Thymocyte apoptosis as a mechanism for tributyltin-induced thymic atrophy in vivo

Tributyltin (TBT) immunotoxicity in rodent species is primarily characterised by T-lymphocyte deficiency resulting from a depletion of cortical thymocytes. In this study, bis(tri-n-butyltin) oxide (TBTO) was administered to male rats as a single oral dose of 30 or 60 mg/kg, and assessments were made of thymic cytopathology and the integrity of cellular DNA. TBTO treatment did not cause severe toxicity or overt clinical signs; however, by 48 h post-dosing relative thymus weights at 30 and 60 mg/kg were reduced to 66 and 43%, respectively, of control values. Increased DNA fragmentation was evident in thymic cell isolates (principally thymocytes) obtained from treated animals during the period of thymic involution. When DNA purified from these cells was visualised by agarose gel electrophoresis a multimeric internucleosomal fragmentation pattern, indicative of supra-physiological levels of apoptosis, was detected. Although unassociated apoptotic or necrotic thymocytes were essentially absent in cell preparations from TBTO-treated rats, significantly increased numbers of mononuclear phagocytic cells were observed. Many of these cells contained either apoptotic thymocytes, with nuclear morphologies exhibiting chromatin condensation, or cell remnants which were characterised as apoptotic bodies. Dibutyltin, which is a major metabolic dealkylation product of tributyltin, failed to significantly stimulate apoptosis when added to isolated thymocytes in vitro. Collectively, these findings suggest that activation of apoptosis contributes to TBT-induced thymocyte depletion in vivo, and indicate that it is unlikely that the metabolite dibutyltin is responsible for this effect.     

Loss of pre-B and IgM(+) B cells in the bone marrow after exposure to a mixture of herbicides

This study determined alterations to bone marrow B-cell populations after in vivo exposure to a mixture containing the herbicides 3,4-dichloropropionanilide (propanil) and 2,4-dichlorophenoxyacetic acid (2,4-D) and compared them to the effects of exposure to the individual herbicides. Propanil and 2,4-D are postemergent herbicides that are sold commercially as a mixture. The individual herbicides or the mixture containing propanil and 2,4-D were administered intraperitoneally to C57Bl/6 female mice at doses from 50 to 200 mg herbicide/kg body weight. The mixtures were given in a 1:1 ratio. Flow cytometric analysis was performed to quantitate bone marrow B-cell populations at 1, 2, 7, and 14d posttreatment. Mixture treatment decreased pre-B and immunoglobulin (Ig) M(+) B-cell populations at all doses by 2 d postexposure. The cell populations were still decreased at 7d posttreatment. In contrast, exposure to the individual herbicides only caused decreases in the pre-B and IgM(+) B-cell populations 7d after exposure to the high doses. Previous studies have demonstrated that corticosterone levels are increased by exposure to propanil. Therefore, the glucocorticoid hormone, corticosterone, was investigated as a possible mediator of cell loss in the bone marrow. Treatment with the glucocorticoid receptor antagonist, RU 486, however, did not prevent cell loss in the bone marrow of mice exposed to the mixture of propanil and 2,4-D. This study demonstrates that pre-B and IgM(+) B-cell populations are decreased after exposure to propanil, 2,4-D, or the mixture containing propanil and 2,4-D. Exposure to the mixture had greater toxic effects than the individual herbicides on bone marrow pre-B and IgM(+) B-cell populations, emphasizing the need to study mixture interactions.     

Thyroid disruption in the lizard Podarcis bocagei exposed to a mixture of herbicides: a field study

Pesticide exposure has been related with thyroid disrupting effects in different vertebrate species. However, very little is known about the effects of these compounds in reptiles. In the Mediterranean area, lacertid lizards are the most abundant vertebrate group in agroecosystems, and have been identified as potential model species for reptile ecotoxicology. The aim of this study was to understand if the herbicides applied in corn fields have thyroid disruptive effects in the lizard Podarcis bocagei. Adult male lizards were captured in north-western Portugal in corn fields treated with herbicides (exposed sites), and in organic agricultural fields (reference sites). Thyroid and male gonad morphology and functionality, and testosterone levels were investigated through histological, immunohistochemical and biochemical techniques. Lizards from exposed locations displayed thyroid follicular lumens with more reabsorption vacuoles and significantly larger follicular area than those from reference fields. Furthermore, testes of lizards from exposed locations had significantly larger seminiferous tubule diameters, significantly higher number of spermatogenic layers and displayed an up-regulation of thyroid hormone receptors when compared with lizards from reference areas. These findings strongly suggest that the complex mixture of herbicides that lizards are exposed to in agricultural areas have thyroid disrupting effects which ultimately affect the male reproductive system. Alachlor, which has demonstrated thyroid effects in mammals, may be largely responsible for the observed effects.     

Alterations in the cortical thymic epithelium of rats after in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): an (immuno)histological study.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces thymic atrophy in rats. The present study was initiated to provide (immuno)histological data on the mechanism of action. Juvenile male Wistar rats were orally intubated once with 50 or 150 micrograms/kg TCDD. They were euthanized 4 or 10 days thereafter, or were allowed to stay alive until Day 20 or 26. Growth retardation occurred rapidly in all TCDD-treated animals. Lethality was demonstrated within 20-21 days after administration. At Days 4 and 10 after intubation, thymic atrophy was shown by reduction of thymic weight and cortex/medulla ratio. Staining patterns for T-cell markers in the atrophic thymuses coincided with the reduction of cortical areas. There was no evidence indicating that the effects were indirectly caused by stress. TCDD-induced thymic atrophy persisted until Day 26 after administration. Immunohistochemical analysis revealed prominent changes in the cortical thymic epithelium at the 150-micrograms/kg dose level. First, in the cortex epithelial cell aggregates were observed both at Day 4 and at Day 10 after administration. Apparently, the architecture of the epithelium had changed in these animals. Second, at 10 days after administration epithelial cells were found with the simultaneous expression of markers that in the normal uninvoluted thymus only occur either in the subcapsular/medullary area or in the cortex. This phenotype points to an unusual stage of differentiation. We conclude that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Apparently, it disturbs the epithelial network and interfers with the differentiation of epithelial cells.     

Persistent developmental toxicity in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides

There is growing concern of permanent damage to the endocrine and nervous systems after developmental exposure to endocrine disrupting chemicals. In this study the permanent reproductive and neurobehavioral effects of combined exposure to five endocrine disrupting pesticides, epoxiconazole, mancozeb, prochloraz, tebuconazole and procymidone, were examined. Pregnant and lactating rat dams were dosed with a mixture of the five pesticides at three different doses, or with the individual pesticides at one of two doses. Adverse effects were observed in young and adult male offspring from the group exposed to the highest dose of the mixture. These included reduced prostate and epididymis weights, increased testes weights, altered prostate histopathology, increased density of mammary glands, reduced sperm counts, and decreased spatial learning. As no significant effects were seen following single compound exposure at the doses included in the highest mixture dose, these results indicate cumulative adverse effects of the pesticide mixture.     

Diethylstilbestrol (DES)-induced fetal thymic atrophy in C57BL/6 mice: inhibited thymocyte differentiation and increased apoptotic cell death

Treatment of pregnant C57Bl/6 mice with 48 mu g/kg diethylstilbestrol (DES) on gestation days (GDs) 14 and 16 resulted in both decreased day 18 fetal thymic cellularity as well as alterations in thymocyte phenotype. Histopathologic examination of GD 18 fetal thymi from DES-exposed dams demonstrated a decrease in thymic size and cellularity and an increase in pyknotic nuclei, indicative of apoptosis, relative to control thymi. Thymic architecture was also altered by DES treatment with a decrease in the distinction between the cortical and medullary regions. Flow cytometric staining of day 18 thymocyte suspensions with the apoptotic marker 7-aminoactinomycin D showed a decrease in thymocyte viability after DES, and a concomitant increase of thymocytes in early apoptosis. When thymocytes were co-identified by CD4 and CD8 cell surface antigen expression, trends toward increased apoptosis were present in the CD4+CD8+ and CD4+CD8- subpopulations, and significantly increased apoptosis occurred in the CD4-CD8- and CD4-CD8+ subpopulations. These histopathologic and flow cytometric findings support enhanced apoptosis of thymocytes as a contributing factor to fetal thymic atrophy caused by DES.     

Patterns of gene expression in carp liver after exposure to a mixture of waterborne and dietary cadmium using a custom-made microarray

Gene expression changes in carp liver tissue were studied after acute (3 and 24h) and subchronic (7 and 28 days) exposure to a mixture of waterborne (9, 105 and 480 microg/l) and dietary (9.5, 122 and 144 microg/g) cadmium, using a custom-made microarray. Suppression subtractive hybridization-PCR (SSH-PCR) was applied to isolate a set of 643 liver genes, involved in multiple biological pathways, such as energy metabolism (e.g. glucokinase), immune response (e.g. complement C3) and stress and detoxification (e.g. cytochrome P450 2F2, glutathione-S-transferase pi). These genes were subsequently spotted on glass-slides for the construction of a custom-made microarray. Resulting microarray hybridizations indicated a highly dynamic response to cadmium exposure. At low exposure concentrations (9 microg/l through water and 9.5 microg/g dry weight through food) mostly energy-related genes (e.g. glucokinase, elastase) were influenced, while a general stress response was obvious through induction of several stress-related genes, including hemopexin and cytochrome P450 2F2, at high cadmium concentrations. In addition, fish exposed to the highest cadmium concentrations showed liver damage after 7 days of exposure, as measured by elevated alanine transaminase activity in plasma and increased liver water content (wet-to-dry weight ratio). Moreover, decreased hematocrit and growth were found at the end of the experiment. Altogether this study clearly demonstrated the importance of varying exposure conditions for the characterization of the molecular impact of cadmium and showed that microarray results can provide important information, required to unravel the molecular events and responses related to cadmium exposure.     

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo.     

Metabolic changes in rat brain synaptosomes after exposure to sulfide in vivo

The effects of exposure of rats to sulfide and the action of exogenous heme were studied in rat-brain synaptosomes. Exposure to sulfide impaired the respiration of synaptosomes which was reversed by heme (4 mg/kg body weight). Sodium sulfide caused partial inhibition of gamma-aminobutyric acid (GABA) and dopamine uptake, strongly inhibited veratridine-dependent release of these neurotransmitters and reduced veratridine-dependent changes in transmembrane electrical potential. Heme treatment did not reverse these changes.     

Pulmonary macrophage phagocytosis and enzyme production after in vivo exposure to silica dust

Guinea pigs were exposed to silica dust (SiO2) aerosols at 6, 13, 28, and 46 mg/m3 for 3 weeks and examined 4, 8, 16, and 23 weeks later. Twenty-four milligrams of TiO2 per cubic meter served as the control dust. Lung weights were increased at 8 weeks and later. Free lung cells showed a tendency to decrease in phagocytosis capacity at 8 weeks after cessation of exposure and later. Alveolar macrophage production of N-acetyl-beta-D-glucosaminidase, cathepsin D, and acid phosphatase was decreased at 8 weeks after exposure and later. The relationship between the depressant effect on macrophages and the absence of an exposure-related polymorphonuclear neutrophil response for the development of fibrosis could be part of the mechanism behind fibrosis.     

Effects on fetal thymocyte populations and postnatal T-cell-dependent immune functions after maternal exposure to 5-fluorouracil during pregnancy in mice

5-Fluorouracil (5-FU) is a cytostatic anti-tumor drug which is known to have immunosuppressive activities. To assess the immunotoxic effects of 5-FU on fetal thymocyte populations and immune functions after birth, pregnant C57BL/6 mice were orally administered vehicle or 17 mg/kg/day of 5-FU during gestational days (GD) from 6 to 14. The fetal thymocyte populations were analyzed with flow cytometry (CD4/CD8 double staining), and immune functions (a mixed lymphocyte reaction, in vitro cytotoxic T-cell response, in vitro antibody-forming response) after birth were measured. Fetal thymus weight and thymocyte numbers were decreased by 5-FU administration. The decrease of the thymocytes was due mainly to the decrease of small CD4CD8 double positive (DP) thymocytes. The thymocyte numbers and populations recovered to the normal level 1 week after birth. The mixed lymphocyte response at the 6th week after birth tended to be slightly lower than the control levels, but the cytotoxic T-cell response and the antibody-forming response were the same as the control levels. These results suggest that immune functions might recover after birth, although maternal administration of 5-FU has a suppressive effect on fetal thymocyte maturation.     

Adverse effects on sexual development in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides

The present study investigated whether a mixture of low doses of five environmentally relevant endocrine disrupting pesticides, epoxiconazole, mancozeb, prochloraz, tebuconazole and procymidone, would cause adverse developmental toxicity effects in rats. In rat dams, a significant increase in gestation length was seen, while in male offspring increased nipple retention and increased incidence and severity of genital malformations were observed. Severe mixture effects on gestation length, nipple retention and genital malformations were seen at dose levels where the individual pesticides caused no or smaller effects when given alone. Generally, the mixture effect predictions based on dose-additivity were in good agreement with the observed effects. The results indicate that there is a need for modification of risk assessment procedures for pesticides, in order to take account of the mixture effects and cumulative intake, because of the potentially serious impact of mixed exposure on development and reproduction in humans.     

In vivo cytokine production and resistance to infection after acute exposure to 3,4-dichloropropionaniline

3,4-Dichloropropionaniline (propanil) is an extensively used postemergent herbicide that has been shown to produce toxic and immunotoxic effects. The present report examined if acute exposure to propanil altered in vitro or in vivo cytokine production in response to antigenic stimulation. Studies to determine resistance to infection by the intracellular bacterium Listeria monocytogenes after exposure to propanil were also conducted. Our experiments demonstrate that in vivo exposure to propanil during bacterial infection reduced the subsequent in vitro production of interferon-gamma (IFN-gamma) by splenocytes and liver nonparenchymal cells in response to antigenic and mitogenic stimulation. Additional experiments examined the production of cytokines in vivo after propanil exposure alone or combined propanil exposure and L. monocytogenes infection. It was found that the endogenous levels of cytokines in the liver, spleen, and blood were similar in control and propanil-treated mice. The levels of cytokines were also similar in control and exposed mice that were infected with L. monocytogenes. Initial resistance to the infection was not affected by exposure to propanil. These results demonstrate that in vivo exposure to propanil during a bacterial infection suppresses the subsequent in vitro production of cytokines but that the endogenous levels are not affected during the initial stages of infection.     

Early developmental neurotoxicity of a PCB/organochlorine mixture in rodents after gestational and lactational exposure.

The developmental and neurobehavioral effects of gestational and lactational exposure to a mixture of 14 polychlorinated biphenyls (PCBs) and 11 organochlorine pesticides was examined and compared against the commercial PCB mixture Aroclor 1254. The mixture was based on blood levels reported in Canadian populations living in the Great Lakes/St. Lawrence basin. Pregnant Sprague-Dawley rats were dosed orally with 0.013, 0.13, 1.3, or 13 mg/kg of the chemical mixture or 15 mg/kg of Aroclor 1254 from gestation day (GD) 1 to postnatal day (PND) 23. The highest mixture dose decreased maternal gestation and lactation body weight, and produced high mortality rates (80% overall) and reductions in offspring weight that persisted to adulthood. Aroclor 1254 produced smaller but persistent decreases in offspring weight without affecting maternal weight or offspring mortality. Aroclor 1254 and 13 mg/kg of the mixture produced comparable decreases in maternal and offspring serum T4 levels and comparable alterations to maternal thyroid morphology. Aroclor 1254 delayed the righting reflex and ear opening, accelerated eye opening, and reduced grip strength at PNDs 10-14. The mixture at 13 mg/kg delayed negative geotaxis in addition to delaying righting reflex and ear opening and reducing grip strength but had no effect on eye opening. Lower mixture doses (0.13 and 1.3 mg/kg) also delayed ear opening but affected no other parameters. Developmental exposure to the chemical mixture was found to be more toxic than exposure to Aroclor 1254 and produced a different profile of effects on early neurodevelopment. This PCB/organochlorine pesticide mixture affects mortality, growth, thyroid function, and neurobehavioral development in rodents.     

Rat lung inflammatory responses after in vivo and in vitro exposure to various stone particles

Rat lung alveolar macrophages and type 2 cells were exposed for 20 h in vitro to various stone particles with differing contents of metals and minerals (a type of mylonite, gabbro, feldspar, and quartz). The capability to induce the release of the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2) was investigated. We found marked differences in potency between the various particles, with mylonite being most potent overall, followed by gabbro, and with feldspar and quartz having an approximately similar order of lower potency. The results also demonstrated differences in cytokine release pattern between the two cell types. For all particle types including quartz, type 2 cells showed the most marked increase in MIP-2 and IL-6 secretion, whereas the largest increase in TNF-alpha release was observed in macrophages. To investigate possible correlations between in vitro and in vivo inflammatory responses, rats were instilled with the same types of particles and bronchoalveolar lavage (BAL) fluid was collected after 20 h. The results demonstrated a correlation between the in vitro cytokine responses and the number of neutrophilic cells in the BAL fluid. The BAL fluid also showed a strong MIP-2 response to mylonite. However, this was the only particle type to give a significant cytokine response in the BAL fluid. We further examined whether a similar graded inflammatory response would be continued in type 2 cells and alveolar macrophages isolated from the exposed animals. Again a differential cytokine release pattern was observed between type 2 cells and macrophages, although the order of potency between particle types was altered. In conclusion, various stone particles caused differential inflammatory responses after both in vitro and in vivo exposure, with mylonite being the most potent stone particle. The results suggest the alveolar type 2 cell to be an important participant in the inflammatory response following exposure to particles.     

Immunomodulatory role of piperine in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium being a potent immunotoxicant, affects both humoral and cell mediated immunity. In rodents, it is primarily characterized by marked thymic atrophy and splenomegaly. Cadmium induces apoptosis in mice and suppresses the immune functions. Piperine, major alkaloid of Piper longum Linn. and Piper nigrum Linn. with a long history of medicinal value, has shown anti-apoptotic activity in vitro. Thus, to delineate its role in vivo, piperine (2.5mg/kg/day, oral, 7 days) treated Balb/C mice were administered Cd as CdCl(2) (1.8mg/kg, i.p., once, 4th day). The various biochemical indexes of cell damage such as cytotoxicity (MTT assay), oxidative stress (glutathione, reactive oxygen species), apoptosis (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) along with lymphocyte phenotyping, cell proliferative response and cytokine secretion (IL-2 and IFNγ) were assessed in thymic and splenic single cell suspensions. Lowering of body weight gain and cellularity and a loss in cell viability seen in Cd group, were abrogated by piperine treatment. Similarly, oxidative stress and apoptotic markers altered by Cd were also modulated by this alkaloid. In addition, a pronounced inhibition of cell proliferative response, alterations in T- and B-cell phenotypes, cytokine release and morphological changes were restored to normalcy. The present in vivo data corroborating with our previous in vitro findings, provide confirmatory evidence of the immuno-protective efficacy of piperine.     

Reduced levels of 1,25-dihydroxyvitamin D(3) in rat dams and offspring after exposure to a reconstituted PCB mixture.

Previous studies revealed effects of polychlorinated biphenyls (PCBs) and other polyhalogenated hydrocarbons on steroid hormone levels and hormone-dependent functions including behavior. In the present study serum concentrations of the vitamin D(3) metabolites 25-hydroxycholecalciferol (25-D) and 1,25-dihydroxycholecalciferol (1,25-D) were determined in rat dams and offspring after exposure to a PCB mixture that was reconstituted according to the congener pattern found in human breast milk. Unmated females were exposed to diets adulterated with 0; 5; 20; or 40 mg PCBs/kg diet. Exposure started 50 days prior to mating and was terminated at birth. Gestational exposure reduced serum concentrations of 1,25-D in dams in a dose-dependent manner. Concentration of 25-D was also decreased at the time of delivery, but not at weaning. Determination of 1,25-D in offspring at weaning revealed reductions in both high-exposure groups. Levels of 25-D were diminished only at the highest exposure level. Internal PCB concentrations in adipose tissue and brains exhibited a linear relation to dosages in diet. Concentrations of PCBs in brains were similar in dams and offspring at birth, but decreased at the end of lactation in dams. In offspring, values increased during this period because of continued exposure via the milk. In the adipose tissue, PCB levels were much lower in offspring than in dams. To our knowledge, this is the first report of PCB-induced effects on vitamin D(3) metabolites. In dams, reductions were seen even at the lowest exposure level used. Further studies are needed to evaluate the biological significance of these reductions in pregnant dams and possible consequences for the developing offspring.     

Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice

Cadmium immunotoxicity in rodents is primarily characterized by marked thymic damage and splenomegaly. To understand the toxicity of Cd on lymphoid cells in vivo, a single dose of Cd as CdCl2 (1.8 mg/kg, i.p.) was administered to male BALB/c mice and cytotoxicity (MTT assay), oxidative stress indicators (glutathione, reactive oxygen species) and apoptotic markers (mitochondrial membrane potential, caspase-3 activity, phosphatidylserine externalization, apoptotic DNA, intranucleosomal DNA fragmentation) were assessed in thymic and splenic single cell suspensions, at various time intervals. Lowering of body weight gain and cellularity and a loss in cell viability was seen in the Cd treated mice. The earliest significant increase in ROS at 18 h, followed by mitochondrial membrane depolarization, caspase-3 activation and GSH depletion at 24h in spleen and later at 48 h in thymus, strongly implicate the possible involvement of ROS. A pronounced inhibition of cell proliferative response at 48 h and 72 h may also be linked to Cd induced apoptosis. The morphological alterations including thymic cortical cell depletion and an increase in red pulp with diminished white pulp in spleen were observed at 48 h and beyond. The splenic cells appeared more susceptible than thymus cells to the adverse effects of Cd. The present study, therefore, demonstrates potentiation of oxidative stress followed by mitochondrial-caspase dependent apoptotic pathway. This may, in part, be responsible for causing suppression of cell proliferative response, thymic atrophy and splenomegaly.     

Immunological responsiveness of mouse spleen cells after in vivo or in vitro exposure to 3-acetyldeoxynivalenol

The effects of 3-acetyldeoxynivalenol (3-AcDON) on mitogen-induced lymphocyte proliferation and antibody production were studied in male CD-1 mice exposed to 0, 2.5, 5 or 10 ppm 3-AcDON in the diet for 35 days. Mitogen-induced lymphocyte proliferation and T-cell-independent antibody responses to dinitrophenyl-ficoll or Escherichia coli were not altered by dietary exposure to 3-AcDON. The T-cell-dependent antibody response to sheep red blood cells was increased in the group fed 10 ppm 3-AcDON. In vitro, 3-AcDON inhibited lymphocyte proliferation in a dose-dependent manner. Inhibition was observed when the toxin was present during the first 8 hr in phytohaemagglutinin-stimulated cultures and during the first 24 hr in lipopolysaccharide-stimulated cultures. This suggests that 3-AcDON blocks an early step in lymphocyte activation. This inhibition was not restored by thiol reducing agents (dithiothreitol, L-cysteine or 2-mercaptoethanol). Similarly, the addition of lymphokines, including interleukin-1 or interleukin-2, did not alter the inhibitory effects of 3-AcDON. These results suggest that the in vitro effects of 3-AcDON may not reflect its in vivo immunotoxicity. However, 3-AcDON may serve as a chemical probe for examining the activation process of lymphocyte proliferation.