosis. Ultrastructural analysis revealed that the primary site of damage was the mitochondrion. By day 5, even though dosing was continued, the toxic lesions in the heart began to resolve, and by study day 16, the heart appeared histologically normal. RNA was extracted from whole hearts after 2 or 5 days of CEM dosing. After a screen for transcript change by microarray analysis, dose-response trends for selected transcripts were analyzed by qRT-PCR. The selected transcripts code for proteins involved in energy production, control of calcium levels, and maintenance of heart function. The down-regulation of ATP subunit transcripts (Atp5j, ATP5k), which reside in the mitochondrial membranes, indicated a decrease in energy supply at day 2 and day 5. This was accompanied by down-regulation of transcripts involved in high-energy consumption processes such as membrane transport and ion channel transcripts (e.g., abc1a, kcnj12). The up-regulation of transcripts encoding for temperature regulation and calcium binding proteins (ucp1 and calb3) only at the 2 low exposure levels, suggest that these adaptive processes cannot occur in association with severe cardiotoxicity as seen in hearts at the high exposure level. Transcript expression changes occurred within 2 days of CEM exposure, and were dose-and time-dependent. The heart transcript changes suggest that CEM cardiotoxicity activates protective processes associated energy conservation and maintenance of heart function.

o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis.

In the previous 500 2-year chemical bioassays within the National Toxicology Program, large intestinal tumors (cecal carcinomas) related to chemical exposure have not been observed in B6C3F1 mice. The recently completed o-nitrotoluene study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. Morphologically, the carcinomas were gland-forming tumors lined by tall columnar epithelial cells that were positive for cytokeratin 20 and negative for cytokeratin 7. Using immunohistochemistry beta-catenin (encoded by Catnb) protein accumulation was detected in 80% (8/10) of the cecal carcinomas, while increased cyclin D1 and p53 protein expression was detected in 73% (8/11), respectively. There was no difference in adenomatous polyposis protein expression between normal colon and cecal carcinomas. All tumors examined exhibited mutations in exon 2 (corresponds to exon 3 in humans) in the Catnb gene. Mutations in p53 were identified in nine of 11 carcinomas, and all were in exon 7. Analysis of the K-ras gene revealed mutations in 82% (9/11) of carcinomas; all had specific G --> T transversions (Gly --> Val) at codons 10 or 12. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations that activate signal transduction pathways (K-ras and Catnb) and changes that disrupt the cell-cycle and bypass G(1) arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene exposure.

Chemical-specific alterations in ras, p53, and beta-catenin genes in hemangiosarcomas from B6C3F1 mice exposed to o-nitrotoluene or riddelliine for 2 years.

The most prominent neoplastic lesions in mice in the 2-year studies of o-nitrotoluene and riddelliine were hemangiosarcomas. Fifteen o-nitrotoluene-induced hemangiosarcomas of the skeletal muscle, subcutaneous tissue, and mesentery; 12 riddelliine-induced hemangiosarcomas of the liver; and 15 spontaneous subcutaneous hemangiosarcomas were examined for genetic alterations in ras, p53, and beta-catenin genes. Mutations in at least one of these genes were identified in 13 of 15 (87%) of the o-nitrotoluene-induced hemangiosarcomas with missense mutations in p53 exons 5-8 detected in 11 of 15 (73%) of these neoplasms. Seven of 15 (47%) hemangiosarcomas from mice exposed to o-nitrotoluene had deletions at exon 2 splice sites or smaller deletions in the beta-catenin gene. K-ras mutation was detected in only 1 of the 15 (7%) o-nitrotoluene-induced hemangiosarcomas. In contrast to the o-nitrotoluene study, 7/12 (58%) riddelliine-induced hemangiosarcomas had K-ras codon 12 GTT mutations and, when screened by immunohistochemistry, 9/12 (75%) had strong staining for the p53 protein in malignant endothelial cells, the cells of origin of hemangiosarcomas. Riddelliine-induced hemangiosarcomas were negative for the beta-catenin protein. Spontaneous hemangiosarcomas from control mice lacked both p53 and beta-catenin protein expression and ras mutations. Our data indicated that p53 and beta-catenin mutations in the o-nitrotoluene-induced hemangiosarcomas and K-ras mutations and p53 protein expression in riddelliine-induced hemangiosarcomas most likely occurred as a result of the genotoxic effects of these chemicals. It also suggests that these mutations play a role in the pathogenesis of the respective hemangiosarcomas in B6C3F1(1) mice.

P-Nitrobenzoic acid alpha2u nephropathy in 13-week studies is not associated with renal carcinogenesis in 2-year feed studies.

The objective of this study was to characterize the renal toxicity and carcinogenicity of p-nitrobenzoic acid in F344 rats. Dose levels in 13-week and 2-year studies ranged from 630-10,000 ppm and 1,250-5,000 ppm, respectively. At 13 weeks, renal lesions included minimal to mild hyaline droplet accumulation in male rats and karyomegaly in male and female rats. At 2 years, renal lesions included proximal tubule epithelial cell hyperplasia in male rats and oncocytic hyperplasia in high-dose male and female rats, and a decreased severity of nephropathy in males and females. The hvaline droplets in renal tubular epithelial cells of male rats at 13 weeks were morphologically similar to those described in alpha2u-globulin nephropathy. Using immunohistochemical methods, alpha2u-globulin accumulation was associated with the hyaline droplets. In addition, at 13 weeks, cell proliferation as detected by PCNA immunohistochemistry was significantly increased in males exposed to 5,000 and 10,000 ppm when compared to controls. Cytotoxicity associated with alpha2U-globulin nephropathy such as single-cell necrosis of the P2 segment epithelium or accumulation of granular casts in the outer medulla did not occur in the 13-week study. In addition, chronic treatment related nephrotoxic lesions attributed to accumulation of alpha2u-globulin such as linear foci of mineralization within the renal papilla, hyperplasia of the renal pelvis urothelium and kidney tumors were not observed. Although there was histologic evidence of alpha2u-globulin accumulation in male rats at 13 weeks, the minimal severity of nephropathy suggests that the degree of cytotoxicity was below the threshold, which would contribute to the development of renal tumors at 2 years.

Chromosome 11 loss from thymic lymphomas induced in heterozygous Trp53 mice by phenolphthalein.

C57BL/6 p53 (+/-) N5 mice heterozygous for a null p53 allele were given phenolphthalein to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. DNA samples isolated from 10 phenolphthalein-induced thymic lymphomas were analyzed for loss of heterozygosity (LOH) at the Trp53 locus and simple sequence length polymorphic (SSLP) loci. The initial screening revealed remarkable results from only chromosome 11. Allelotyping at approximately five centiMorgan intervals, we found SSLP heterozygosity for C57BL/6 and 129Sv over much of chromosome 11. In the tumors, treatment-related LOH was apparent on chromosome 11 at each of the 28 informative loci examined. The strain-specific polymorphism lost from individual tumors allowed us to deduce the distribution of alleles along the length of the maternal and paternal chromosomes 11. The allelic patterns indicate that mitotic homologous recombination occurred during embryogenesis if breeding protocols were carried out as described. The mitotic recombination observed may be attributable to p53 haploinsufficiency for normal suppression of mitotic recombination.

Chromosome 11 allelotypes reflect a mechanism of chemical carcinogenesis in heterozygous p53-deficient mice.

Mice heterozygous for a null p53 allele were administered three well-characterized carcinogens to learn more about mechanisms of carcinogenesis and to evaluate the p53-deficient mouse as a tool for identifying potential human carcinogens. Benzene-induced sarcomas, p-cresidine-induced bladder carcinomas and phenolphthalein-induced thymic lymphomas were allelotyped at the Trp53 locus and chromosome 11 simple sequence length polymorphic (SSLP) loci. Loss of Trp53 and loss of one copy of chromosome 11 occurred in each of 10 lymphomas examined and each of the eight sarcomas examined. Loss of Trp53 and loss of heterozygosity (LOH) at SSLP loci were sporadic in the bladder carcinomas. However, LOH was detected at two or more SSLP loci in six of the eight bladder tumors examined. Loss of one complete copy of chromosome 11 was implicated in three of the bladder tumors where LOH occurred at seven or more widely dispersed SSLP loci. Loss of one copy of chromosome 11 likely occurred through a p53-mediated selection process since Trp53 is located on mouse chromosome 11 and only one copy harbored a functional gene. The data suggest that loss occurred through a mechanism common among the three tumor types. Allelotype patterns of the maternal chromosome 11 were inconsistent with those expected from a nullizygous C57BL/6-Trp53 (N4) x inbred C57BL/6 cross which was reported for production of the mice under investigation. However, comparison with individual control tissues still allowed deduction of maternal chromosome loss. If the breeding protocols were carried out as described, the unexpected allelotype patterns observed in histologically normal tissues might be due to mitotic homologous recombination during embryogenesis.

Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/-) mouse tumors is carcinogen-and tissue-dependent.

Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.

Toxicokinetics of phenolphthalein in male and female rats and mice.

Phenolphthalein (PTH), which has been used as the active ingredient in a number of prescription and over-the-counter laxative products, is a rodent chemical carcinogen in multiple organs in the NTP 2-year bioassay at doses of 291-2927 mg/kg. This paper describes the toxicokinetics and estimates the internal dose of PTH administered as a single iv or gavage dose, or ad libitum for 14 days in feed to F344 rats, B6C3F1 mice, p53 (+/-) mice, and C57BL mice at doses that bracketed those used in the bioassay. Plasma concentrations for free phenolphthalein (PTH-F) and phenolphthalein glucuronide (PTH-G) were obtained for each dose regimen. Total phenolphthalein (PTH-T) was calculated as the sum of the molar concentrations of PTH-F and PTH-G. Noncompartmental pharmacokinetic models were used to calculate the area under the curve (AUC) from 0 h to infinity (AUC(infinity)), clearance (Cl), and oral bioavailability (F) for PTH-F; and were used to calculate AUC(infinity), t((1/2)), and relative absorption (Q) for PTH-T. After iv administration, PTH-F rapidly declined in rats and mice; PTH-T rose rapidly to Cmax and slowly declined 6-8 h after dosing, with no sex-related differences for rats or mice. For feed studies, mean plasma concentration (f1.gif" BORDER="0">(infinity)) and 24-h area under the curve (AUC(24h)) values were calculated. Results from feed studies showed no dose response in rat plasma PTH-F above approximately 50 mg/kg. Rat PTH-T AUC(24h) and f1.gif" BORDER="0">(infinity) were linear with doses up to approximately 650 mg/kg. In B6C3F1 mice, PTH-F and PTH-T AUC(24h) increased nonlinearly with doses above approximately 165 mg/kg. PTH is well absorbed and readily converted to PTH-G when administered in feed to rats and mice, except at the highest bioassay doses, where PTH absorption may be saturated.

Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.

Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG-p53 mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time-weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, peripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN-PCE and MN-NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6-month treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose-dependent increase in the frequency of MN-PCE and MN-NCE and in %PCE. Maximal induction of MN-PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration-dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not exclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN-NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans.

Regenerative hyperplasia is not required for liver tumor induction in female B6C3F1 mice exposed to trihalomethanes.

Chloroform (TCM), a water disinfection by-product, induced liver tumors in female mice when administered by gavage in corn oil but not when given in drinking water at comparable daily doses. Because short-term studies showed that the gavage doses also induced liver toxicity, it has been suggested that the liver tumor response occurs secondary to cytotoxicity and consequent regenerative hyperplasia induced by oxidative metabolism of TCM to the toxic dihalocarbonyl intermediate. This study compares dose-response relationships of gavage-administered chlorinated/brominated trihalomethanes for hepatotoxicity, replicative DNA synthesis, and hepatocarcinogenicity in female B6C3F1 mice. The liver tumor data were obtained from previously published studies. Because bromine is a better leaving group than chlorine, metabolism of bromodichloromethane (BDCM) should produce the same intermediates as would be formed from TCM. Hence, the toxicity and carcinogenicity of BDCM was expected to be qualitatively similar to that of TCM. Dose responses for liver weight, serum sorbitol dehydrogenase and alanine aminotransferase (ALT) activities, hepatocyte degeneration, and hepatocyte labeling index (LI, a measure of replicative DNA synthesis) in female mice were similar following 3 weeks of gavage administration (once per day, 5 days per week) with TCM, BDCM, or chlorodibromomethane (CDBM). Fits of composite data for these trihalomethanes to a Hill equation model revealed sigmoidal dose responses for ALT activity and hepatocyte LI and a nearly linear low-dose response for liver tumor incidence. For this family of chemicals, the mouse liver tumor response was not associated with an elevated hepatocyte LI at doses of approximately 1 mmol/kg or less. High incidences of liver tumors were observed with BDCM and CDBM at doses that had a marginal effect or no effect on the hepatocyte LI. Thus, the carcinogenic effects of trihalomethanes are not simply a consequence of cytotoxicity and regenerative hyperplasia. The possible contributions from other activation pathways, including GSH conjugation and reductive metabolism, need to be considered in assessments of the carcinogenicity of the trihalomethanes.

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.