Neil A. Littlefield

Commercial laboratory animal diets: toxicant and nutrient variability.

A commercial rodent feed was analyzed for a series of nutrients and potential contaminants during a 5-yr period. Annual average Cu and vitamin A concentrations were generally at least 12% lower than the approximate concentrations listed by the manufacturer, whereas Ca, protein, and vitamin B1 were within +/- 5% and fat and Zn within +/- 8% of the manufacturers specifications. Frequently, Se was found at concentrations at which it has been shown to interact with the process of chemical carcinogenesis. DDT, dieldrin, Cd, and Pb were occasionally close to concentrations known to have biological effects.

Influence of oral administration of sulfamethazine on thyroid hormone levels in fischer 344 rats

Fischer 344 rats (810 of each sex) were divided into treatment groups and fed diets containing 0, 10, 40, 600, 1200, or 2400 ppm sulfamethazine. Serum samples were analyzed for levels of thyroid-stimulating hormone (TSH), total thyroxine (T4), total triiodothyronine (T3), and T3 uptake after 12, 18, or 24 mo of continuous dosing. There were no statistically significant differences in T3 levels or percent T3 uptake for either sex after any of the exposure periods. The serum T4 levels were lower (p less than 0.05) for females dosed at 1200 and 2400 ppm for 18 mo and for males dosed at 600, 1200, or 2400 ppm sulfamethazine for 24 mo than for those dosed at levels of 40 ppm or less. Serum TSH levels showed a general increasing trend (but not statistically significant) among animals receiving 600 ppm or more sulfamethazine. There was a significant dose-related reduction in (T3 + T4)/TSH ratio for both sexes (p less than 0.05) after 18 and 24 mo of exposure at dose levels of 600 ppm or more. A lack of response at 12 mo may have been due to the shorter treatment time. At each sacrifice period both sexes of rats fed sulfamethazine at 1200 and 2400 ppm had significantly heavier (p less than 0.05) thyroid weights than animals fed control diet. The heavier thyroid weights in the dosed animals may have resulted from increased TSH levels. The cause of reduction in serum T4 was not clearly evident. Therefore, the thyroid hormone to pituitary feedback mechanism apparently compensated for sulfamethazine effects in most animals. This would suggest that the thyroid gland was not irreversibly affected.

Influence of total dose and dose rate in carcinogenicity studies

One element of the ED01 Study contained a group of animals that were dosed with 2-acetylaminofluorene for 9, 12, 15, 18, or 24 mo and then sacrificed at 18 or 24 mo. This provided data to compare the relative effects on carcinogenicity of dose rate versus total dose. The prevalence of liver and bladder tumors were used as the comparison. Animals receiving similar total doses but over a different length of time (different dose rates) were compared at the 18- and 24-mo sacrifices. When the total doses were similar, the higher dose rates for shorter time periods induced a higher prevalence of tumors. Results were more consistent for bladder tumors than for liver tumors, although the same trends were noted for both endpoints. Those groups dosed at higher rates but for fewer months had a generally higher prevalence than those receiving similar total doses but at lower rates for more months. This data from the ED01 Study illustrates the importance of experimental design, dosing regime, length of study time, and age of the animals at time of dosing in respect of calculation of risk.

Protective effect of magnesium on DNA strand breaks induced by nickel or cadmium

Magnesium, an essential metal that is important in the normal functioning of DNA, has been shown to interact with some of the toxic heavy metals in respect to biochemical and molecular mechanisms and in altering the tumorigenic process. This study examined the influence of magnesium in combination with nickel and cadmium in respect to damage of the DNA molecule. The purpose of this study was to evaluate the influence of magnesium on the amelioration of the toxic metals nickel and cadmium in respect to sustaining DNA damage. Two types of lymphocytes were used, i.e., primary Fischer 344 rat splenocytes and AHH-1 TK+/-, a human B-lymphoblastoid cell line that has been spontaneously transformed. These cells were grown in either a magnesium-free or magnesium-supplemented RPMI 1640 medium that was specifically formulated for this study. A 2x2 factorial design was employed with magnesium and either nickel or cadmium serving as the two factors. The experimental groups were as follows: +Mg+Ni, +Mg−Ni, −Mg+Ni, −Mg−Ni, with cadmium alternating for the nickel in the subsequent studies. The nickel or cadmium was added at a concentration of 50 μmol/L. The presence of double-stranded DNA was determined in each of the respective treatment groups with the two types of cell lines. Based on the results of this study, nickel is not directly toxic to DNA, whereas cadmium produces damage directly on the DNA molecule. The magnesium has little or no direct influence on the occurrence of DNA damage from nickel since the toxicity of nickel appears to be manifested in areas other than the DNA molecule, such as in the heterochromatin. The presence of cadmium in the treatment group resulted in DNA damage, which in turn was reduced significantly by the presence of magnesium.

Chronic dose-response studies in mice fed 2-AAF.

Large numbers of mice were exposed to 2‐acetylaminofluorene (2‐AAF) in the diet in order to provide quantitative information on carcinogenic dose‐response relationships. Weanling BALB/c and C57BL/6 mice of both sexes were fed 2‐AAF ad libitum in their diet for 18 months at concentrations of 0, 100, 250, and 500 ppm. Occurrence of a bladder tumor was considered as the endpoint for determination of dose‐response curves. Data were accumulated from mice that died during the 18‐month period, from moribund mice that were necropsied, and from mice necropsied at the terminal sacrifice after 18 months of exposure to the carcinogen. A dose response was noted for the body weights of all animals and for the mortality rate and bladder tumor development of the females. In the males the dose of 500 ppm of 2‐A A F appeared to be above the maximum tolerated dose and resulted in early death of the animals, a decreased tumor incidence, and the lack of a dose‐related response. Bladder tumor development in the females followe...

Benzidine dihydrochloride: toxicological assessment in mice during chronic exposures

Although benzidine is recognized as a bladder carcinogen in humans and a liver carcinogen in laboratory animals, its toxicological effects appear to be extended to several other endpoints. This economically important chemical is the base for over 200 dyes and is used extensively in manufacturing. In a chronic lifespan study lasting 33 months, both sexes of F1 hybrid (genetically homogeneous) and monohybrid cross (genetically heterogeneous) mice from BALB/c male and C57BL/6 female crosses were exposed to benzidine dihydrochloride in their drinking water at concentrations of 0, 20, 30, 40, 60, 80, and 120 ppm for the females, and 0, 30, 40, 60, 80, 120, and 160 ppm for males. Animals were removed from the study when they were dead or moribund. In addition to hepatocellular carcinomas, there were several other toxicological end-points identified that appeared to be related to the administration of benzidine. Dose-response trends were noted for pigmentation of the spleen, hepatic cytological alterations, hyperplasia of the bile ducts, megakaryocytosis of the bone marrow, vacuolization of the brain, adenoma of the Harderian gland, atrophy of the ovaries, and angioma of the uterus. Also, dose-related effects were noted with respect to time to lung tumor and time to mortality due to reticulum-cell sarcomas.

High Glucose Concentration Alters Cell Proliferation Dynamics in Human Hepatoma Cells

The aim of this study was to develop an in vitro model to investigate the molecular mechanisms of glucose-induced inhibition of cell proliferation. HuH7 cells were grown in the presence or absence of glucose for 7 days and cell proliferation was stimulated by exposure to thioacetamide. Lactate dehydrogenase leakage and 3H-thymidine incorporation were used as indices of toxicity and DNA synthesis, respectively. Cell cycle progression and protooncogene expression was monitored by flow cytometry and slot-blot analyses. Toxicity caused by thioacetamide regressed with time in the presence of 11 mM glucose (control). However, in the presence of 28 mM glucose, sustained toxicity was evident as mirrored by lactate dehydrogenase leakage. Peak DNA synthesis noted at 48 hours in the thioacetamide-treated group (11 mM glucose) was significantly diminished in the presence of 28 mM glucose. Increased c-myc expression was observed as early as 30 minutes in the thioacetamide-treated group. When cells were exposed to 28 mM glucose, c-myc expression was delayed and diminished. Methylation profile studies revealed no appreciable changes, but c-myc was significantly amplified in the control, thioacetamide-, and in the presence of 28 mM of glucose-treated groups which correlated with mRNA changes in these groups. In the glucose-pretreated group (28 mM) significant amplification of the c-myc gene was observed at later time points but there was no change in the mRNA expression, indicating that the expression was delayed. This study shows that high glucose concentrations diminish DNA synthesis and cell cycle progression normally stimulated by thioacetamide. It is concluded that high glucose concentration causes cell cycle arrest via perturbation in protooncogene expression and hence the use of high glucose concentrations in therapy should be carefully examined in situations where postsurgical healing and healing after xenobiotic-induced injury are encountered.

EFFECT OF MAGNESIUM ON THE GROWTH AND CELL CYCLE OF TRANSFORMED AND NON-TRANSFORMED EPITHELIAL RAT LIVER CELLS IN VITRO

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G1 and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G1 with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G1 nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.

Influence of genetic population structure on the results of chronic toxicity studies

The importance of the genetic makeup of animals used for toxicological testing has been recognized, although there are few data regarding this. In the study discussed here offspring from a BALB/cStCrlfC3Hf/Nctr male and C57BL/6jfC3Hf/Nctr female cross were used to produce a homogeneous (F1) strain, and brother-sister mating of the F1 population was used to produce a heterogeneous (F2) strain of mice, both having essentially the same gene pool but with different distributions. The end points of mortality and body weights were used to demonstrate relative variability about a mean resulting from genetic constitution when the mice were exposed to benzidine dihydrochloride. This life-span study demonstrated that genetic constitution in research animals should be an important consideration in toxicological research. There were significant differences in body weight and mortality between the two populations, with the F2 mice exhibiting wider body weight ranges and greater susceptibility to the test chemical.

Influence of genetic composition of test-animal populations on chronic toxicity studies used for risk estimation

A lifespan exposure of mice to benzidine dihydrochloride was conducted for 33 m using both sexes of two populations of mice with the same gene pool. One population was the genetically homogeneous F1 hybrid produced by crossing BALB/cStCrlC3Hf/Nctr males with C57BL/6jfC3Hf/Nctr females. The second population consisted of genetically heterogenous monohybrid cross (MC) offspring produced by mating the F1 hybrids inter se. Data comparisons were made to determine if gene distribution among members of a population affects the response to a toxic insult. Endpoints tested consisted of mortality, liver tumor incidence and time of tumor onset, mortality from reticulum-cell sarcoma, and body weights. In most instances it was noted that among animals not dosed (controls), the F1 population had lower background incidence of lesions and lived longer than the MC population. However, among the dosed animals, the F1 mice were generally more susceptible to the toxic agent and developed higher incidences of the chemically induced lesions than did the MC population. The F1 hybrid population gave a more conservative estimate of risk than did the MC population. The calculation of the liver tumor risk for these two populations showed that lifespan exposure to benzidine would be predicted to result in a larger number (higher risk) when using the F1 data. A 4.5-fold difference in the toxic response was observed between the F1 females and the MC males. This emphasizes the importance of gene distribution in risk estimation studies.