Jeffrey L. Larson

Acute Hepatotoxic and Nephrotoxic Effects of Chloroform in Male F-344 Rats and Female B6C3F1 Mice

Previous studies demonstrated that chloroform given by oral gavage in corn oil caused an increased incidence of liver tumors in male and female mice and kidney tumors in male rats, while administration in drinking water resulted in an increased tumor incidence only in the kidneys of the male rats. The tumorigenicity of this nongenotoxic agent has been postulated to be linked with cytolethality and cell proliferation. This study examined the organ-specific toxicity of acute doses of chloroform. Male F-344 rats were given chloroform by gavage in corn oil at the bioassay doses of chloroform of 0 and 180 mg/kg body wt as well as 34 and 477 mg/kg and necropsied 24 hr later. Additional rats were given a single dose of 180 mg chloroform/kg and administered bromodeoxyuridine (BRDU) 2 hr prior to necropsy at 0.5, 1, 2, 4, and 8 days after chloroform treatment. Female B6C3F1 mice were given chloroform by gavage at the bioassay doses of 0, 238, and 477 mg/kg as well as 34 mg/kg and necropsied at 24 hr after treatment. Additional mice were given a single dose of 350 mg chloroform/kg, labeled with BRDU, and necropsied at 0.5, 1, 2, 4, and 8 days after treatment. The kidneys of male rats administered 34, 180, and 477 mg chloroform/kg exhibited mild to severe proximal tubular necrosis in a dose-dependent manner. A 20-fold increase in the labeling index (LI, the percentage of nuclei in S-phase) in the proximal tubule cells was observed 2 days after treatment with the bioassay dose of 180 mg/kg. The livers of male rats exhibited only slight to moderate multifocal centrilobular necrosis at 180 and 477 mg/kg. A 10-fold increase in the LI was observed in the liver of male rats given 477 mg/kg, but no increase was observed at the bioassay dose of 180 mg/kg. In contrast to male rats, female mice developed a dose-dependent centrilobular hepatic necrosis at 238 and 477 mg/kg. No renal lesions were observed in female mice at any dose. A peak increase in LI of 38-fold was observed in hepatocytes in the livers of female mice 2 days after treatment with 350 mg chloroform/kg, with only a 2-fold increase in LI observed in the kidneys. These data indicate that acute chloroform-induced cytolethality leads to increased cell proliferation and that the organ-specific pattern of toxicity is the same as the organ-specific pattern of tumor formation.(ABSTRACT TRUNCATED AT 400 WORDS)

A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxicity and Relevance to Cancer Studies

Chloroform acts via a nongenotoxic-cytotoxic mode of action to produce cancer if given in doses and at dose rates sufficiently high to produce organ-specific toxicity. In a recent study, chloroform failed to induce cancer in male or female F-344 rats when administered by inhalation for 2 years at 90 ppm, 5 days/week. The present study was undertaken to define the concentration-response curves for chloroform-induced lesions and regenerative cell proliferation in the F-344 rat when exposed by inhalation and to correlate those patterns of toxicity with the results from the inhalation cancer bioassay. Male and female F-344 rats were exposed to airborne concentrations of 0, 2, 10, 30, 90, or 300 ppm chloroform 6 hr/day, 7 days/week for 4 days or 3, 6, 13 weeks. Additional treatment groups were exposed 5 days/week for 13 weeks or were exposed for 6 weeks and held until Week 13. Bromodeoxyuridine was administered via osmotic pumps implanted 3.5 days prior to necropsy and the labeling index (LI, percentage of nuclei in S-phase) was evaluated immunohistochemically. A full-screen necropsy identified the kidney, liver, and nasal passages as the only target organs. This study confirmed that 300 ppm is extremely toxic and would be inappropriate for longer-term cancer studies. The primary target in the kidney was the epithelial cells of the proximal tubules of the cortex, with significantly elevated increases in the LI at concentrations of 30 ppm and above. However, only a marginal increase in the renal LI in the males was seen after exposures of 90 ppm, 5 days/week. Chloroform induced hepatic lesions in the midzonal and centrilobular regions with increases in the LI throughout the liver, but only at 300 ppm exposures. An additional liver lesion seen only at the highly hepatotoxic concentration of 300 ppm was numerous intestinal crypt-like ducts surrounded by dense connective tissue. Enhanced bone growth and hypercellularity in the lamina propria of the ethmoid turbinates of the nose occurred at the early time points at concentrations of 10 ppm and above. At 90 days there was a generalized atrophy of the ethmoid turbinates at concentrations of 2 ppm and above. Cytolethality and regenerative cell proliferation are necessary but not always sufficient to induce cancer because of tissue, sex, and species differences in susceptibility. A combination of a lack of direct genotoxic activity by chloroform, only a marginal induction of cell proliferation in the male rat kidney, and lower tissue-specific susceptibility in the female rat is apparently responsible for the reported lack of chloroform-induced cancer in a long-term inhalation bioassay with F-344 rats.

Induced regenerative cell proliferation in livers and kidneys of male F-344 rats given chloroform in corn oil by gavage or ad libitum in drinking water

These studies were designed to establish the dose response relationships for the induction of cytolethality and regenerative cell proliferation in the liver and kidneys of male F-344 rats given chloroform by gavage or in drinking water. Rats were administered oral doses of 0, 10, 34, 90 or 180 mg/kg/day chloroform dissolved in corn oil by gavage for 4 days or for 5 days/week for 3 weeks. A second group of rats was given chloroform ad libitum in the drinking water at concentrations of 0, 60, 200, 400, 900 or 1800 ppm for 4 days or 3 weeks. Bromodeoxyuridine (BrdU) was administered via an implanted osmotic pump 3.5 days prior to necropsy to label cells in S-phase. Cells having incorporated BrdU were visualized in tissue sections immunohistochemically and the labelling index (LI) evaluated as the percentage of S-phase cells. Rats treated with 90 or 180 mg/kg/day by gavage for 4 days had mild to moderate degeneration of renal proximal tubules and centrilobular hepatocytes. These alterations were absent or slight after 3 weeks of treatment. LI were increased in the kidney cortex only in the rats treated with 180 mg/kg/day for 4 days. A dose-dependent increase in LI was seen in rat liver after 4 days of treatment with 90 and 180 mg/kg/day by gavage, but the LI remained elevated after 3 weeks of treatment only at the 180 mg/kg/day dose. When chloroform was administered in the drinking water, no microscopic alterations were seen in the kidneys after 4 days of treatment. As a general observation, rats treated for 3 weeks with 200 ppm chloroform and greater had slightly increased numbers of focal areas of regenerating renal proximal tubular epithelium and cell proliferation than were noted in the controls, but no clear dose response relationship was evident. However, the overall renal LI was not increased at any dose or time point. Similarly, only mild hepatocyte vacuolation was observed in rats given 1800 ppm chloroform in the water for 3 weeks with no increase in the hepatic LI at any time point, even though the rats were consuming chloroform at a rate of 106 mg/kg/day at the 1800 ppm drinking water concentration. These data indicate more severe hepatic and renal toxicity when chloroform is administered by gavage than in the drinking water and a different pattern of regenerative proliferation in the kidney.(ABSTRACT TRUNCATED AT 400 WORDS)

The toxicity of 1-week exposures to inhaled chloroform in female B6C3F1 mice and male F-344 rats.

Detailed quantitative descriptions of the toxicity of inhaled chloroform are lacking, despite the fact that the majority of environmental exposures occur by this route. We investigated the ability of chloroform vapors to produce toxicity and regenerative cell proliferation in the livers and kidneys, the principal target organs for carcinogenicity of female B6C3F1 mice and male F-344 rats, respectively. Nasal passages were also examined for toxic responses. Rodents were exposed to chloroform vapors at concentrations of 0, 1, 3, 10, 30, 100, or 300 ppm for 6 hr/day for 7 consecutive days and necropsied on Day 8. Animals were administered bromodeoxyuridine (BrdU) via implanted osmotic pump for the previous 3.5 days before necropsy. Cell proliferation was quantitated as the percentage of cells in S-phase (labeling index; LI) measured by immunohistochemical detection of BrdU-labeled nuclei. Mice exposed to 100 or 300 ppm exhibited centrilobular hepatocyte necrosis and severe vacuolar degeneration of midzonal and periportal hepatocytes, while exposure to 10 or 30 ppm resulted in mild to moderate vacuolar changes in centrilobular hepatocytes. Slight, dose-related increases in the hepatocyte LI were observed for exposure concentrations of 10 and 30 ppm, while the LI was increased more than 30-fold in the 100 and 300 ppm groups. The kidneys of mice were affected only at the 300 ppm exposure, with approximately half of the proximal tubules lined by regenerating epithelium and an increased LI of tubule cells of 8-fold over control. In rats, mild centrilobular vacuolation was observed only in the livers of rats exposed to 300 ppm. The hepatocyte LI in rats were increased only at 100 and 300 ppm, 3- and 7-fold over control, respectively. In the kidneys of the male rats exposed to 300 ppm, about 25 to 50% of the proximal tubules were lined by regenerating epithelium. The LI for tubule cells in the cortex was increased at 30 ppm and above. In the nasal passages of rats, chloroform concentrations of 10 ppm and above induced histopathological changes that exhibited clear concentration-related severity. These lesions consisted of respiratory epithelial goblet cell hyperplasia and degeneration of Bowmans glands in olfactory mucosa with an associated osseous hyperplasia of the endo- and ectoturbinates in the periphery of the ethmoid region. These nasal lesions were not observed in mice. Knowledge of the dose-dependent responses in rats and mice will be valuable in assessing the potential risks to humans posed by inhaled chloroform and in setting exposure concentrations for longer-term studies.

Toxicity and cell proliferation in the liver, kidneys and nasal passages of female F-344 rats, induced by chloroform administered by gavage

Dose-response relationships were determined for the induction of cytolethality and regenerative cell proliferation in the established target organs (liver, kidneys, and nasal passages) of female F-344 rats given chloroform daily by gavage. Rats were administered chloroform dissolved in corn oil at doses of 0, 34, 100, 200 or 400 mg/kg/day for 4 consecutive days or for 5 days/wk for 3 wk. Bromodeoxyuridine (BrdU) was administered through an implanted osmotic pump 3.5 days prior to autopsy to label cells in S-phase. Cells in S-phase were visualized immunohistochemically in tissue sections and the labelling index (LI) calculated as the percentage of cells in S-phase. Mild degenerative centrilobular changes and dose-dependent increases in the hepatocyte LI were observed after administration of 100 mg or more chloroform/kg/day. Rats given 200 or 400 mg/kg/day for 4 days or 3 wk had degeneration and necrosis of the proximal tubules of the renal cortex. Regenerating epithelium lining proximal tubules was seen histologically and as an increase in LI. Dose-dependent increases in LI were observed in the kidneys at doses of 100 mg or more chloroform/kg/day at both 4 days and 3 wk. Two distinct treatment-induced responses were observed in specific regions of the olfactory mucosa lining the ethmoid region of the nose. A peripheral lesion was seen at all doses used and included new bone formation, periosteal hypercellularity and increased cell replication. A central lesion was seen at doses of 100 mg or more chloroform/kg/day and was characterized by degeneration of the olfactory epithelium and superficial Bowmans glands. These observations define the dose-response relationships for the liver, kidneys and nasal passages as target organs for chloroform administered by gavage in the female F-344 rat.

Phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein DAS181

BACKGROUND influenza viruses (IFVs) frequently achieve resistance to antiviral drugs, necessitating the development of compounds with novel mechanisms of action. DAS181 (Fludase), a sialidase fusion protein, may have a reduced potential for generating drug resistance due to its novel host-targeting mechanism of action. METHODS IFV strains B/Maryland/1/59 and A/Victoria/3/75 (H3N2) were subjected to >30 passages under increasing selective pressure with DAS181. The DAS181-selected IFV isolates were characterized in vitro and in mice. RESULTS despite extensive passaging, DAS181-selected viruses exhibited a very low level of resistance to DAS181, which ranged between 3- and 18-fold increase in EC(50). DAS181-selected viruses displayed an attenuated phenotype in vitro, as exhibited by slower growth, smaller plaque size and increased particle to pfu ratios relative to wild-type virus. Further, the DAS181 resistance phenotype was unstable and was substantially reversed over time upon DAS181 withdrawal. In mice, the DAS181-selected viruses exhibited no greater virulence than their wild-type counterparts. Genotypic and phenotypic analysis of DAS181-selected viruses revealed mutations in the haemagglutinin (HA) and neuraminidase (NA) molecules and also changes in HA and NA function. CONCLUSIONS results indicate that resistance to DAS181 is minimal and unstable. The DAS181-selected IFV isolates exhibit reduced fitness in vitro, likely due to altered HA and NA functions.

A Safety Evaluation of DAS181, a Sialidase Fusion Protein, in Rodents

DAS181 is a novel inhaled drug candidate blocking influenza virus (IFV) and parainfluenza virus (PIV) infections through removal of sialic acid receptors from epithelial surface of the respiratory tract. To support clinical development, a 28-day Good Laboratory Practices inhalation toxicology study was conducted in Sprague-Dawley rats. In this study, achieved average daily doses based on exposure concentrations were 0.47, 0.90, 1.55, and 3.00 mg/kg/day of DAS181 in a dry powder formulation. DAS181 was well tolerated at all dose levels, and there were no significant toxicological findings. DAS181 administration did not affect animal body weight, food consumption, clinical signs, ophthalmology, respiratory parameters, or organ weight. Gross pathology evaluations were unremarkable. Histological examination of the lungs was devoid of pulmonary tissue damage, and findings were limited to mild and transient changes indicative of exposure and clearance of a foreign protein. DAS181 did not show any cytotoxic effects on human and animal primary cells, including hepatocytes, skeletal muscle cells, osteoblasts, or respiratory epithelial cells. DAS181 did not cause direct or indirect hemolysis. A laboratory abnormality observed in the 28-day toxicology study was mild and transient anemia in male rats at the 3.00 mg/kg dose, which is an expected outcome of enhanced clearance of desialylated red blood cells resulting from systemic exposure with DAS181. Another laboratory observation was a transient dose-dependent elevation in alkaline phosphatase (ALP), which can be attributed to reduced ALP clearance resulting from increased protein desialylation due to DAS181 systemic exposure. These laboratory parameters returned to normal at the end of the recovery period.

Levels of myc, fos, Ha-ras, met and hepatocyte growth factor mRNA during regenerative cell proliferation in female mouse liver and male rat kidney after a cytotoxic dose of chloroform

Chloroform is a liver carcinogen in mice and a kidney carcinogen in rats. It is thought to act through a non-genotoxic-cytotoxic mode of action. Changes in expression of growth control genes accompanying chloroform-induced cytolethality and regeneration may play a part in the development of chloroform-induced tumors. In this experiment, we examined the levels of the myc, fos, Ha-ras, met and hepatocyte growth factor mRNA in livers of female B6C3F(1) mice and kidneys of male F-344 rats to detect changes in gene expression following a single, cytotoxic gavage dose of chloroform in corn oil. Poly A+ RNA was purified from homogenates of livers of mice treated with 350mg/kg chloroform and kidneys of rats treated with 180 mg/kg chloroform and used for Northern blot analysis. Livers of female mice showed large transient increases in levels of myc and fos mRNA while levels of Ha-ras, met and the hepatocyte growth factor gene mRNA remained near control levels. In the male rat kidney, levels of myc mRNA increased after treatement, while levels of mRNA of all other genes examined remained near control levels. This pattern of gene expression is consistent with that induced by other cytotoxic carcinogens and suggest that alteration of the myc and fos genes could be involved in the regenerative cell proliferation that ultimately could play a role in chloroform-induced tumors.