Bonnie J. Smith

Distribution of intestinal enzyme activities along the intestinal tract of cultured Nile tilapia, Oreochromis niloticus L.

Regional distribution of intestinal enzymes in Nile tilapia was investigated using enzyme histochemistry. Samples from adult fish were obtained from the five major intestinal segments. Activities of maltase, leucine aminopeptidase, dipeptidyl aminopeptidase IV, lipase, non-specific esterases, and alkaline phosphatase were examined in each segment. All enzymes were present at specific sites along the first four intestinal segments. Strong reaction for maltase was present in the third intestinal segment, while aminopeptidases and alkaline phosphatase were detected in the first three parts. The most intense activity for lipase was present in the first two parts, while non-specific esterases were observed in the first four portions. Activities of all these enzymes were demonstrated in the brush border. Non-specific esterases were also present in the cytoplasm of the enterocytes. In addition to its brush border localization in the cranial segments, dipeptidyl aminopeptidase IV was also observed in the basal lamina of all segments, including the terminal segment. These results suggest that the first four regions play the most important role in both digestion and absorption of nutrients. In addition, the great length of the portions of the intestinal tract participating in digestion provides abundant surface area for nutrient absorption, and is likely one factor involved in the rapid growth rate characteristic of tilapian fish.

Analysis of apoptosis of lymphoid cells in fish exposed to immunotoxic compounds

BACKGROUND Chemical induction of apoptosis in cells is believed to contribute to toxicity. Techniques for measuring apoptosis have increased in both sensitivity and number and in many cases can be readily extended to nontraditional research species. A comparison of established assays for measuring apoptosis of lymphoid cells has thus far not been performed in the fish and thus would be efficacious in assessing immunotoxicity. METHODS The present study evaluated chemical-induced immune cell apoptosis in fish (tilapia, Oreochromis niloticus) exposed to two known immunotoxic chemicals, azathioprine and T-2 toxin. Cytocentrifugation and light microscopy of leukocyte-enriched cell samples from the pronephros (i.e., the fish primary hematopoietic compartment) demonstrated chemical-related increases in apoptotic bodies. This observation was examined further with the ApoAlert Annexin V Apoptosis kit and two DNA-binding dyes employed for detecting apoptosis, 7-aminoactinomycin D (7-AAD) and propidium iodide (PI). RESULTS The apoptotic probes confirmed the microscopic observations of increased apoptosis in the chemical-exposed fish. The ApoAlerttrade mark annexin V and 7-AAD assays, which discriminate early and late apoptosis/necrosis, compared well in identifying apoptotic populations. PI staining in Vindelovs solution was unable to detect early apoptosis. CONCLUSIONS The present data suggest that apoptotic immune cells may be a useful marker for certain immunotoxicant exposures in fish. These findings agree with those of previous reports that fish may respond immunologically in a manner similar to mammals after immunotoxicant challenge.

Ontogenic development of the intestinal enzymes of cultured Nile tilapia, Oreochromis niloticus L.

Ontogenic development of intestinal enzymes in Nile tilapia was investigated using enzyme histochemistry. Intestinal samples of larvae were obtained from day of hatching. Tissue was cryomicrotome-sectioned and fixed using accepted procedures appropriate for the substituted naphthol method of enzyme detection. Prepared sections were incubated, mounted and then examined using a light microscope. Most enzymes demonstrated activities along the brush border, while non-specific esterases were found in the cytoplasm of the epithelial cells. Positive reactions of maltase, leucine aminopeptidase, dipeptidyl aminopeptidase IV, non-specific esterases, and alkaline phosphatase were already present in the brush border of the intestine at hatching (day 0). Only lipase demonstrated a delayed appearance, being first detected in the brush border of the enterocytes at day 3 posthatch. The early temporal appearance and broad anatomic distribution of activities of all intestinal enzymes likely indicates the functional importance of these enzymes (digestion and absorption of food nutrients) at the first feeding.

Nonspecific stimulation of the maternal immune system. I. Effects on teratogen-induced fetal malformations

BACKGROUND Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freunds complete adjuvant (FCA). RESULTS Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Topical permethrin exposure inhibits antibody production and macrophage function in C57Bl/6N mice.

Permethrin was applied to the shaved dorsal interscapular region of C57Bl/6N mice at doses of 0.5, 1.5 or 5.0 microl/day. These doses corresponded to approximately 22-220 mg/kg/day topical insecticide. Mice were exposed to permethrin in this manner daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. The splenic macrophage chemiluminescent response was depressed in a dose-dependent manner at 2 and 10 days post-exposure to permethrin. Phagocytic ability of macrophages was not inhibited. Antibody production as shown by plaque-forming cell (PFC) assay decreased significantly after 10 consecutive days of exposure to permethrin. These data indicate that topical permethrin exposure may produce systemic immune effects.

Exposure of tilapian fish to the pesticide lindane results in hypocellularity of the primary hematopoietic organ (pronephros) and the spleen without altering activity of phagocytic cells in these organs

Tilapia were dosed by intraperitoneal injection for 5 consecutive days with either 20 or 40 mg/kg of the environmental contaminant hexachlorocyclohexane (lindane). The effects of this organochlorine pesticide on morphology and total cellularity of the spleen and pronephros were examined on the second day following termination of dosing. The functional capacity of phagocytic cells isolated from both spleen and pronephros was also evaluated as possible additional indicators of chemical-induced immunotoxicity. A dose-related reduction was found in spleen and pronephros total white blood cell counts in the fish exposed to lindane. In addition, hypocellularity of lymphoid regions in the spleen and pronephros was evident in chemical-exposed animals upon histopathological examination. However, phagocytosis of fluorescent microspheres by phagocytic cells isolated from the spleen and pronephros was not inhibited by the exposure to lindane. Similarly, no decrease in phorbolmyristate acetate (PMA)-stimulated hydrogen peroxide production was observed in phagocytic cells collected from lindane-exposed fish. These results suggest that cellular depletion in tilapia spleen and pronephros may represent a more sensitive indicator of lindane exposure than does the functional capacity of phagocytic cells isolated from these hematopoietic organs. Ultrastructural observations support this hypothesis and, further, suggest that lymphocytic cells may be targeted at the present exposure levels.

Subacute immunotoxic effects of the polycyclic aromatic hydrocarbon 7,12-dimethylbenzanthracene (DMBA) on spleen and pronephros leukocytic cell counts and phagocytic cell activity in tilapia (Oreochromis niloticus)

Tilapia (Oreochromis niloticus) were exposed by intraperitoneal injection of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenzanthracene (DMBA). The relative effects of this carcinogenic PAH on total cellularity and histology of the spleen and pronephros, as well as on the functional capacity of phagocytic cells isolated from both spleen and pronephros, were evaluated as possible indicators of chemical-induced immunotoxicity. Spleen and pronephros total white blood cell counts were reduced, in most cases, in a dose-related manner by DMBA. Marked hypocellularity was also observed histologically in the lymphoid regions of the spleen of DMBA-exposed fish. However, the ability of pronephros and spleen phagocytic cells to phagocytize foreign particles (fluorescent microspheres) was not altered by exposure to DMBA unless dose levels associated with signs of chemical toxicity (reduced swimming and feeding activity, increased cutaneous pigmentation, and increased mortality) were used. When the phagocyte chemiluminescent response was evaluated, there was again no depression of superoxide radical (hydrogen peroxide) production unless other signs of chemical toxicity were apparent. These data suggest that total leukocyte counts in tilapia hematopoietic organs may represent more sensitive indicators of environmental PAH exposure than does phagocytic activity or chemiluminescent response of phagocytes isolated from these organs.

Tilapia (Oreochromis niloticus) dosed with azathioprine display immune effects similar to those seen in mammals, including apoptosis

Azathioprine, an anti-neoplastic drug and therapeutic immunosuppressant, was administered intraperitoneally at 10.0 and 50.0 mg/kg to 3-6-month-old tilapia (Oreochromis niloticus). Consistent alterations in immune cellular parameters of the blood, pronephros (hematopoietic kidney) and spleen were observed. Peripheral blood total cellularity decreased as the azathioprine dose increased, to approximately half that of the control. Differential analysis of white blood cells indicated a decline in lymphocyte number, in particular, with increased dosage of azathioprine. Pronephric total cellularity was depressed in fish receiving the 10.0 or 50.0 mg/kg dose. In contrast, both splenic weight and splenic total cellularity increased proportionately with the increase in the drug dosage. Histopathologic examination of the spleens showed normal patterns for both control and 10.0 mg/kg dose groups. At 50.0 mg/kg, spleens were characterized by marked expansion of the white pulp, although lymphocytes were rare. Melanomacrophage centers at the higher dose were also larger and more numerous than in the control group. Evaluation of splenic and pronephric leukocytes with apoptotic markers showed an increase in apoptotic cells in the pronephros with increasing drug dose. These changes in fish are consistent with those seen in humans and laboratory rodents dosed with azathioprine, suggesting that fish may be potentially useful as preliminary models for detecting immunosuppressive compounds.

Hematopoietic alterations after exposure to T-2 mycotoxin

Adult mice were exposed by oral gavage to 0.75, 1.25, or 1.75 mg/kg body weight T-2 mycotoxin for 5 consecutive days. Thymic atrophy on the 2nd day following cessation of dosing was profound, and was characterized by significant decreases in the total number of cells within all phenotypes defined by CD4 and CD8 cell-surface antigen expression. Further, the distribution of thymocytes within these phenotypes was significantly altered. Increased percentages of CD4-8- (DN) and decreased percentages of CD4+8+ (DP) cells in thymuses from treated animals suggested that T-2 toxin may inhibit thymocyte maturation. In addition to thymus, the bone marrow of treated animals showed a highly significant hypocellularity, indicating that this hematopoietic compartment may also be targeted by T-2 toxin. A trend toward reduced splenic cellularity was additionally observed in exposed animals, but failed to reach significance. A significant decrease in the total number of both B and T-lymphocytes present within the spleen was observed, however. These data, taken together, indicate that effects at multiple hematopoietic compartments involved in the production of T-lymphocytes may contribute to the peripheral T-cell lymphocytopenia and T-cell mediated immunosuppression produced by T-2 toxin.

Maternal immune stimulation reduces both placental morphologic damage and down-regulated placental growth-factor and cell cycle gene expression caused by urethane: are these events related to reduced teratogenesis?

Activation of the maternal immune system in mice decreased cleft palate caused by the chemical teratogen, urethane. Direct and indirect mechanisms for this phenomenon have been suggested, including maternal macrophages that cross the placenta to find and eliminate pre-teratogenic cells, or maternal immune proteins (cytokines) that cross placenta to alleviate or partially alleviate toxicant-mediated effects in the developing fetus. A third mechanism to explain improved fetal developmental outcome in teratogen-challenged pregnant mice might involve beneficial effects of immune stimulation on the placenta. In the present experiments, urethane treatment altered placental morphology and impaired placental function, the latter indicated by down-regulated activity of cell cycle genes and of genes encoding cytokines and growth factors. Maternal immune stimulation with either Freunds complete adjuvant (FCA) or interferon-gamma (IFNgamma) reduced morphologic damage to the placenta caused by urethane and normalized expression of several genes that were down-regulated by urethane. Urethane treatment also shifted placental cytokine gene expression toward a T cell helper 1 (Th1) profile, while immunostimulation tended to restore a Th2 profile that may be more beneficial to pregnancy and fetal development. These data suggest that the beneficial effects of maternal immune stimulation on fetal development in teratogen-exposed mice may, in part, result from improved placental structure and function.