High in vitro endotoxin responsiveness of macrophages from an endotoxin-resistant wild rodent species, Sigmodon hispidus.

It has been reported that macrophages primarily mediate endotoxin shock and cell death by synthesizing and releasing cytokines, largely tumor necrosis factor (TNF) and interleukin-1 (IL-1). However, macrophages from some laboratory mouse strains such as C3h/HeN are unresponsive to endotoxin both in vivo and in vitro. We found members of a wild rodent species, Sigmodon hispidus, to also be extremely resistant to bacterial endotoxin challenge. Intravenous administration of up to 100,000 micrograms/kg body mass of Escherichia coli O26:B6 endotoxin did not cause lethality in adult S. hispidus. In contrast to the endotoxin-resistant mouse strain, peritoneal macrophages derived from S. hispidus were responsive to in vitro endotoxin challenge as measured by high levels of TNF and IL-1 activity in supernatants of macrophage cultures. Thus, in vitro macrophage responsiveness to endotoxin does not always indicate high host sensitivity to endotoxin challenge.

Protective effects of glucose-6-phosphate and NADP against alpha-chaconine-induced developmental toxicity in Xenopus embryos.

In previous studies a metabolic activation system (MAS) composed of Aroclor 1254-induced rat liver microsomes led to an apparent reduction of potato glycoalkaloid developmental toxicity in the frog embryo teratogenesis assay-Xenopus (FETAX). The reasons for this reduction were investigated in this study. The effect of the exogenous MAS on glycoalkaloid developmental toxicity was examined in two experiments in which a concentration series of alpha-chaconine was tested with a MAS with and without a reduced nicotinamide adenine dinucleotide (NADPH) generator system consisting of NADPH, oxidized nicotinamide adenine dinucleotide (NADP), glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase. The NADPH generator system and each of its individual components were tested at a single high concentration of alpha-chaconine to evaluate their potential effects on toxicity. The findings indicated that the protective effect of the MAS was not the result of detoxification by microsomal enzyme systems, but was caused by two components of the NADPH generator system, namely NADP and G6P. G6P was more protective of alpha-chaconine-induced toxicity than NADP at the concentrations tested. Thus, FETAX with a MAS must be performed with appropriate controls that take into account the possible interactions with individual components of the system.

The acute and subchronic toxicity of BRB-I-28, a novel class Ib antiarrhythmic agent, in CD-1 mice.

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.

Evaluation of myelotoxicity in cotton rats (Sigmodon hispidus) exposed to environmental contaminants. I. In vitro bone-marrow progenitor culture.

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 microg/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.

Widespread risks of dental fluorosis in cotton rats (Sigmodon hispidus) residing on petrochemical waste sites.

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.

Evaluation of myelotoxicity in cotton rats (Sigmodon hispidus) exposed to environmental contaminants. II. Myelotoxicity associated with petroleum industrial wastes.

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.

Immunotoxicity risks associated with land-treatment of petrochemical wastes revealed using an in situ rodent model.

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.

Efficacy of histopathology in detecting petrochemical-induced toxicity in wild cotton rats (Sigmodon hispidus).

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.

Cytochrome P450 induction in feral Cricetid rodents: a review of field and laboratory investigations.

The constitutive and inducible hepatic cytochromes P450 of various feral Cricetid rodents (family Cricetidae, comprising various New World rats and mice, hamsters, gerbils and voles), have been examined in a relatively limited number of field and laboratory investigations. These studies, reviewed herein, have employed substrates and immunochemical reagents that are diagnostic for individual P450 subfamilies of Rattus norvegicus (the common laboratory species derived from the Norway rat, a member of the family Muridae). The results have demonstrated that the feral rodents display hepatic responses to prototypic CYP1A inducers (3-methylcholanthrene, beta-naphthoflavone) similar to those displayed by R. norvegicus and Mus musculus (the common laboratory species derived from the house mouse, another member of the family Muridae). At least one study has demonstrated the induction, by ethanol, of a protein immunochemically similar to CYP2E1 in a Cricetid rodent. In Cricetid rodents, phenobarbital-type inducers cause the induction of a hepatic protein immunologically similar to that primarily induced (CYP2B) in R. norvegicus and M. musculus. The proteins induced in the Cricetid rodents, however, exhibit striking differences in substrate specificity, compared to the proteins induced in R. norvegicus. These results indicate that the previously described differences between the P450 induction responses exhibited by the commonly utilized laboratory species R. norvegicus and M. musculus (family Muridae) and the Syrian hamster and gerbil (family Cricetidae) are observed as a generality for members of the Cricetid family of rodents.

In situ (mesocosm) assessment of immunotoxicity risks to small mammals inhabiting petrochemical waste sites.

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.

Calcification of the urinary bladder in the wild cotton rat (Sigmodon hispidus).

Calcification of the urinary bladder epithelium was observed in 19 of 30 and 18 of 30 wild cotton rats from control and petrochemical-contaminated sites, respectively. The rats in the two sites did not differ significantly in respect of serum calcium and phosphorus concentrations. The calcification was considered to be dystrophic in nature. An unidentified factor common to both control and petrochemical-contaminated sites was considered to be responsible for this syndrome.

Spontaneous altered hepatocellular foci (AHF) in captive aged male cotton rats (Sigmodon hispidus).

Spontaneous altered hepatocellular foci (AHF) of basophilic type were observed in adult male cotton rats. The histological features of these foci were similar to those observed in Fischer 344 rats. However, an immunohistochemical technique with antibody to glutathione S-transferase placental form (GST-P) failed to stain these foci. Also normal bile ducts were not immunoreactive for GST-P. The presence of a gall bladder and of non-GST-P immunoreactive liver foci and bile ducts suggests that these cotton rats are phylogenetically closer to mice than rats.

Effect of pyrrolizidine alkaloid-induced hepatic disease on plasma amino acid patterns in the horse.

Plasma amino acid patterns were studied in 6 clinically normal adult horses during the course of hepatic disease induced by feeding them plants containing pyrrolizidine alkaloids. At death, there were significant (P less than 0.01) increases in glutamine, proline, tyrosine, asparagine, lysine, histidine, alanine, phenylalanine, methionine, aspartic acid, and ornithine values. There were no significant changes in glycine, valine, isoleucine tryptophan, and arginine values. There were significant (P less than 0.01) decreases in citrulline. Ammonia increased 4-fold. Alpha-Aminoadipic acid and alpha-aminobutyric acid were not detectable in the plasma of clinically normal horses, but were in the plasma of horses with severe hepatic disease. The ratio of branched chain amino acids (isoleucine + leucine + valine) to phenylalanine plus tyrosine progressively decreased from a normal of +/- 0.5 to 1.3 +/- 0.3 (mean +/- SD) just prior to death. The relative changes in plasma amino acid pattern in horses with hepatic diseases seem similar to those changes occurring in man and other animals with hepatic disease.

Sensitivity of condition indices to changing density in a white-tailed deer population.

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.

Strongyloidiasis in cotton rats (Sigmodon hispidus) from central Oklahoma.

Thirty-one of 40 cotton rats (Sigmodon hispidus) collected from central Oklahoma were infected with Strongyloides sp. (78% prevalence). Larvae of Strongyloides sp. (rhabditiform or filariform) were not demonstrable in intestinal contents and scrapings. Female nematodes recovered from intestinal contents and scrapings had morphological similarities with Strongyloides sigmodontis. Cotton rats infected with Strongyloides sp. were indistinguishable clinically from non-infected hosts. Infected animals had no significant gross lesions, but the presence of Strongyloides sp. in the intestinal mucosa was associated with villus atrophy and mild to moderate infiltration of the lamina propria by lymphocytes, plasma cells and occasional eosinophils. Other organs or tissues examined were free from lesions induced by Strongyloides sp.

Environmental, age, and sex effects on cotton rat (Sigmodon hispidus) hematology.

We determined the effects of sex, age, and environment (inbred, captive-wild, and wild animals) on selected blood parameters of the cotton rat (Sigmodon hispidus) in central Oklahoma (USA) from 1990 to 1994. Male and female cotton rats had similar blood profiles. Age-related differences were confined to differential white blood cell counts where adults possessed greater numbers of neutrophils and lower numbers of lymphocytes compared to juveniles. Environment had a strong influence on many hematological parameters. Hematocrit, hemoglobin concentration, RBC count, and eosinophil number were generally greater for wild stocks compared to inbred animals, and differences were more pronounced for adults than juveniles.

Cryptosporidiosis in a cotton rat (Sigmodon hispidus).

Cryptosporidiosis, previously unreported in cotton rats (Sigmodon hispidus), was observed in one of nine cotton rats from Pryor, Oklahoma (USA). Infection was confined to the large intestine. Microscopically, numerous cryptosporidians measuring 2 to 3 microns in diameter were intimately associated with the luminal surface of colonic and rectal mucosae. The affected lamina propria of the large intestine was diffusely infiltrated by eosinophils, lymphocytes and macrophages. Ultrastructurally, numerous trophozoites and a single schizont were observed. Microvilli were displaced by the presence of cryptosporidians at the attachment site.

Sensitivity of selected immunological, hematological, and reproductive parameters in the cotton rat (Sigmodon hispidus) to subchronic lead exposure.

The effect of lead exposure on cellular immunity, hematology, and reproductive and body condition in mature cotton rats (Sigmodon hispidus) was examined. Two groups of 36 cotton rats each were exposed to 0, 100, or 1,000 ppm lead in drinking water for either 7 or 13 weeks, between 31 August and 2 December 1990. Specific and non-specific cell-mediated immunity was assessed by measuring splenocyte proliferative responses to polyclonal mitogens (Concanavalin A and Pokeweed mitogen), in vivo 24-hr delayed-type hypersensitivity, metabolic activity of peritoneal macrophages, spleen mass and cellularity, and immune organ development. General physiological condition was assessed from hematological, morphological, and reproductive measures. Immune function was sensitive to lead exposure based on depressed proliferative responses of cultured splenocytes, smaller popliteal lymph nodes, and larger spleens among cotton rats receiving 1,000 ppm lead. Spleen mass was reduced in cotton rats receiving 100 ppm lead. Total leukocytes, lymphocytes, neutrophils, eosinophils, total splenocyte yield, packed cell volume, hemoglobin, and mean corpuscular hemoglobin were sensitive to lead exposure. Effects of lead exposure on general condition and reproductive parameters included reduced mass of liver, seminal vesicles, and epididymes in males following a 7-week exposure. Histopathologic changes reflected lead toxicity and included altered renal proximal tubular epithelium, renal intranuclear inclusions, and in some cases, lowered numbers of sperm and developing follicles. In general, lesions were more pronounced with increased lead concentration and longer exposure.

Bone fluoride concentrations in beluga whales from Canada.

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 (mean +/- 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.

Cause-specific mortality of white-tailed deer as influenced by military training activities in southwestern Oklahoma.

Radio-telemetry was used to monitor movements and mortality of 56 white-tailed deer (Odocoileus virginianus) in response to intensive military training activities on West Range (18,000 ha), Fort Sill Military Reservation, Oklahoma. Cause-specific mortality was determined for 22 radio-collared deer, including adults (greater than or equal to 2.0-yr-old), yearlings (0.6-1.9-yr-old), and fawns (less than or equal to 75-day-old age group) from 1987 to 1989. Winter home ranges were largely confined to a 14,411 ha impact area centrally located on West Range. The mean annual mortality rate was 0.50 for adults and yearlings combined. Fifty percent of all adult and yearling mortality was attributed to military training activities, 28% to hunting, 16% to collisions with automobiles, and 6% to unknown causes. The mean monthly mortality rate was 0.61 for neonatal fawns and predation accounted for three of four mortalities. All captured deer in the greater than or equal to 2.6-yr-old, 82% in the 1.6-yr-old, 10% in the 0.6-yr-old, and all deer in the less than 7-day-old age groups were seropositive for bluetongue virus (BTV). Our study strongly suggests that the consequences of military training activities should be considered in the management of white-tailed deer herds on military installations.