Assessment of the immunotoxic potential of trichloroethylene and perchloroethylene in rats following inhalation exposure.

The immunotoxic potential of trichloroethylene (TCE) and perchloroethylene (PERC) was assessed after inhalation exposure through the evaluation of the antibody forming cell (AFC) response to sheep red blood cells (SRBC). Female Sprague-Dawley rats were exposed to TCE or PERC vapor at 0, 100, 300, or 1000 ppm for 6 h/day, 5 days/week for 4 weeks (20 exposure days). Additional 0 ppm control groups were included and were dosed with cyclophosphamide via intraperitoneal injection to serve as positive immunosuppressive controls in the SRBC assay. Additional end-points evaluated included liver, kidney, spleen, and thymus weights, hematology, cellular differentials in bronchoalveolar lavage fluid, histopathology of select tissues, and assessment of the phagocytic activity of pulmonary alveolar macrophage (PERC only). Exposure to the high concentration of TCE (1000 ppm) resulted in increases in relative liver and kidney weights and suppression of AFC responses (AFC/spleen and AFC/10(6) spleen cells) by ≈ 70%, while no treatment-related effects were noted at 100 and 300 ppm. Animals exposed to PERC at levels of 300 or 1000 ppm had statistically significant increases in relative liver weights that were accompanied by very slight hypertrophy of the centrilobular hepatocytes. Animals exposed to PERC did not demonstrate a treatment-related effect on the AFC response and no effect was noted on the phagocytic activity of pulmonary alveolar macrophages. The results of these studies indicate that TCE had immunotoxic potential only at high exposure concentrations (1000 ppm), while PERC, at similar exposure concentrations, did not display any evidence of immunotoxicity.

Initial insights regarding the role of p53 in maintaining sperm DNA integrity following treatment of mice with ethylnitrosourea or cyclophosphamide.

If p53 is essential to eliminate damaged spermatogenic cells, then mutagen exposure in the absence of p53 would increase sperm containing damaged DNA. p53 knockout (-/-, NULL) and wild-type (+/+, WT) mice (five/group) were exposed to ethylnitrosourea (ENU) or cyclophosphamide (CP). In phase I, mice were exposed by gavage to 0 or 60 mg/kg/day ENU or CP for four days and examined on test day (TD) 4, and in phase II, mice were exposed to 0, 6, 20, or 60 mg/kg/day ENU or CP for four days and evaluated on TD 36 when exposed spermatocytes matured. In phase I, mutagens were not directly cytotoxic to mature sperm. In phase II, WT mice were more sensitive to decreases in reproductive organ weights, whereas both genotypes had decreased sperm counts. Testicular histology revealed similar CP responses, but genotype-specific ENU responses (WT mice had depletion of elongating spermatids; NULL mice had late-stage spermatocyte/early stage spermatid loss). Ethylnitrosourea increased DNA strand breaks in WT mice. Thus, mice responded similarly to CP, suggesting a primarily p53-independent response, whereas the ENU response differed by zygosity, suggesting a role for p53. As DNA damage increased at higher ENU doses, compensatory repair pathways may operate in NULL mice.

Automated dose-response analysis of the relative hepatic gene expression potency of TCDF in C57BL/6 mice.

Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF), relative to TCDD with complementary histopathology, TCDD and TCDF tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay data. Immature ovariectomized C57BL/6 mice were gavaged with 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 microg/kg TCDD, the World Health Organization TEF-adjusted doses (10 x TCDD dose) of TCDF (0.3, 1, 3, 10, 30, 100, or 300 microg/kg), or sesame oil vehicle and killed at 72 h. Two thousand two hundred eighty-eight and 1347 genes were differentially expressed (P1(t) > 0.90) at one or more doses by TCDD and TCDF, respectively. Automated dose-response modeling (ToxResponse Modeler) identified a total of 1027 and 837 genes with either a sigmoidal, exponential, linear, Gaussian, or quadratic dose-response relationship 72 h after treatment in TCDD and TCDF, respectively. Two hundred seventy genes exhibited a sigmoidal TCDD-induced dose-response (ED(50s) from 0.08 to 42.2 microg/kg) compared to only 179 sigmoidal responsive genes (ED(50s) from 0.74 to 299.9 microg/kg) elicited by TCDF. Of the 1027 TCDD dose-responsive genes, 654 were not examined further due to the lack of a dose response elicited by TCDF. Of the 373 genes that exhibited a TCDD and TCDF dose response, REPs were calculated for the 83 genes that exhibited comparable sigmoidal curve shapes and slopes. The median REP for these 83 genes was 0.10, with a maximum REP of 0.56 and a minimum of 0.01. REPs of 0.04 were also calculated for EROD and increase in relative liver weight (RLW) at 72 h. Collectively, the lower number of TCDF-induced genes compared to TCDD and the 0.04 REPs for EROD activity and increased RLW are not consistent with the TEF of 0.10 for the hepatotoxicity of TCDF in C57BL/6 mice at 72 h.

Comparative temporal toxicogenomic analysis of TCDD- and TCDF-mediated hepatic effects in immature female C57BL/6 mice.

Temporal analyses were performed on hepatic tissue from immature female C57BL/6 mice in order to compare the gene expression profiles for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibzofuran (TCDF). Time course studies conducted with a single oral dose of 300 microg/kg TCDF or 30 microg/kg TCDD were used to compare differential gene expression on complementary DNA microarrays containing 13,361 features, representing 8194 genes at 2, 4, 8, 12, 24, 72, 120, and 168 h. One hundred and ninety-five genes were identified as differentially regulated by TCDF, of which 116 genes were in common with TCDD, with 109 exhibiting comparable expression profiles (correlation coefficients > 0.3). In general, TCDF was less effective in eliciting hepatic vacuolization, and differential gene expression compared with TCDD when given at an equipotent dose based on a toxic equivalence factor (TEF) of 0.1 for TCDF, especially 72-h postadministration. For example, the induction of Cyp1a1 messenger RNA by TCDF was less when compared TCDD. Moreover, TCDF induced less severe hepatocyte cytoplasmic vacuolization consistent with lower lipid accumulations which significantly subsided by 120 and 168 h when compared with TCDD. TCDF-elicited responses correlated with their hepatic tissue levels which gradually decreased between 18 and 168 h. Although both compounds elicited comparable gene expression profiles, especially at early time points, the TCDF responses were generally weaker. Collectively, these results suggest that the weaker TCDF responses could be attributed to differences in pharmacokinetics. However, more comprehensive dose-response studies are required at optimal times for each end point of interest in order to investigate the effect of pharmacokinetic differences on relative potencies that are important in establishing TEFs.

Factors affecting grip strength testing.

The rodent grip strength test was developed decades ago and is a putative measure of muscular strength. This test has been included in the functional observational battery (FOB) to screen for neurobehavioral toxicity, and changes in grip strength have been interpreted as evidence of motor neurotoxicity. Despite its widespread use, questions remain about what the grip strength test actually measures. In this study, potential confounders of the grip strength test were identified and tested, including operational parameters, disruption of peripheral sensory function and changes in body weight. Operational parameters (sampling rate, system type and trial angle but not trial speed) had dramatic effects on grip strength data. Doxorubicin (DX, 10 mg/kg iv) was used to cause sensory impairment. It decreased forelimb and hindlimb grip strength (by 27% and 32%, respectively, compared with controls), an effect that was correlated with degeneration of peripheral and central sensory components (distal tibial and sural nerves, dorsal funiculus of the spinal cord and dorsal, but not ventral, spinal roots). Feed restriction-induced loss of body weight (26% compared with controls) and muscle mass (20% compared with controls) reversibly decreased both forelimb and hindlimb grip strength (18% and 17%, respectively, compared with controls). Ignoring these confounding factors could potentially lead to increased data variability and inconsistency within single studies, across studies and in historical control data sets. To assist in data interpretation and evaluation of grip strength results, it is suggested that exact conditions of application of the test be reported in greater detail. Furthermore, given that the grip strength test can be influenced by factors other than true muscular strength, use of the term grip performance is proposed to better reflect the apical nature of this test.

Preliminary study to test the feasibility of using a local lymph node type of approach to characterize respiratory allergens.

There is a critical need to develop animal models that can characterize the potential of respiratory allergy. Dust-mite allergens are one of the major etiological agents in the induction of allergy and asthma in humans. In this study, the effects of intratracheal injection with dust-mite allergen were investigated by analyzing the in vivo proliferative response of lung-draining hilar lymph nodes and histopathological changes in the lung parenchyma. Balb/c mice were inoculated intratracheally with dust-mite allergens, a mixture of Dermatophagoides farinae and D. pteronyssinus dissolved in phosphate-buffered saline, or with an equal volume of saline alone. After 1 week, all the mice were injected intravenously with radioactive (3)H-thymidine and sacrificed 5 h later so as to assess the radioactivity incorporated into the hilar lymph nodes. The results indicated a marked increase in the proliferative response in the hilar lymph nodes of the animals treated with the dust-mite allergen as compared to the response of the control group. Treatment with dust-mite allergen also caused perivascular and interstitial eosinophilic inflammation of the lungs, hyperplasia of bronchus-associated lymphoid tissue, and an increase in the eosinophil peroxidase activity in the lungs. These results indicate that intratracheal injection with dust-mite allergen can trigger a number of changes consistent with respiratory allergy, including an increased proliferation in the draining lymph nodes.