Alan G. E. Wilson

Optimization of Cryopreservation Procedures for Rat and Human Hepatocytes

1. Rat hepatocytes were cryopreserved using a number of procedures and the viability, attachment, and metabolic activity of the cryopreserved cells were compared to freshly isolated hepatocytes. Several cryopreservation agents (dimethylsulphoxide [DMSO], glycerol, polyvinylpyrrolidone [PVP], dextrans), and combinations of these agents, were examined. Other variables tested included the freezing rate, thawing rate, and the concentration of serum in the freezing medium. 2. Recovery of viable attached cells was optimal using DMSO at concentrations of 10% or higher, a slow stepwise cooling procedure, and a quick thaw. The concentration of serum in the freezing medium (0% to 90%) did not affect cryopreservation results. Using this procedure the recovery of viable hepatocytes was 70%. 3. Levels of hepatocyte ethoxycoumarin-O-deethylase (ECOD) activity did not change following cryopreservation. The rate of decline of ECOD activity with time in culture was similar in freshly isolated and cryopreserved hepatocytes. 4. Hepatocytes isolated from three human livers were cryopreserved and recovered with viabilities similar to those obtained with the rat. A preliminary experiment also showed no loss of metabolic activity in human hepatocytes following cryopreservation.

Mode of Action of Thyroid Tumor Formation in the Male Long–Evans Rat Administered High Doses of Alachlor☆

Chronic administration of alachlor in the diet at a level of 126 mg/kg/day has previously been shown to cause an increase in benign thyroid follicular cell tumors in male Long-Evans rats. Studies were conducted to elucidate the mechanism of the alachlor-induced thyroid tumors in the male rat by evaluating changes in parameters that are collectively associated with a hormonally mediated mode of action for thyroid neoplasia. Male Long-Evans rats were administered 126 mg alachlor/kg body wt/day via the diet for up to 120 days. One group of animals was removed from alachlor-treated diet after 60 days and received untreated diet for an additional 60 days. Liver and thyroid weights and serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH), as well as hepatic uridine diphosphate glucuronosyl transferase (UDPGT) activity, were determined at 7, 14, 28, 60, and 120 days of treatment. Liver and thyroid weights, hepatic UDPGT activity, and circulating levels of TSH were significantly increased in animals administered alachlor. These increases were seen as early as 7 days after alachlor administration. Circulating levels of T4 in alachlor-treated animals were significantly decreased compared with controls at 7 days, but had returned to control levels by 60 days. T3 levels were elevated at all time points except at 28 days. The changes in TSH and T3 levels, hepatic UDPGT activity, and liver weights were all reversible on elimination of alachlor from the diet. Thyroid weights did not completely return to control levels after removal of alachlor from the diet, although some recovery was evident. The results of this study clearly suggest that alachlor-induced thyroid neoplasia, observed in previous chronic bioassays with alachlor, was associated with increases in circulating TSH levels. Increased metabolism of T4 via hepatic enzymatic conjugation (i.e., T4-UDPGT) appeared to be responsible for the increased TSH levels. These effects were shown to be reversible on cessation of exposure to alachlor. In summary, evidence is presented for a hormonally mediated process for the development of thyroid follicular cell tumors.

A study of the mechanism of butachlor-associated gastric neoplasms in Sprague-Dawley rats.

Long term administration of butachlor to Sprague-Dawley rats in a previous bioassay, resulted in the induction of gastric neoplasms which occurred only in the highest dose group (3000 ppm in the diet), primarily in females and specifically in the fundic region. The tumors were a composite of highly undifferentiated enterochromaffin-like (ECL) cells and mucus producing cells with morphologic characteristics unlike those previously described in the rat stomach. Mucosal atrophy of marked intensity was a consistent feature of the gastric mucosa in animals from the highest dose group. An additional long term study was conducted in female Sprague-Dawley rats at dietary levels of 0, 100, 1000 and 3000 ppm to explore the mechanism(s) involved in the formation of these neoplasms. Cell proliferation was evaluated in both fundic and pyloric regions of the stomachs of rats at multiple time periods from 14 days to 26 months. Mucosal thickness was determined in the fundic region at the same time intervals as were used for cell proliferation studies. Gastric pH and gastric acid production were measured after approximately 21 months of exposure. Serum gastrin levels were analyzed at 14, 60, and 120 days and at 6, 18 and 20 months. Cholecystokinin (CCK)/gastrin receptor binding studies were conducted on samples of four tumors and pooled fundic mucosa from five animals in the control group. Cell proliferation was increased in both the neck and base regions of the fundic mucosa at nearly all time points measured from 14 days to 26 months. The magnitude of the changes in the base region were substantially greater than those in the neck region. Fundic mucosal thickness was decreased beginning at the 30-day time point and continued at all intervals, being less than one half that of controls at 20 and 26 months. Gastric pH in rats from the highest dose was elevated to nearly twice control levels at 21 months. Gastric acid secretion was dramatically decreased in animals from the 3000 ppm group and was moderately decreased in the 1000 ppm group at 21 months. Hypergastrinemia was observed at the 3000 ppm level only, beginning at 120 days with progression to extremely high levels by 18 months. CCK/gastrin receptor binding was demonstrated in all tumors studied, at levels comparable to or higher than that of the pooled control sample. All changes involved only the fundic region, the site of tumor formation. Tumors occurred only in animals from the 3000 ppm level, the only level at which hypergastrinemia occurred.(ABSTRACT TRUNCATED AT 400 WORDS)

Genotoxic potential of glyphosate formulations: mode-of-action investigations.

A broad array of in vitro and in vivo assays has consistently demonstrated that glyphosate and glyphosate-containing herbicide formulations (GCHF) are not genotoxic. Occasionally, however, related and contradictory data are reported, including findings of mouse liver and kidney DNA adducts and damage following intraperitoneal (ip) injection. Mode-of-action investigations were therefore undertaken to determine the significance of these contradictory data while concurrently comparing results from ip and oral exposures. Exposure by ip injection indeed produced marked hepatic and renal toxicity, but oral administration did not. The results suggest that ip injection of GCHF may induce secondary effects mediated by local toxicity rather than genotoxicity. Furthermore, these results continue to support the conclusion that glyphosate and GCHF are not genotoxic under exposure conditions that are relevant to animals and humans.

Technology Review: Application of Whole-Body Autoradiography in Toxicology

Whole-body autoradiography (WBA) is a technique that has been available for many years for studying the biological fate of chemicals. This technique, which is most frequently employed with radiolabeled chemicals, provides a visual analysis of tissue distribution. In recent years, there has been an increasing interest in using this technique in studies of the absorption, distribution, metabolism, and elimination (ADME) of chemicals. An important factor in the increasing use of WBA is the ability to utilize image analysis technology to quantitate WBA data. In this article, the status of quantitative WBA is discussed. In addition, this article discusses recent developments in the application of WBA in the field of toxicology.

Lack of in vivo DNA binding of mercaptobenzothiazole to selected tissues of the rat

In this study, the in vivo binding of 14C-labelled 2-mercaptobenzothiazole (MBT) to DNA was investigated. Male and female Fischer 344 rats were gavaged with 375 mg MBT/kg body weight and killed 8 hours later. DNA was extracted from the liver, adrenal glands, pituitary gland, pancreas, and bone marrow and the amount of radioactivity associated with the DNA was determined. Results from this study indicate that MBT does not significantly bind to DNA from any of the tissues examined. CBI values for liver for the 3 methods of purification were -1-3 which are on the low end of the covalent binding index. The CBI values for the other tissues were always less than 1. Other chemicals with similar CBI values include estrone and diethylstilbesterol. Strong hepatocarcinogens such as dimethylnitrosamine and aflatoxin have CBI values ranging from 6000 to greater than 20000.

Toxicity of Calcium Sodium Metaphosphate Fiber I. In Vitro and in Vivo Degradation and Clearance Studies

In an experiment to ascertain the degradability of calcium sodium metaphosphate (CSM) fiber in vitro, 32P-labeled CSM fiber was incubated in media with or without rat lung epithelial cells (LEC) or rat alveolar macrophages (RAM). The amount of radioactivity appearing in the filtrate of the media in the presence of cells minus the radioactivity in the media in the absence of cells was considered to reflect cell-aided dissolution of the fiber. LEC and RAM cells increased the degree of dissolution two- and sevenfold, respectively, compared to their respective media controls in a 7-day time period. In a separate experiment, male Fischer rats were given 32P-labeled CSM fiber either by intraperitoneal injection or by intratracheal instillation and the amount of radioactivity appearing in the urine and feces was measured over a period of 60 days. Selected animals from this experiment were also subjected to whole-body autoradiography 0, 1, 5, 15, 30, and 60 days postexposure. After intraperitoneal injection, approximately 0.9% of the administered dose appeared in the urine. A similar percentage of the dose was eliminated in the urine when the fibers were administered by intratracheal instillation; however, the amount of radioactivity in the feces after intratracheal instillation, i.e., 11.6% of the administered dose, was much higher than that after intraperitoneal dose, i.e., 0.24% of the administered dose. Whole-body autoradiographs showed a time-related increase in radioactivity at a site other than the site of administration, and the location of this radioactivity appeared to be exclusively associated with mineralized tissue. The clearance of nonradiolabeled CSM fiber (approximately 200,000 fibers) from rat lung after intratracheal inhalation (IH) and intratracheal instillation (IT) was monitored. Approximately 93% of the initial fiber load after IH and approximately 84% of the initial fiber load after IT was cleared from the lung in 6 months. Histological and biochemical evaluation of the rat lungs did not reveal any indication of fibrosis up to a period of 6 months. All the studies discussed indicate that CSM is degradable in biological systems and is cleared from the lung after IT and IH administration. These attributes of CSM fiber should reduce the potential for chronic adverse effects in the lung after inhalation.

Chapter 69 – Chloracetanilides

This chapter describes the toxicology of chloracetanilide herbicides such as alachlor, acetochlor, butachlor, metolachlor, and propachlor. Eye and skin irritation studies conducted in rabbits showed alachlor to be non-irritating to the eye and slightly irritating to the skin. Alachlor produced skin sensitization in guinea pigs. Acetochlor has been shown to be practically nonirritating to the eyes and skin of rabbits. A dermal sensitization study in guinea pigs was positive. In studies with rabbits, butachlor was practically non-irritating to the skin and moderately irritating to the eye; dermal sensitization was observed in a study with guinea pigs. Metolachlor did not produce eye or skin irritation in rabbits; the material was positive in a dermal sensitization study with guinea pigs. Eye and skin irritation studies conducted in rabbits showed propachlor to be severely irritating to the eye and slightly irritating to the skin. Propachlor produced skin sensitization in guinea pigs. The herbicidal mode of action for chloracentanilides is not totally understood. It is known that this class of herbicides inhibits the biosynthesis of lipids, alcohols, fatty acids, proteins, isoprenoids, and flavonoids. By inhibiting synthesis of various terpenoid precursors, these herbicides appear to interfere with the production of gibberellins.

Biochemical toxicology and disposition of therminol 66 heat transfer fluid after inhalation or after dietary administration to male sprague‐dawley rats

The objectives of this study were to determine the disposition of Therminol 66 in rats and to determine the effects of this heat-transfer fluid on liver and kidney microsomal drug-metabolizing enzymes. Therminol 66 was administered to male Sprague-Dawley rats at various doses as either a single oral administration at 0, 100, or 300 mg/kg, or as a single 6-h inhalation exposure at 0 or 350 mg/m3. Animals were killed 48 h after gavage or after termination of inhalation exposure. Additional groups of animals were exposed to Therminol 66 via the diet at 0, 100, 500, or 5000 ppm for 14 d, or via repeated inhalation exposure at 0, 25, 250, or 1200 mg/m3 for 6 h/d for 14 d. These exposure scenarios represent approximately equivalent doses of Therminol 66 by the different routes of administration. No change in body weight was observed after acute oral or inhalation exposure, and little change in body weight was observed in animals administered Therminol 66 via the diet except at the highest dose. There was no change in kidney weight, and liver weights were increased only at the higher doses of Therminol 66. The body weight gain of animals exposed to Therminol 66 via inhalation decreased in a dose-dependent manner over the 2-wk exposure period. Results from the disposition study indicated that Therminol 66 did not appear to accumulate in the tissues examined and did not appear to be extensively absorbed after a single oral dose of 300 mg/kg. The whole-body elimination half-life was approximately 14 h and occurred primarily via the feces. There was no significant induction of hepatic aryl hydrocarbon hydroxylase (AHH) activity after single oral or inhalation exposures to Therminol 66. Ethoxycoumarin O-deethylase (ECOD) was significantly induced only in animals exposed to 350 mg/m3 via inhalation. Repeated dietary and inhalation exposures resulted in AHH and ECOD induction only at the highest doses, and the kidney appeared to be less sensitive than the liver. Animals exposed via inhalation demonstrated a greater hepatic inductive effect than did animals exposed via the diet, which may be due to absorption differences.(ABSTRACT TRUNCATED AT 400 WORDS)