Multiple environmental stressors induce complex transcriptomic responses indicative of phenotypic outcomes in Western fence lizard.

BACKGROUND: The health and resilience of species in natural environments is increasingly challenged by complex anthropogenic stressor combinations including climate change, habitat encroachment, and chemical contamination. To better understand impacts of these stressors we examined the individual- and combined-stressor impacts of malaria infection, food limitation, and 2,4,6-trinitrotoluene (TNT) exposures on gene expression in livers of Western fence lizards (WFL, Sceloporus occidentalis) using custom WFL transcriptome-based microarrays. RESULTS: Computational analysis including annotation enrichment and correlation analysis identified putative functional mechanisms linking transcript expression and toxicological phenotypes. TNT exposure increased transcript expression for genes involved in erythropoiesis, potentially in response to TNT-induced anemia and/or methemoglobinemia and caused dose-specific effects on genes involved in lipid and overall energy metabolism consistent with a hormesis response of growth stimulation at low doses and adverse decreases in lizard growth at high doses. Functional enrichment results were indicative of inhibited potential for lipid mobilization and catabolism in TNT exposures which corresponded with increased inguinal fat weights and was suggestive of a decreased overall energy budget. Malaria infection elicited enriched expression of multiple immune-related functions likely corresponding to increased white blood cell (WBC) counts. Food limitation alone enriched functions related to cellular energy production and decreased expression of immune responses consistent with a decrease in WBC levels. CONCLUSIONS: Despite these findings, the lizards demonstrated immune resilience to malaria infection under food limitation with transcriptional results indicating a fully competent immune response to malaria, even under bio-energetic constraints. Interestingly, both TNT and malaria individually increased transcriptional expression of immune-related genes and increased overall WBC concentrations in blood; responses that were retained in the TNT x malaria combined exposure. The results demonstrate complex and sometimes unexpected responses to multiple stressors where the lizards displayed remarkable resiliency to the stressor combinations investigated.

Interspecific effects of 4A-DNT (4-amino-2,6-dinitrotoluene) and RDX (1,3,5-trinitro-1,3,5-triazine) in Japanese quail, Northern bobwhite, and Zebra finch.

The purpose of this study was to assess the toxicological effects of two munition compounds, 4-amino-2,6-dinitrotoluene (4A-DNT) and 1,3,5-trinitro-1,3,5-triazine (RDX), on three different bird species: two common toxicological model species-the Northern Bobwhite (Colinus virginianus) and the Japanese Quail (Coturnix japonica), and a representative passerine-the Zebra Finch (Taeniopygia guttata). Bobwhite were exposed to 4A-DNT at 0, 8, 15, 30, 60, or 150 mg/kg body weight (bw) d by oral gavage for seven days; because the high dose of 4A-DNT was lethal to bobwhite, the maximum dose was changed to 100 mg/kg bw d for Japanese quail and finches to ensure tissue could be used for future toxicogenomic work. RDX was similarly administered at 0, 0.5, 1.5, 3, 6, or 12 mg/kg bw d. Blood was drawn prior to euthanasia for blood cellularity and chemistry analyses. Finches were clearly least affected by 4A-DNT as evidenced by a lack of observable effects. Bobwhite appeared to be the most sensitive species to 4A-DNT as observed through changes in blood cellularity and plasma chemistry effects. Bobwhite appeared to be more sensitive to RDX than Japanese Quail due to increased effects on measures of plasma chemistries. Finches exhibited the greatest sensitivity to RDX through increased mortality and seizure activity. This study suggests that sensitivity among species is chemical-specific and provides data that could be used to refine current avian sensitivity models used in ecological risk assessments.

Multiple environmental stressors elicit complex interactive effects in the western fence lizard (Sceloporus occidentalis).

Evaluation of multiple-stressor effects stemming from habitat degradation, climate change, and exposure to chemical contaminants is crucial for addressing challenges to ecological and environmental health. To assess the effects of multiple stressors in an understudied taxon, the western fence lizard (Sceloporus occidentalis) was used to characterize the individual and combined effects of food limitation, exposure to the munitions constituent 2,4,6-trinitrotoluene (TNT), and Plasmodium mexicanum (lizard malaria) infection. Three experimental assays were conducted including: Experiment I--TNT × Food Limitation, Experiment II--Food Limitation × Malaria Infection, and Experiment III--TNT × Malaria Infection. All experiments had a 30 day duration, the malaria treatment included infected and non infected control lizards, food limitation treatments included an ad libitum control and at least one reduced food ration and TNT exposures consisting of daily oral doses of corn oil control or a corn oil-TNT suspension at 5, 10, 20, 40 mg/kg/day. The individual stressors caused a variety of effects including: reduced feeding, reduced testes mass, anemia, increased white blood cell (WBC) concentrations and increased mass of liver, kidney and spleen in TNT exposures; reduced cholesterol, WBC concentrations and whole body, testes and inguinal fat weights given food limitation; and increased WBC concentrations and spleen weights as well as decreased cholesterol and testes mass in malaria infected lizards. Additive and interactive effects were found among certain stressor combinations including elimination of TNT-induced hormesis for growth under food limitation. Ultimately, our study indicates the potential for effects modulation when environmental stressors are combined.

Toxic effects of oral 2-amino-4,6-dinitrotoluene in the Western fence lizard (Sceloporus occidentalis).

The compound 2-amino-4,6-dinitrotoluene (2A-DNT) was evaluated under laboratory conditions in the Western fence lizard (Sceloporus occidentalis) to assess the potential for reptile toxicity. Oral LD(50) values were 1406 and 1867 mg/kg for male and female lizards, respectively. Based on responses from a 14-day subacute study, a 60-day subchronic experiment followed where lizards were orally dosed at 0, 5, 15, 20, 25, 30 mg/kg-d. At day 60, number of days and survivors, food consumption, and change in body weight were inversely related to dose. Signs of toxicity were characterized by anorexia and generalized cachexia. Significant adverse histopathology was observed in hepatic tissue at ≥ 15 mg/kg-d, consistent with hepatocellular transdifferentiation. Based on survival, loss of body weight, diminished food intake, changes in liver, kidney, and testes, and increased blood urea nitrogen, these data suggest a LOAEL of 15 mg/kg-d and a NOAEL of 5 mg/kg-d in S. occidentalis.

Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus).

2-Amino-4,6-dinitrotoluene (2A-DNT) is a metabolite of the explosive 2,4,6-trinitrotoluene (TNT) which is present in the soil at numerous U.S. Army installations as the result of TNT manufacture or training activities. Although many avian species are known to inhabit areas where 2A-DNT has been found in the environment, no published studies of the effects of 2A-DNT exposure in birds are available. In this study, we conducted an evaluation of the oral toxicity of 2A-DNT in a representative ground foraging species of management concern, the northern bobwhite (Colinus virginianus). Subacute (14 days) and subchronic (60 days) oral gavage exposure studies were conducted following determination of the median acute lethal dose (LD50 = 1167 mg/kg). In the subacute study, survival occurred at 50 mg/kg/day. This helped to determine dose groups for the subchronic study: 0, 0.5, 3, 14, and 30 mg 2A-DNT/kg body weight-d in corn oil. The lowest observed adverse effects level (LOAEL) was determined to be 14 mg/kg/day based on mortality, and the no observed adverse effects level (NOAEL) was determined to be 3 mg/kg/day based on lack of effects at this exposure level.

Toxicity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) in three vertebrate species.

The explosive, octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine or high-melting explosive (HMX), has been found in soils in areas used for testing and training by the military. Many of these areas contain habitat for valued wildlife species. In an effort to better understand the environmental consequences from exposure, a reptilian (western fence lizard [Sceloporus occidentalis]), an amphibian (red-backed salamander [Plethodon cinereus]), and a mammalian species (rabbit [Oryctolagus cuniculus]) were exposed to HMX under controlled laboratory conditions. Lizards and rabbits were exposed to HMX by way of corn oil through gavage, and salamanders were exposed to HMX in soil. Two deaths occurred from acute oral exposures to lizards to 5000 mg HMX/kg BW. Histological and gross pathologic assessment suggested gut impaction as a possible cause of death. Salamanders exposed to concentrations of HMX in soil < or = 1970 mg HMX/kg soil for 10 days did not show adverse effects. Rabbits, however, showed neurologic effects manifested as hyperkinetic events with convulsions at > 24 h after oral exposures. An LD(50) for rabbits was calculated as 93 mg/kg (95% confidence interval 76-117). A subacute 14-day testing regime found a lowest observed effect level of 10 mg/kg-d and a no observed adverse effect level of 5 mg/kg-d based on hyperkinesia and seizure incidence, although changes suggesting functional hepatic alterations were also found. These data suggest that physiologic differences between species, particularly in gastrointestinal structure and function, can affect the absorption of HMX and hence lead to marked differences in toxicity from exposure to the same compound.

Unsuitability of the northern bobwhite as a model species for the assessment of reproductive behavior in toxicological studies.

The northern bobwhite (Colinus virginianus) is used in numerous wildlife toxicity studies, however no published reports could be located that mention the measurement of reproductive behavior in this species. Changes in reproductive behavior can be potentially more sensitive to environmental contaminant exposures and less resilient than more traditional physiological responses. Male bobwhite copulatory behaviors were measured similarly to those that are well established for use in Japanese quail (Coturnix japonica). Time to initiate mating, time to achieve a successful copulation, the number of mating attempts, and the number of successful copulations were recorded daily for four consecutive days over a period of 3 min for each male/female pair of birds per day. When females were introduced to male cages, males were more occupied with shows of aggression towards neighboring males than attempts to mate with the female sharing their space. Only one male successfully mated with a female over the entire 4 days of the test. Future attempts at assessing reproductive behavior in this species may be more successful if birds are separated from the rest of the group when paired. The Japanese quail seems to be a more appropriate species for overall reproductive tests due to: willingness of males to copulate in the presence of other males, consistent egg laying ability, and the short time required for embryonic development and reproductive maturity.

A preliminary assessment of relative sensitivities to foreign red blood cell challenges in the northern bobwhite for potential evaluation of immunotoxicity.

Many environmental toxins have been shown to suppress the immune system across taxa. The foreign red blood cell (RBC) challenge is an important part of a complement of tests used to assess immunocompetence in the laboratory because it can assess an individual's humoral response without impacting its health. This challenge is used commonly across species and measures antibody titers in response to an intraperitoneal, intravenous, or subcutaneous injection of foreign RBCs. Determination of the best appropriate foreign RBC challenge is therefore important when designing tests for evaluation of humoral responses. The northern bobwhite (Colinus virginianus) is a commonly used species for avian toxicity tests, however little is known about the relative sensitivities of its humoral responses to foreign erythrocytes. In this pilot study, we exposed adult quail to intravenous injections of 5% solutions of sheep, rat, rabbit, bovine, or chicken erythrocytes and performed antibody titers [hemagglutination assay for total immunoglobulin (Ig), IgG, and IgM] for primary and secondary responses. Although the bobwhites appeared to respond strongly to rat RBCs, high variability in responses were observed among individuals. Chicken RBCs elicited the poorest responses for both primary and secondary challenges. Sheep and bovine RBCs were adequate antigens for this test in bobwhites. We found that rabbit erythrocytes elicited the strongest responses with the least amount of variability between individuals. Rabbit RBCs, therefore, appear to be the ideal antigen for this test of the humoral response in this species.

Reproductive and developmental effects and physical and chemical properties of pentaerythritol tetranitrate (PETN) in the rat.

Pentaerythritol tetranitrate (PETN) is an explosive chemical that has been detected in environmental media. Although previous toxicology studies have shown PETN to be relatively benign, a lack of available information concerning developmental and reproductive effects from oral PETN exposure was needed. Sprague-Dawley rats were exposed to oral daily adjusted volumetric doses of 0, 100, 500, or 1,000 mg PETN/kg body mass in a corn oil vehicle for up to 56 days. Mating, duration of gestation, body weight, feed consumption, overall condition of adults, and the number, sex, and condition of pups were recorded. Histological examinations were also performed on the ovaries, testes, and epididymides of animals from the control and the highest dose groups. Other environmental criteria, water solubility, octanol/water partition coefficient, and biodegradation rates of neat PETN were also determined. Only body weights and feed consumption were affected by treatment; however, these differences may be attributed more to volumetric adjustments of vehicle in the control and high-dose groups than to PETN toxicity. No adverse effects on development or reproduction from PETN exposure were observed. Water solubility, octanol water partition coefficient, and water suspension and biodegradation rates suggest PETN is unlikely to transport or bioaccumulate in the environment to any appreciable extent. Additionally, biotic processes are most likely faster in breaking down PETN than the abiotic processes involved in dissolving PETN in water.

Sublethal effects of subacute exposure to RDX (1,3,5-trinitro-1,3,5-triazine) in the northern bobwhite (Colinus virginianus).

Northern bobwhite (Colinus virginianus) were orally exposed via gavage to 0, 0.5, 3, 8, 12, or 17 mg/kg of RDX (1,3,5-trinitro-1,3,5-triazine) in corn oil daily for 14 d to evaluate sublethal effects of this explosive in birds. Mortality occurred at a rates of 100, 67, and 25% for the 17, 12, and 8 mg/kg/d dose groups, respectively. Death was preceded by clonic and tonic convulsions and weight loss caused by gastrointestinal effects. Increases in serum globulin and total leukocytes were observed in the two highest-dose groups. Degeneration of testicular and splenic tissue also was observed. The no-observed-adverse-effects and lowest-observed-adverse-effects levels were determined as 3.0 and 8.0 mg/kg/d, respectively.

Toxic effects of oral hexahydro-1,3,5-trinitro-1,3,5-triazine in the western fence lizard (Sceloporus occidentalis).

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been widely used as an explosive in munition formulations, resulting in contamination of wildlife habitat on military installations. To estimate health effects for reptilian species, acute, subacute, and subchronic oral toxicity studies were conducted using the Western fence lizard (Sceloporus occidentalis). Estimated oral median lethal doses were 72 (95% confidence interval [CI], 49-106) mg/kg body weight (slope, 3.754) for males and 88 (95% CI, 65-119) mg/kg (slope, 4.525) for females. Toxicity from RDX suggested the neurological system as the critical target tissue. A 14-d subacute study followed with males dosed orally with RDX (corn oil) at 0, 10, 20, 25, 30, 45, and 60 mg/kg/d. Signs of toxicity frequently included a characteristic body posture. A significant dose-survival relationship was seen over the range of doses, with a significant decrease in survival at 20 mg/kg/d. Males in the 60-d subchronic study were dosed at 0, 1, 2.5, 5, 8, and 11 mg/kg/d, and signs of toxicity included lethargy, cachexia, and anorexia. Survival was decreased at 8 and 11 mg/kg/d. Reduced growth rate and food consumption occurred at 5 mg/kg/d. Brain tissue was assayed for RDX when seizures were observed at a residue concentration of at least 18 microg/g. No abnormalities were observed in the hematologic indices, whereas plasma proteins were reduced. Hepatic enlargement and decreased testes mass occurred at 8 and 11 mg/kg/d. Plasma testosterone concentrations, sperm counts, and motility measures were variable for all treatment levels. Based on survival, growth rate, food intake, and testes to brain weight ratios, these data suggest a lowest-observed-adverse effect level of 5 mg/kg/d and a no-observed-adverse effect level of 2.5 mg/kg/d.

Toxicity of oral exposure to 2,4,6-trinitrotoluene in the western fence lizard (Sceloporus occidentalis).

Contamination of the soil with the explosive 2,4,6-trinitrotoluene (TNT) has been found at military sites, many of which are habitats used by reptiles. To provide data useful in assessing ecological risk for reptilian species, acute, subacute, and subchronic oral toxicity studies were conducted with the western fence lizard (Sceloporus occidentalis). Oral median lethal dose (LD50) values for TNT in corn oil were 1,038 and 1,579 mg/kg of body weight for male and female lizards, respectively. Overt signs of toxicity included chromaturia, abdominal enlargement, and tremors. A 14-d subacute study followed in which male lizards were orally dosed with TNT (corn oil) at 0, 33, 66, 132, 263, 525, and 1,050 mg/kg of body weight each day. Clinical signs of toxicity, while similar to the LD50 study, were more subtle and noted in lizards receiving TNT amounts of at least 66 mg/kg/d. Chromaturia was an early consistent sign, often preceding the onset of adverse effects. Male lizards in the 60-d subchronic study were dosed at 0, 3, 15, 25, 35, and 45 mg/kg/d with nearly complete survival (>90%) for lizards in all treatments. Changes in food consumption and body weight were observed at 35 and 45 mg/kg/d. Alterations in hematological end points; blood chemistries (albumin, total protein, alkaline phosphatase, calcium); kidney, spleen, and liver weights; and adverse histopathology were observed in lizards exposed at 25 to 45 mg/kg/d. Testosterone concentration, sperm count, and motility were variable between treatments. Although not significant, incidences of hypospermia and testicular atrophy were observed in some individuals. Together, these data suggest a lowest-observed-adverse effect level of 25 mg/kg/d and a no-observed-adverse effect level of 15 mg/kg/d in S. occidentalis.

Effects of subchronic exposure to 2,6-dinitrotoluene in the northern bobwhite (Colinus virginianus).

Explosives and their breakdown products are commonly found in soils at U.S. military installations. Many areas where these compounds are found represent useful habitat for ground-foraging birds. Because these birds likely are exposed to such compounds, we evaluated the oral toxicity of 2,6-dinitrotoluene (DNT) in a representative ground-foraging species of management concern. Adult male and female northern bobwhite (Colinus virginianus) were exposed to either 0, 5, 10, 40, or 60 mg/kg/d via gavage for 60 d (subchronic) following determination of the median acute lethal dose (320 mg/kg). Circulating levels of red blood cells and hemoglobin were significantly decreased in a dose-dependent manner; however, levels remained within normal ranges. Plasma concentrations of total protein, albumin, globulin, aspartate aminotransferase, and potassium, sodium, and chlorine ions were significantly decreased, and circulating levels of uric acid were significantly increased. Decreased body weight, enlarged gallbladders, edematous gastrointestinal tracts, pale kidneys, pale and fibrous livers, and loose stools were consistent observations. The effects found in the clinical chemistries taken together with histopathological abnormalities observed in sections of hepatic and renal tissue suggest that the liver and kidneys are major targets for 2,6-DNT. Oral exposures to 2,6-DNT appear to affect northern bobwhite in a manner similar to that of the other main DNT isomer, 2,4-DNT, but in more subtle ways, adversely affecting the gastrointestinal system and leading to diarrhea and, ultimately, emaciation. The lowest-observed-adverse-effect level is 40 mg/ kg/d based on hematological measures, and the no-observed-adverse-effect level is 10 mg/kg/d based on the absence of results indicative of adverse effects.

Northern pocket gophers (Thomomys talpoides) as biomonitors of environmental metal contamination.

We live-trapped 40 northern pocket gophers across two years from the Anaconda Smelter Superfund Site, Anaconda, Montana, USA, to determine their exposure to five metal contaminants and effects of exposure on selected measurements. Soil, gopher blood, liver, kidney, and carcass samples were analyzed for arsenic, cadmium, lead, copper, and zinc. Hematological parameters, kidney and liver porphyrins, and red blood cell delta-aminolevulinic acid dehydratase (ALAD) activity were also measured. Micronutrients Cu and Zn were detected in all tissues analyzed, and Cd, Pb, and As were detected less frequently. We report differences in metal distribution among different tissues and differences in bioaccumulation for different metals within the same tissue. No significant differences were observed in concentrations of Zn or Cu in any tissue across the study site, but relationships between lead in soil and lead in carcass proved especially strong (r2 = 0.80; p < 0.001; n = 18). Among biomarker data, we observed a negative relationship between concentration of lead in the soil and ALAD activity in gophers with detectable concentrations of lead in their blood (r2 = 0.45; p = 0.006; n = 15). Results of this study suggest that northern pocket gophers are useful biomonitors of environmental Pb, Cd, and As contamination, and their broad geographic range across North America could allow them to be an important component of site-specific metals assessments.