The acute and subchronic toxic effects of GLG-V-13 (3-[4-(1H-imidazol-1-yl)benzoyl]-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane dihydroperchlorate. CAS 155029-33-7), a novel class III with some class Ib antiarrhythmic activity, were investigated in mice. The estimated LD50 for GLG-V-13 given orally were 419 mg/kg for male mice and 383 mg/kg for female mice, respectively. The acute toxic signs appeared to be of the central nervous system in origin. Four groups of mice (15 per sex, group and dose) were fed daily with diets containing GLG-V-13 for 90 consecutive days. The equivalent daily doses were 0, 22, 50 and 121 mg/kg/day and 0, 27, 60 and 136 mg/kg/day for male and female mice, respectively. All of the mice survived. Food consumption was decreased. However. mean body weight and body weight gain were not significantly changed. Gross pathological changes, especially in the lungs and liver, were found in the middle and high dose groups. Consistent increased mean corpuscular hemoglobin concentration and decreased mean corpuscular hemoglobin were observed in all dose groups. Hepalocellular necrosis was found in both male and female mice treated with the drug and was dose-dependent. Marked vacuolation of the X zone in the adrenal gland with mild to moderate deposition of ceroid pigments (brown degeneration) was observed in female mice. Lesions in the kidneys and adrenal glands may be a possible reason for changes in serum sodium and potassium ions concentrations leading to an increase in water intake. A significant reduction in cholesterol in the high dose group may be a favorable pharmacological effect of GLG-V-13. The data from the 90-day subchronic toxicity studies indicate that GLG-V-13 appears to have limited systemic toxicity potential.

1,3,5-Trinitrobenzene (TNB) is a munitions chemical that causes gliovascular lesions in the brain stem of rats similar to those produced by thiamine deficiency and nitroaromatic compounds, including m-dinitrobenzene. To identify neuropathic indices of toxicity, the effects of varying concentrations (0 to 2 mM) of TNB on cytotoxicity and cellular metabolic activity were examined using cultured astrocytes from Fischer-344 rats. The cytotoxicity was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. Astrocyte metabolic activity was assessed by measuring the conversion of a tetrazolium salt to a formazan product. Additionally, the effects of oxidative stress on cellular metabolic activity - were determined by varying oxygen tension via alteration of culture media depth. In vitro, the toxic concentration 50% (TC50) of TNB, which induced cell death, was 16 μM following a 24-h exposure. The concentration of TNB that reduced cellular metabolic activity by 50% was 29 μM following a 24-h exposure. Varying the depth of the culture media did not influence the cellular metabolic activity in control or TNB-treated astrocytes. These results support the hypothesis that TNB induced neurotoxicity could partially be mediated via injury to astrocytes, a major component of the blood-brain barrier.

Bone marrow is extremely sensitive to toxicants, and in vitro culture of bone-marrow progenitor cells has been shown to be a sensitive indicator of bone-marrow injury in laboratory rodents. The ability of a bone-marrow progenitor cell assay to detect myelotoxicity in a wild rodent model (cotton rat, Sigmodon hispidus) that inhabits many contaminated ecosystems in the southern United States was examined. Responsiveness of progenitor cells to recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and cotton rat lung-conditioned medium (LCM) was determined to optimize culture conditions for cotton rats. Myelotoxicity was induced in cotton rats by treating animals with either cyclophosphamide (8 or 80 mg/kg) or dexamethasone (500 µg/kg) over a 5-d period. Administration of a high dose of cyclophosphamide caused nearly total suppression of colony formation of granulocyte-macrophage progenitor cells (CFU-GM). Marked histological changes in both the bone marrow and spleen were also observed in cotton rats treated with a high dose of cyclophosphamide. Although histological lesions were not apparent, the number of CFU-GM in the bone marrow of low-dose cyclophosphamide- and dexamethasone-treated cotton rats was significantly suppressed compared to controls. The number of CFU-GM was consistently higher using LCM than recombinant murine GM-CSF. This reproducible, quantitative, in vitro bone-marrow progenitor cell culture system was a sensitive indicator of myelotoxicity in wild cotton rats and should be useful for monitoring chronic exposures to low levels of environmental toxicants in wild rodent populations.

Various chemical mixtures exist in soil contaminated with petrochemical wastes, yet no comprehensive assessment of their impact on terrestrial ecosystems has been conducted. The purpose of this study was to evaluate hematotoxicity risks to wild populations of cotton rats (Sigmodon hispidus) residing in habitats previously contaminated by petroleum industrial wastes. Resident cotton rats were monitored on nine contaminated sites and nine ecologically matched reference sites in Oklahoma. The possible toxicological interactions of petrochemical wastes on bone marrow was investigated by using the assay of colony formation of granulocyte-macrophage progenitor cells. There was a consistent significant 21 to 39% decrease in the number of colony-forming units of granulocyte-macrophage (CFU-GM) in cotton rats from petrochemical-contaminated sites compared to matched reference sites, with no marked changes in hematological or histopathological parameters. These results suggest that bone-marrow progenitor cell culture is a sensitive indicator for the assessment of ecotoxicity risks associated with petrochemical wastes that are generated by the oil refining industry. Long-term exposure to hazardous wastes associated with the petroleum industry may represent a subtle risk to the hematopoietic system in humans.

Fluoride has been identified as a ubiquitous contaminant of soils where petrochemical wastes have been disposed. The purpose of this study was to assess how widespread toxicity risks are to resident vertebrates from chronic exposure to fluoride in the soil of petrochemical-contaminated waste sites. In total, 573 wild cotton rats (Sigmodon hispidus) were examined. The rats that were seasonally collected from 12 contaminated and 12 ecologically matched reference sites across Oklahoma over a 3-yr period. The risks of cotton rats exposed to fluoride were analyzed by means of gross examination, histopathology, and scanning electron microscopy of rat incisors. Cotton rats from reference sites showed no pathologic changes in incisors (98%). In comparison, 46% of cotton rats from contaminated sites had various degrees of dental lesions. The prevalence and severity of dental lesions in cotton rats from contaminated sites were significantly influenced by season. There was a 45% increase in prevalence and a 65% increase in severity of dental lesions from summer to winter. This study demonstrated that cotton rats are very sensitive biomonitors for assessing toxicity risks from soils contaminated with fluoride and that such assessments should consider seasonal influences.

A variety of chemical mixtures exist in the soil of petrochemical waste sites, and many of these compounds are known immunotoxicants that have been observed to induce immune alterations in wild rodents inhabiting many of these petrochemical waste sites. Conventional histopathological assessments have been widely used with considerable success to investigate immunotoxicity of various agents under laboratory conditions. We hypothesized that histopathologic assessments would be equally sensitive for detecting exposure to complex mixtures of toxicants in cotton rats (Sigmodon hispidus) residing in contaminated habitats. Histopathological parameters were examined from a total of 624 cotton rats that were seasonally collected from 13 petrochemical-contaminated waste sites and 13 ecologically matched reference sites in Oklahoma over a 3-year period. Histopathological examination did not reveal any lesion associated with exposure to petrochemical wastes except renal inclusion bodies. Prevalence and severity of histologic lesions in liver and kidneys of cotton rats were significantly influenced by season, where prevalence and severity were lower in winter than summer on all study sites. These results suggest that the evaluation of toxicity from exposure to contaminants in the soil of industrial waste sites using histopathological assessments is not sensitive enough to detect exposure to the low levels of environmental contaminants present on most waste sites.

Land-treatment of petrochemical wastes is a widely used method to dispose of hazardous and non-hazardous waste by biodegradation. However, no comprehensive assessment of the impact of such disposal techniques on terrestrial ecosystems has been conducted. Despite the presence of suspected immunotoxicants in the soil, wild rodents frequently reside on these waste sites after closure or abandonment. We explored the seasonal sensitivity of the immune system of the hispid cotton rat (Sigmodon hispidus) to in situ exposures on sites land-treated with petrochemical wastes. Animals were monitored on five contaminated land-treatment sites and five ecologically matched-reference sites in Oklahoma, USA, over two seasons (summer and winter). Most hematological parameters were not adversely affected by land-treatment; however, platelet counts were 26% greater in cotton rats from land-treatment sites compared to reference sites in winter. Significant treatment-related differences were observed in total serum protein concentrations, organ mass and organ cellularity, but these differences were not consistent across the five land-treatment units. Lymphoproliferative responses of cotton rat splenocytes stimulated in vitro were elevated for a T-cell mitogen and depressed for a B-cell mitogen in animals from land-treatment compared to reference sites. The ability of splenocytes to proliferate in response to interleukin-2 receptor-binding was not influenced by treatment. Total yields of peritoneal cells, yield of peritoneal macrophages, and yield of peritoneal lymphocytes were influenced to varying degrees by land-treatment. Functionally, in vitro metabolic activity of peritoneal macrophages was 114% greater in cotton rats from land-treatment sites compared to reference sites during summer. These results indicate that petrochemical wastes applied to soils on these five land-treatment sites had variable immunomodulatory effects in resident cotton rats. Immune alterations for some assays were indicative of enhancement on some land-treatment sites while suppressive on other land-treatment sites, which could have been a function of type and concentration of immunotoxicants present on each site and highlights the uniqueness of each land-treatment site.

The acute and subchronic toxic effects of BRB-I-28 (7-benzyl-3-thia-7-azabicyclo[3.3.1]nonane HCl), a novel class Ib antiarrhythmic agent, were investigated in male and female mice. The estimated oral LD(50) for BRB-I-28 was 128 mg/kg (male mice) and 131 mg/kg (female mice). In subchronic oral studies, four groups of mice (15/sex/group/dose) were fed daily with diets containing BRB-I-28 for 90 consecutive days. The equivalent daily doses were approximately 0, 16, 32, 76 (male) and 0, 18, 37, 89 mg/kg (female). All mice survived. Food consumption per day was decreased, but water consumption per day was increased (in a non-dose-dependent manner). However, both mean body weight and mean body weight gain were not significantly changed as were true for hematological and clinical chemistry profiles, except for serum Na(+) concentration (male) and serum K(+) concentration in male and female mice (high dose levels). Hepatocellular necrosis occurred in male and female mice (in a dose-dependent fashion). Renal cortical vacuoles and myocardial necrosis with low numbers of lymphocytic infiltrations were present in female mice (middle and high doses). Lesions in the liver, kidney and heart were mild with (very small) changes in serum biochemical values. These data suggest that BRB-I-28 has limited toxic potential, and coupled with low proarrhythmic and other desirable cardiovascular effects, makes BRB-I-28 worthy of further development.

Petrochemical waste contains inorganic contaminants that can pollute soil and pose ecological risks to wildlife. Few studies have evaluated bioaccumulation of inorganic contaminants by small mammals from soil contaminated with petrochemical waste. In this study, we determined the extent of soil contamination with inorganics, the bioaccumulation of metals and F in bone of cotton rats (Sigmodon hispidus), and the relationship between contaminants in soil and in bone of cotton rats on petrochemical waste sites. Cotton rats and soils from 12 petrochemical‐contaminated and matched reference sites were analyzed over a three‐year period. The number of petrochemical‐contaminated sites with soil contamination (in parentheses) were Zn (12), Pb (9), Cr (9), Cu (8), F (7), Ni (7), Sr (6), Ti (5), V (5), Co (3), Ba (3), and Cd (2). Lead and F were the most frequently bioaccumulated contaminants in cotton rat bones. Bone Pb of 3.8 to 63.4 mg/kg was 2‐ to 42‐fold and bone F of 830 to 3,680 mg/kg was 5‐ to 23‐fold greater than mean bone Pb and F from reference sites. Bone Pb and F showed a seasonal trend with winter greater than summer levels. Bone F was an accurate predictor of dental fluorosis when bone F was low (< 1,000 mg/kg, no fluorosis) or high (>3,000 mg/kg, fluorosis) but was inaccurate for intermediate bone F (1,000–3,000 mg/kg). The prevalence of dental fluorosis on F‐contaminated sites was 50% higher in winter than summer. Strong relationships were found between bone F and HCl‐extractable F (r = 0.70) and bone F and total content of F (r = 0.85) in F‐contaminated soils. Land disposal of petrochemical wastes should limit the amount of inorganic contaminants applied to soil.

The contamination of soil and water with munitions chemicals and their degradation products has been reported at certain munitions production waste disposal sites and at certain Army installations. The effects of 2,4,6-trinitrotoluene (TNT) on wild cotton rats ( Sigmodon hispidus) were evaluated to identify target organ toxicity that could be used to develop biomarkers for exposure assessment for ecological and health risks. The oral LD 50 values for TNT in corn oil were 607 and 767 mg/kg body weights for male and female cotton rats respectively. Hematological, pathological, and biochemical effects of TNT were determined after daily oral gavage of TNT in corn oil at doses of 0, 75.9, 151.8, and 303.5 (males) or 0, 96, 192, and 384 mg/kg (females) for 7 days. Cotton rats treated with TNT showed an increase in spleen weights in males (303.5 mg/kg) and in females (192 and 384 mg/kg). Cotton rats of both sexes treated for 7 days with TNT had marked hemolytic anemia with reduced erythrocytes, hemoglobin, and hematocrit in high-dose groups; methemoglobin levels were elevated significantly in males at mid and high dose. Hepatic drug metabolizing enzyme analysis revealed that microsomal O-dealkylation of methoxy, ethoxy, and pentoxy resorufin were elevated in male (high dose) cotton rats. The activity of hepatic glutathione S-transferases (GST) was significantly elevated in male (mid and high dose) and female (all doses) cotton rats exposed to TNT. Histopathological analysis of spleen revealed mild to marked splenic congestion with mild extramedullary hematopoiesis, hemosiderosis, and lymphoid hyperplasia in male and female cotton rats treated with TNT (all doses). Liver weights were increased in males (mid and high dose) and in females (high-dose group). In the high-dose groups, histological changes in liver (mild to moderate hepatocellular hypertrophy, increased hemosiderin pigment in Kupffer cells) in both sexes, and in testis (premature exfoliation of spermatozoa from dilated seminiferous tubules) were observed (mid and high dose). These results suggest that hepatic GST and hemolytic anemia may be biomarkers in cotton rats of terrestrial contamination with TNT or other nitroaromatic explosive compounds.

Land-treatment of petroleum wastes is a widely used industrial practice, yet there has been no comprehensive evaluation of the long-term risks to human or terrestrial ecosystems from such practices. We evaluated cotton rat (Sigmodon hispidus) populations on three sites in Oklahoma (USA) that historically used land-treatment for disposal of various petroleum wastes (July 1995-March 1997). Average concentrations of fluoride in soil from these sites ranged from 878 to 4317 mg/kg. A census of resident cotton rats on land-treatment sites revealed a high incidence (40% overall) of dental lesions compared to reference populations (<1% dental lesions). During winter there was a 34% to 65% increase compared to summer in frequency of dental lesions in cotton rats on two of the three land-treatment sites. Incidence of dental lesions on two land-treatment sites was greater (9-16%) in female cotton rats compared to males. Cotton rats from land-treatment sites had higher concentrations of fluoride in bone and greater severity of dental lesions compared to reference animals. Dental lesions were considered to be most consistent with dental fluorosis because of elevated fluoride in bone. Neither concentration of fluoride in soil nor level of fluoride in bone was a good predictor of severity of dental lesions in cotton rats on land-treatment sites.

Total fluoride (F) content of soils, vegetation, and cotton rats (Sigmodon hispidus) was measured on a landfarm contaminated with petrochemical wastes. A potentially bioavailable form of F was determined by HCl extraction of soils and vegetation. Cotton rats from the landfarm were examined for dental lesions indicative of fluorosis. Exposure pathways including dietary ingestion of unwashed vegetation and nondietary ingestion of soil associated with grooming and burrowing were evaluated. Mean bone F (1,515 mg/kg) and mean soil total F (1,954 mg/kg) from the landfarm site were greater than bone F (121 mg/kg) and soil total F (121 mg/kg) at a reference site. The HCl-extractable F was elevated in landfarm soil (326 mg/kg) compared to the reference site (2.3 mg/kg). About 80% of the cotton rats collected from the landfarm had dental fluorosis. During winter, the dietary pathway consisted of 78.9% of the potential bioavailable exposure (HCl-extractable F) of the two exposure pathways. However, in the summer, the nondietary pathway consisted of 87.9% of the potential bioavailable exposure of the two pathways. incidental ingestion of soil associated with grooming and burrowing is more important than consumption of unwashed vegetation for cotton rats on the landfarm site in the summer. Fluoride accumulation in the soil from landfarming of petroleum wastes may pose a risk to terrestrial vertebrates. In addition to monitoring petroleum hydrocarbons, land application of petrochemical wastes should consider F and other inorganic contaminant loadings to the soil system.

Oil refineries inadvertently deposit a variety of complex mixtures of organic hydrocarbons and heavy metals in the soil, many of which are thought to be potent immunotoxicants. Terrestrial ecosystems such as this have not been adequately investigated with respect to wild rodent populations. The primary objective of this study was to use mesocosms to assess the immunotoxicity risks to feral small mammal populations associated with soils contaminated with petroleum refinery wastes. A series of 4-week and 8-week exposure trials using laboratory raised cotton rats (Sigmodon hispidus) were conducted in situ on three contaminated and three reference sites on the Oklahoma Refining Company Superfund Waste Site, Cyril, Oklahoma. Cotton rats exposed to these soils showed significant alterations in selected morphological traits, in vivo humoral immune responses, complement activity, and macrophage activity. However, immune alterations were not great, suggesting that resident small mammals may be a better biomonitoring choice than using mesocosms.

Beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary have been reported to have dental and bone abnormalities. To determine whether these lesions could be caused by high exposure to fluorides, we measured bone fluoride levels in eight beluga whales stranded on the shores of the St. Lawrence Estuary (Quebec, Canada), and in nine beluga whales killed by Inuit hunters in the Hudson Bay (North Western Territories, Canada). In both groups, fluoride concentrations were higher than those found in terrestrial mammals intoxicated by fluorides. Unexpectedly, fluoride concentration was significantly higher in beluga whales from the Hudson Bay (X̄ ± SD: 10,365 ± 1,098 ppm) than in beluga whales from the St. Lawrence Estuary (4,539 ± 875 ppm) and was positively correlated with age in the latter population. Differences in diet might explain the differences in fluoride concentrations found between these two populations.

Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals that have known effects on physiological and biochemical function. We evaluated the induction of major hepatic cytochrome P-450 isoenzymes through O-dealkylation of various resorufin ethers in wild cotton rats (Sigmodon hispidus) inhabiting three reference areas and three contaminated areas at an abandoned oil refinery Superfund waste site in Oklahoma. Isoenzyme activities were monitored in animals from these sites in summer and winter for 2 years. O-dealkylation of ethoxyresorufin and methoxyresorufin was significantly greater (170–180%) in cotton rats from contaminated areas compared to those from reference areas in summer, but not winter. These results indicate that the cotton rat may be a sensitive model species for biomonitoring petrochemical-contaminated ecosystems and demonstrate the importance of multi-season sampling in biomonitoring studies.

Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals, many of which have known effects on physiological and biochemical function. Although sensitivity of the immune system to chemical exposure has been documented in laboratory animal and wildlife species, little work has been conducted on feral wildlife populations inhabiting contaminated sites. Immune function was measured in populations of wild cotton rats (Sigmodon hispidus) inhabiting replicated reference and contaminated study sites at an abandoned oil refinery in Oklahoma four times from 1991 to 1992. Several measures of immunocompetence were examined including immune organ mass and cellularity, hematology,in vivohypersensitivity, macrophage function, killer cell activity, and lymphoproliferative responsiveness.In vitroproliferation of splenocytes, either spontaneous or induced with concanavalin A (Con A), was the most consistent and reliable indicator of immunotoxicity. Spontaneous proliferation of splenocytes was 48 and 24% higher for cotton rats collected from contaminated than reference sites in September 1991 and September 1992, respectively. Likewise, Con A-induced proliferation of splenocytes ranged form 20 to 53% higher in animals collected from contaminated than reference sites in three of four collection periods. The percentage of splenocytes (mean±SE) staining positive for Con A receptors was lower on contaminated sites (73.7±1.2%) than reference sites (77.0±1.4%) in September 1991. Other measures of immune function including macrophage metabolism, hypersensitivity, blood cellularity, and mass and cellularity of immune organs varied between contaminated and reference sites.

1,3,5-Trinitrobenzene (TNB) is a soil and water contaminant at certain military installations. Encephalopathy in rats given 10 daily oral doses of TNB has been reported. The lesion was bilaterally symmetric vacuolation and microcavitation in the cerebellar roof nuclei, vestibular nuclei, olivary nuclei, and inferior colliculi. The contribution of the blood-brain barrier (BBB) in the genesis of these lesions remains uncertain. One of the main goals of the present work was to evaluate the functional state of the BBB. Male Fischer 344 rats (five rats/group) were euthanatized after four, five, six, seven, eight, or 10 daily doses of TNB (71 mg/kg). A different set of rats (five rats/group) was allowed to recover for 10 or 30 days after receiving 10 doses of TNB. Integrity of the BBB was assessed by immunohistochemical staining for extrav-asated plasma albumin on paraffin-embedded sections. Rats euthanatized after four to eight doses had no lesions, and albumin extravasation in the susceptible regions of the brain was minimal. Rats receiving 10 daily doses of TNB had bilaterally symmetric vacuolation and microcavitation in the cerebellar nuclei, vestibular nuclei, and inferior colliculi in association with multifocal, often confluent foci of extravasated albumin in susceptible nuclei. Albumin was present in vascular walls, extracellular space, and neurons. Immunoreactivity in neurons was of two types: cytoplasmic staining representing pinocytic uptake and homogenous staining of the entire neuron (nucleus and cytoplasm) due to uncontrolled albumin leakage through the damaged cell membrane. In rats allowed to recover for 10 days, the microcavitated foci were infiltrated by glial and gitter cells. Albumin immunoreactivity was present as extracellular granular debris, and neuronal staining (for albumin) was mild. In rats allowed to recover for 30 days, immunoreactivity to albumin was not seen. This study demonstrates that TNB-mediated tissue damage is accompanied by breakdown of the BBB. The presence of vacuolation and associated extravasated serum proteins in TNB-treated rats is an indication of vasogenic brain edema, which appears to be a critical event in TNB toxicity. Additional studies are needed to determine the reason for selective regional vulnerability and brain microvasculature susceptibility to TNB.

The ways in which comprehensive condition profiles, incorporating morphometric, histologic, physiologic, and diet quality indices, responded to changes in density of a white-tailed deer (Odocoileus virginianus) population were examined. Changes in these condition indices were monitored in a northeastern Oklahoma deer herd as density declined from peaks of 80 and 72 deer/km2 in 1989 and 1990 (high-density) to lows of 39 and 41 deer/km2 in 1991 and 1992 (reduced-density), respectively. Compared to a reference population (6 deer/km2), deer sampled during high-density exhibited classic signs of nutritional stress such as low body and visceral organ masses (except elevated adrenal gland mass), low fecal nitrogen levels, reduced concentrations of serum albumin, elevated serum creatinine concentrations, and a high prevalence of parasitic infections. Although density declined by one half over the 4-yr study, gross indices of condition (in particular body mass and size) remained largely unchanged. However, selected organ masses, serum albumin and non-protein nitrogen constituents, and fecal nitrogen indices reflected improvements in nutritional status with reductions in density. Many commonly used indices of deer condition (fat reserves, hematocrit, total serum protein, and blood urea nitrogen) were not responsive to fluctuations in density.