Joseph K. Haseman

The comparative toxicity of chlorinated dibenzo-p-dioxins in mice and guinea pigs

Abstract Mice and guinea pigs were given a single oral dose of various chlorinated dibenzo-p-dioxins (CDD) to establish and compare the LD50 and clinical and pathologic manifestations of toxicity. It was apparent that the 2,3,7, and 8 positions must be chlorinated to achieve the greatest degree of toxicity. Additional chlorine atoms at an ortho position reduced toxicity but not nearly to the degree caused by deletion of a chlorine atom at one of the lateral positions. A decrease in body weight gain was the most sensitive clinical parameter and animals severely intoxicated showed a marked weight loss, especially guinea pigs. The median time to death at a LD50 was 17 to 20 days in guinea pigs and 22 to 25 days in mice. Doses 10 times greater did not markedly shorten this period. At the toxic dose the spectrum and severity of lesions and organ weight effects were similar for all homologs and isomers within the same animal species; however, there were interspecies differences. The thymus was greatly reduced in size in both animal species due to a reduction in the number of cortical lymphocytes. Significant macroscopic and histopathologic hepatic effects including porphyria were observed only in the mouse and were found at does levels well below the LD50. Hyperplasia of the transitional epithelium in the urinary tract was found in guinea pigs. There was a reduction of total serum protein in the mouse due to lower levels of α-globulin. Other lesions were generally interpreted to be a secondary response to debilitation.

Use of Historical Control Data in Carcinogenicity Studies in Rodents

This paper considers the use of historical control data in the evaluation of tumor incidences from carcinogenicity studies in rodents. Although the most appropriate control group for interpretative purposes is always the concurrent control, there are instances in which the use of historical control information can aid an investigator in the overall evaluation of tumor incidence data. One example is rare tumors; another is a tumor that shows a marginally significant result relative to concurrent controls. However, before historical control data can be used in a formal testing framework, a number of important issues must first be considered. The nomenclature conventions and diagnostic criteria for each study should be identical to insure unambiguous identification of all relevant tumors in the historical control database. Criteria should be established that will aid in determining whether a particular study should be included in the database. This will assure a homogeneous set of studies upon which to base statistical comparisons. Since study-to-study variability in tumor rates may exceed what would be expected by chance alone, these sources of variability should be identified and controlled. Finally, statistical procedures should be employed that adjust for extra-binomial variability. This paper also summarizes tumor incidence data from untreated Fischer 344 rats and B6C3F1 mice in the National Toxicology Program (NTP) historical control database. All studies in the database are of two years duration, and all neoplasms occurring with a frequency of 0.5% or more are reported.

Spontaneous Neoplasm Incidences in Fischer 344 Rats and B6C3F1 Mice in Two-Year Carcinogenicity Studies: A National Toxicology Program Update

Spontaneous neoplasm rates were determined for control Fischer 344 (F344) rats and B6C3F, mice from 2-yr rodent carcinogenicity studies carried out by the National Toxicology Program (NTP). The most frequently occurring neoplasms in untreated male F344 rats were testicular adenoma (89.1%), mononuclear cell leukemia (50.5%), adrenal gland pheochromocytoma (31.9%), and pituitary gland neoplasms (30.4%). For untreated female F344 rats, the most frequently occurring neoplasms were pituitary gland neoplasms (54.2%), mammary gland fibroadenoma (41.2%), and mononuclear cell leukemia (28.1%). The most frequently occurring neoplasms in untreated male B6C3F, mice were liver adenoma/carcinoma (42.2%), lung adenoma/carcinoma (20.5%), and malignant lymphoma (8.3%). For untreated female B6C3F, mice, the most frequently occurring neoplasms were liver adenoma/carcinoma (23.6%), malignant lymphoma (20.9%), and pituitary gland adenoma/carcinoma (1.4.8%). The tumor rates observed in feeding study (untreated) and inhalation study (chamber) control rats were generally similar. The major exceptions were pituitary gland tumors and testicular adenoma in male F344 rats. The overall incidence of testicular adenoma was much lower in chamber controls (69.4%) than in feeding study controls (89.1%), whereas pituitary gland neoplasms showed the opposite trend (60.7% vs 30.4%). The most likely explanation for this difference is related to the individual housing of chamber controls and the group housing of feeding study controls. Differences in diagnostic criteria may influence reported tumor rates. To ensure consistency and comparability of tumor diagnosis from study to study, the NTP uses rigorous histopathology quality assurance and peer review procedures. Biological factors such as body weight may also affect tumor incidence. For example, increased body weights are associated with increased incidences of certain site-specific neoplasms, especially pituitary gland and mammary gland neoplasms in rats and liver tumors in mice. The presence of Helicobacter hepaticus has been associated with an increased incidence of liver neoplasms in male B6C3F, mice. Other factors that may produce differences in control tumor rates from study to study include diet, environmental factors, genetic drift, study duration, and survival differences. The NTP database provides historical control data that may be useful in the evaluation of possible chemically related changes in tumor incidence. However, it is essential that the study being evaluated be comparable to those in the NTP database with respect to those factors that are known to influence tumor occurrence.

The use of a correlated binomial model for the analysis of certain toxicological experiments.

In certain toxicological experiments with laboratory animals, the outcome of interest is the occurrence of dead or malformed fetuses in a litter. Previous investigations have shown that the simple one-parameter binomial and Poisson models generally provide poor fits to this type of binary data. In this paper, a type of correlated binomial model is proposed for use in this situation. First, the model is described in detail and is compared to a beta-binomial model proposed by Williams (1975). These two-parameter models are then contrasted for goodness of fit to some real-life data. Finally, numerical examples are given in which likelihood ratio tests based on these models are employed to assess the significance of treatment-control differences.

The carcinogenicity of dietary di(2‐ethylhexyl) phthalate (DEHP) in fischer 344 rats and B6C3F1 mice

Groups of 50 male and female Fischer 344 rats and male and female B6C3Fl mice were fed diets containing 6000 or 12,000 (rats) or 3000 or 6000 (mice) mg di(2‐ethylhexyl) phthalate (DEHP)/kg feed for 103 consecutive wk. Concurrent controls (50 of each sex and species) were fed diet without the addition of DEHP. Treatment with DEHP did not affect survival rates for rats or mice, nor did it alter the amount of food consumed. Mean body weight gains of treated male rats (6000 and 12,000 mg/kg), female rats (12,000 mg/kg), and female mice (3000 and 6000 mg/kg) were less than those of the corresponding controls. Seminiferous tubular degeneration and hypertrophy of cells in the anterior pituitary were observed in male rats at 12,000 mg/kg, and chronic inflammation of the kidney and seminiferous tubular degeneration were observed in male mice at 6000 mg/kg. Neither clinical signs of toxicity nor nonneoplastic lesions were detected in the other treated groups at incidences greater than in the corresponding controls....

Growth, Body Weight, Survival, and Tumor Trends in F344/N Rats during an Eleven-Year Period:

Time trends for growth, body weight, survival and tumor prevalences in 144 diet control groups with a total of 5,184 male F344/N rats and 146 diet control groups with a total of 5,289 female rats of NCI-NTP 2-yr chemical carcinogenicity studies started during an 11-yr period (1971 to 1981) in 11 toxicology testing laboratories were evaluated. Male and female rats in more recent studies grew faster and attained a higher body weight than rats from earlier studies. Survival of males showed a significantly decreasing trend over time, which may have been related to diseases associated with increasing body weight, prevalence of leukemia and changes in criteria for euthanasia of moribund animals. The time trend for survival of females was not significant. There were highly significant (p < 0.001) positive time trends for prevalences of leukemia, anterior pituitary tumors and thyroid C-cell tumors in both sexes, adrenal pheochromocytomas in males and mammary tumors and endometrial stromal polyps in females. The prevalence of mammary tumors in females and pituitary tumors in males had a highly significant (p < 0.01) positive association with body weight. Histological reevaluation of tumor prevalences in approximately 250 rats of each sex at each of 4 different time periods indicated that changes in diagnostic criteria may have contributed to but could not totally explain the increased prevalence of leukemia. Changes in diagnostic criteria and the amount of tissue examined may have contributed to the increased prevalence of anterior pituitary tumors in both sexes and adrenal pheochromocytomas in males. Interlaboratory variability and changes in diet may also have contributed to the time-related trends.

p53 +=¡ Hemizygous Knockout Mouse: Overview of Available Data

The performance of the p53 +/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institutes (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17β-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-defi cient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.

Patterns of Tumor Incidence in Two-Year Cancer Bioassay Feeding Studies in Fischer 344 Rats

Patterns of tumor incidence in 25 of the National Toxicology Programs (NTPs) two-year cancer bioassay feeding studies in Fischer 344 rats were investigated. It was found that the overall frequencies of statistically significant (P less than 0.01) increases and decreases in organ-specific tumor incidence in treated groups relative to controls were approximately the same. The decreases were due primarily to mammary gland fibroadenomas in females (which were clearly associated with decreased weight gain in the treated groups) and leukemia/lymphoma in both sexes (which were frequently associated with increased liver tumor incidences in the treated groups). A clear explanation for this latter association was not apparent. The increased tumor incidences in the treated groups relative to controls were due primarily to liver neoplastic nodules. Treated animals of either sex had slightly (but significantly) improved overall survival relative to controls. The intra- and inter-laboratory variability in control tumor incidence frequently exceeded what one would expect to find by chance alone. This investigation helps explain the decreased tumor incidences observed in many NTP feeding studies. It also indicates that attention should be directed toward the determination of underlying mechanisms responsible for the negative correlation between the incidences of liver tumors and leukemia/lymphoma.

Immortalization of Human Uterine Leiomyoma and Myometrial Cell Lines After Induction of Telomerase Activity: Molecular and Phenotypic Characteristics

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-α and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle–specific cytoskeletal protein α-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.

Dose-Additive Carcinogenicity of a Defined Mixture of “Dioxin-like Compounds”

Use of the dioxin toxic equivalency factor (TEF) approach in human risk assessments assumes that the combined effects of dioxin-like compounds in a mixture can be predicted based on a potency-adjusted dose-additive combination of constituents of the mixture. In this study, we evaluated the TEF approach in experimental 2-year rodent cancer bioassays with female Harlan Sprague-Dawley rats receiving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of the three compounds. Statistically based dose–response modeling indicated that the shape of the dose–response curves for hepatic, lung, and oral mucosal neoplasms was the same in studies of the three individual chemicals and the mixture. In addition, the dose response for the mixture could be predicted from a combination of the potency-adjusted doses of the individual compounds. Finally, we showed that use of the current World Health Organization dioxin TEF values adequately predicted the increased incidence of liver tumors (hepatocellular adenoma and cholangiocarcinoma) induced by exposure to the mixture. These data support the use of the TEF approach for dioxin cancer risk assessments.