William T. Stott

Chemically induced proliferation of peroxisomes: Implications for risk assessment

An increasing number of beneficial and economically important drugs, industrial chemicals, and agrichemicals are being found to cause a dose-related hepatomegaly in rodent species which is associated with the proliferation of the subcellular organelle, the peroxisome. The prolonged proliferation of hepatocellular peroxisomes and the enhanced production of the normal peroxisomal metabolic byproduct, hydrogen peroxide, in these animals during chronic bioassays has been hypothesized to account for the tumorigenicity of several of these compounds, most of which lack any measurable genotoxicity in in vitro and in vivo assays. This paper briefly reviews the basic morphology and enzymology of the peroxisome and its relationship to specific pathologic changes in animals. The potential impact of the mechanism of action of peroxisome proliferators upon the design of toxicity studies and, in conjunction with interspecies sensitivity data, upon risk assessment is discussed.

The comparative absorption and excretion of chemical vapors by the upper, lower, and intact respiratory tract of rats.

Upper respiratory tract (URT) absorption of several compounds with differing water solubilities and potentials to cause lesions of the nasal mucosa were studied in rats. Absorption of propylene glycol monomethyl ether (PGME), PGME acetate (PGMEAc), ethyl acrylate (EA), epichlorohydrin (EPI), styrene (STY), nitroethane (NE), ethylene dibromide (EDB), and methylene chloride (MeCl2) vapors by the isolated URT was compared to that by the isolated lower respiratory tract (LRT) and the intact animal. Nearly all PGME and PGMEAc and 30-70% of EA, EPI, STY, NE, and EDB were absorbed when passed through the URT. In general, similar levels were absorbed by both the isolated LRT and intact animal. It was estimated that intact animals received more than 90% of their total dose of PGME and PGMEAc, and 50% of EA, NE, EPI, and EDB via the URT. Further, the dosage per unit of surface area in the URT may be 5000-6000 times that of the LRT. However, the extent of URT absorption was not related to the ability to cause lesions of the nasal mucosa. Absorption of compounds by the URT was not a simple function of water solubility or of blood or water/air partitioning coefficients suggesting that a more complex mechanism for controlling absorption may exist. In one case, it was demonstrated that URT enzymatic activity could influence the absorption of certain compounds by the URT.

Chronic Dietary Toxicity/Oncogenicity Studies on 2,4-Dichlorophenoxyacetic Acid in Rodents

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Doses in the 2-year chronic/oncogenicity rat study were 0, 5, 75, and 150 mg/kg/day. The chronic toxicity paralleled subchronic findings, and a NOEL of 5 mg/kg/day was established. A slight increase in astrocytomas observed (in males only) at 45 mg/kg/day in a previously conducted chronic rat study was not confirmed in the present study at the high dose of 150 mg/kg/ day. Doses in the 2-year mouse oncogenicity studies were 0, 5, 150, and 300 mg/kg/day for females and 0, 5, 62.5, and 125 mg/ kg/day for males. No oncogenic effect was noted in the study. In summary, the findings of these studies indicate low chronic toxicity of 2,4-D and the lack of oncogenic response to 2,4-D following chronic dietary exposure of 2,4-D in the rat and mouse.

Potential mechanisms of tumorigenic action of diethanolamine in mice

Diethanolamine (DEA), a secondary amine found in a number of consumer products, reportedly induces liver tumors in mice. In an attempt to define the tumorigenic mechanism of DEA, N-nitrosodiethanolamine (NDELA) formation in vivo and development of choline deficiency were examined in mice. DEA was administered with or without supplemental sodium nitrite to B6C3F1 mice via dermal application (with or without access to the application site) or via oral gavage for 2 weeks. Blood levels of DEA reflected the dosing method used; oral greater than dermal with access greater than dermal without access. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Choline, phosphocholine and glycerophosphocholine were decreased </=62-84% in an inverse relation to blood DEA levels. These data demonstrated a lack of NDELA formation in vivo at tumorigenic dosages of DEA but revealed a pronounced depletion of choline-containing compounds in mice. It is suggested that the latter effect may underlie DEA tumorigenesis in the mouse.

The chronic toxicity and oncogenicity of inhaled technical-grade 1,3-dichloropropene in rats and mice

Male and female Fischer 344 rats and B6C3F1 mice were exposed by inhalation to target concentrations of 0, 5, 20, or 60 ppm (0, 22.7, 90.8, or 272 mg/m3) technical-grade 1,3-dichloropropene (DCPT) 6 hr/day, 5 days/week, for up to 2 years. Ancillary groups of rats and mice were exposed for 6- and 12-month periods. Significant treatment-related nonneoplastic changes following exposure for 2 years were morphological alterations in the nasal tissues of rats exposed to 60 ppm and mice exposed to 20 or 60 ppm DCPT. In addition, mice exposed to 20 or 60 ppm had hyperplasia of the transitional epithelium lining the urinary bladder. Survival of male and female rats and mice exposed to DCPT was similar to that of the corresponding controls. No statistically increased tumor incidence was observed in treated rats. The only neoplastic response observed in mice was an increased incidence of benign lung tumors (bronchioloalveolar adenomas) in male mice exposed to 60 ppm DCPT (22/50 versus 9/50 in controls).

Inhalation pharmacokinetics of technical grade 1,3-dichloropropene in rats.

Kinetic data on the uptake of vapors of technical grade 1,3-dichloropropene (DCP) and resultant cis- and trans-DCP blood concentrations were obtained in rats exposed to 30, 90, 300, or 900 ppm DCP for 3 hr. The uptake of DCP did not increase proportionately with increasing exposure concentration due to an exposure level-related decrease in the respiratory ventilatory frequency of rats exposed to greater than or equal to 90 ppm DCP and the saturation of metabolism of DCP by rats exposed to greater than or equal to 300 ppm DCP. Absorption of inhaled DCP occurred primarily in the lower respiratory tract, although a small amount of the chemical was absorbed via the nasal mucosa, a known target tissue of inhaled DCP in rats. Following absorption, both isomers of DCP were, at less than or equal to 300 ppm exposure levels, rapidly eliminated from the bloodstream (3-6 min half-life). In addition, data obtained in rats exposed to 300 ppm DCP revealed that this rapid elimination phase was followed by a slower elimination phase having a 33-43 min half-life. Rats exposed to 900 ppm vapors also eliminated DCP in a biphasic manner; however, in this case the blood half-life of DCP during the initial phase of excretion was 14 to 27 min. Exposure to 90 ppm DCP also produced a significant decrease in renal (31%) and hepatic (41%), but not pulmonary, nonprotein sulfhydryl content. Overall, these data demonstrated that a combination of saturable metabolism and chemically induced changes in respiration control DCP uptake and body burden in rats.

Hepatic lacI and cII mutation in transgenic (λLIZ) rats treated with dimethylnitrosamine

Abstract The recent introduction of a transgenic rat in vivo mutation assay is a much needed supplement to the transgenic mouse models and offers the tools necessary for collecting target tissue specific genotoxicity data in this species. The utility of the Big Blue® rat for the detection of in vivo mutations was investigated by studying spontaneous and dimethylnitrosamine (DMN)-induced hepatic mutations. High molecular weight DNA isolated from Big Blue® rat livers typically yielded good transgene rescue efficiency of up to 5×10 5 plaque forming units per packaging reaction. DMN, when administered by oral gavage at dose levels of 0.2, 0.6, 2.0, and 6.0 mg kg −1 day −1 , induced up to a 4.5-fold increase in mutations at the highest dose level. There was no apparent difference between the lacI vs. cII target genes of the shuttle vector in either the background or DMN-induced mutant frequencies. These results suggest that the transgenic rat model is a useful tool for studying potential genotoxicity in target organs and, with further validation, the selectable cII target could be an attractive alternative to the conventional lacI color screening method for the detection of mutations in the λLIZ shuttle vector.

Subchronic toxicity of inhaled technical grade 1,3-dichloropropene in rats and mice

In order to provide a comprehensive subchronic inhalation toxicity study of the soil fumigant, technical grade 1,3-dichloropropene (DCPT), male and female Fischer 344 rats and B6C3F1 mice were exposed to 0, 10, 30, 90, or 150 ppm DCPT vapors 6 hr/day, 5 days/week for 13 weeks. The primary target tissues of inhaled DCPT were identified as the nasal mucosa of both sexes of rats and mice, and the urinary bladder of female mice. In addition, depressed growth rates of all animals exposed to 90 or 150 ppm DCPT (up to 20% in rats and 12% in mice) resulted in a variety of alterations in hematologic and clinical chemistry parameters, and changes in organ weights relative to controls. Nasal mucosal effects consisted of a dose-related slight degenerative effect of nasal olfactory epithelium or a mild hyperplasia of the respiratory epithelium or both in all animals exposed to 90 or 150 ppm and 2 of 10 male rats exposed to 30 ppm DCPT. Some focal areas of respiratory metaplasia were also noted in high exposure group mice. Urinary bladder effects consisted of a diffuse, moderate hyperplasia of the transitional epithelium in female mice exposed to 90 or 150 ppm DCPT. No treatment-related effects were observed in rats or mice exposed to 10 ppm DCPT vapors.

The pharmacokinetics of diethanolamine in Sprague–Dawley rats following intravenous administration

In order to better understand the potential toxicity of diethanolamine (DEA) and preparatory to physiologically-based pharmacokinetic model development, the pharmacokinetics of DEA at high and low internal dose through 96-h post-dosing were determined in female Sprague-Dawley rats administered 10 or 100 mg/kg uniformly labeled 14C-DEA via intravenous injection. Clearance of DEA from blood was calculated to be approximately 84 ml/h/kg at the low dose, increasing to approximately 242 ml/h/kg at the high dose. The primary route of excretion of administered radioactivity, approximately 25-36%, was via the urine as parent compound. A majority of the administered radioactivity was recovered in the tissues of treated rats, especially in the liver and kidneys, suggesting a propensity of DEA or its metabolites for bioaccumulation. An accumulation of radioactivity also occurred gradually in the red blood cells from about 6-96 h post-dosing. Some evidence of dose dependency in the fate of iv-administered DEA was observed, suggesting that saturation of the bioaccumulation process(es) occurred at a dose level of 100 mg/kg.

Gene Expression Dose-Response of Liver with a Genotoxic and Nongenotoxic Carcinogen

Tumorigenic mechanisms due to chemical exposure are broadly classified as either genotoxic or nongenotoxic. Genotoxic mechanisms are generally well defined; however nongenotoxic modes of tumorgenesis are less straightforward. This study was undertaken to help elucidate dose-response changes in gene expression (transcriptome) in the liver of rats in response to administration of known genotoxic or nongenotoxic liver carcinogens. Male Big Blue Fischer 344 rats were treated for 28-days with 0, 0.1, 0.3, 1.0, or 3.0 mg/kg/day of the genotoxin 2-acetylaminofluorene (AAF) or 0, 10, 30, 60, or 100 mg/kg/day of the nongenotoxin phenobarbital (PB). Transcriptome analysis was performed using the relatively focused Clontech Rat Toxicology II microarray (465 genes) and hybridized with 32P-labeled cDNA target. The analysis indicated that after 28 days of treatment, AAF altered the expression of 14 genes (9 up-and 5 down-regulated) and PB altered the expression of 18 genes (10 up- and 8 down-regulated). Of the limited genes whose expression was altered by AAF and PB, four were altered in common, two up-regulated, and two down-regulated. Several of the genes that show modulation of transcriptional activity following AAF and PB treatment display an atypical dose-response relationship such that the expression at the higher doses tended to be similar to that of control. This high-dose effect could potentially be caused by adaptation, toxicity, or tissue remodeling. These results suggest that the transcriptional response of the cells to higher doses of a toxic agent is likely to be different from that of a low-dose exposure.