Doutoramento em Toxicologia / PhD Degree - Toxicology

Doutoramento / PhD Degree / The immune evasion mechanisms of pathogenic trypanosomatids involve a multitude of phenomena such as the polyclonal activation of lymphocytes, cytokine modulation and the enhanced detoxification of oxygen reactive species. A trypanothione-cascade seems to be involved in the detoxification process. We have recently described and characterized a tryparedoxin (LiTXN1) involved in the Leishmania infantum cytoplasmatic hydroperoxide metabolism. LiTXN1 is a secreted protein upregulated in the infectious form of the parasite, suggesting that it can play an important role during infection. In the present study, we investigated whether the recombinant LiTXN1 (rLiTXN1) would affect T and B cell functions in a murine model. We observed a significant increase of CD69 surface marker on the B-cell population in total spleen and on isolated B-cells from BALB/c mice after in vitro rLiTXN1 stimulus. Activated Bcells underwent further proliferation, as measured by an increased [3H]-thymidine incorporation. Cytokine quantification showed a dose-dependent upregulation of IL-10 secretion. B-cells were identified as a source. Furthermore, intraperitoneal injection of rLiTXN1 into BALB/c mice triggered the production of elevated levels of rLiTXN1 specific antibodies, predominantly of the IgM, IgG1 and IgG3 isotypes, with a minimum reactivity against other heterologous antigens. Taken together, our data suggest that rLiTXN1 could participate in the immunopathological processes by targeting the B-cells effector functions with subsequent IL-10 secretion and specific antibodies production.

Doutoramento em Toxicologia / PhD Degree - Toxicology

Environmental toxicologists adopted QSARs from pharmacology fairly early on to predict organic contaminant toxicity. In contrast, models relating metal ion characteristics to their bioactivity remain poorly explored and underutillized. Quantitative Ion Character-Activity Relationships (QICARs) have recently been developed to predict metal toxicity. The QICAR approach based on metal-ligand binding tendencies has been applied to a wide range of effects, species, and media on a single metal basis. In previous single metal studies, a softness parameter and the ; log of KOH ; were the ion qualities with the highest predictive value for toxicity. Here, QICAR modeling was brought a step further to predict toxicity of binary metal mixtures. Using the MicrotoxRTM bioassay, the interaction of binary mixtures of metals (Co, Cu, Mn, Ni, and Zn) is quantified using a linear model with an interaction term. A predictive relationship for metal interaction between pairs of metals and the difference in the softness parameter was developed. The interaction of binary mixtures of Co, Cu, Ni, and Zn was quantified using a linear model for nematode (Caenorhabditis elegans) exposures. Contrasting with earlier studies, the difference in polarizing power (Z2/r) between metal ions was the best ion characteristic for predicting interaction coefficients. Current risk assessment methods sum toxic units, assuming that all mixtures act in an additive fashion. General problems with this method are demonstrated utilizing data from the MicrotoxRTM metal mixture tests. An alternative, more accurate, method for summing toxicities with proportions instead of toxic units is illustrated. This study supports the hypothesis that general prediction of metal interactions from ion characteristics is possible. It is important to realize that even with the preliminary success of these models that additional work with metals of different valences and sizes might affect the accuracy of metal interaction predictions. Careful thought and examination of known modes of single metal toxicity should be considered when developing future quantitative metal interaction models.

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It has been suggested that the CB1 G-protein-coupled receptor is internalized following agonist binding and activation of the second messenger pathways. The process of desensitization and resensitization is intimately involved with receptor internalization. Phosphorylation alters tolerance to cannabinoids thus contributing to tolerance. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor. These findings led us to look at which kinase(s) may be involved in cannabinoid tolerance. We therefore hypothesize that by preventing phosphorylation of the CB1 receptor, we may reverse tolerance. We evaluated our hypothesis by testing the role of several kinases in tolerance: protein kinase A (PKA), protein kinase C (PKC), protein kinase C (PKG). Beta Adrenergic Receptor Kinase (β-ARK), Phosphatidylinositol 3-kinase (PI3K) and the src family tyrosine kinase. We also looked at cAMP and cGMP analogs. We evaluated PKA using KT5720, a PKA inhibitor; PKC using bisindolylmaleimide I, HCI] (bis), a PKC inhibitor; PKG using KT5823, a PKG inhibitor; β-ARK using Low molecular weight heparin (LMWH), a β-ARK inhibitor; PI3K using LY294002, a PI3K inhibitor and PP1 a src family tyrosine kinase inhibitor. The cAMP analog was dibutyryl-cAMP and the cGMP analog was dibutyryl-cGMP. ICR mice were rendered tolerant to △9- tetrahydrocannabinol (△9-THC) by administering injections of 20mg/kg △9-THC s.c. every 12 hours for 6.5 days. The mice were subsequently challenged 24 hours later with an ED8O of △9-THC at 20μg/mouse (i.t.). Antinociception was measured by the tail-flick test, %MPE’s and ED5O’s were calculated. The PKG inhibitor, KT5823, showed no significant change in %MPE. The β-ARK inhibitor, LMWH, showed no significant change in the %MPE. The PI3K inhibitor, LY294002, showed no significant change in the %MPE. Inhibition of PKC, by bis had no effect on tolerance, but at a higher dose attenuated the antinociceptive effect of △9-THC in non-tolerant mice. PPl, the src family tyrosine kinase inhibitor, reversed tolerance. KT5720, the PKA inhibitor reversed △9- THC tolerance. These data support a role for PKA and tyrosine kinase in phosphorylation events in THC tolerant mice. (Supported by NIDA grants K02DA00186 and P5ODA05274).

The activity of protein kinase C (PK-C) has been implicated in regulating the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C activators, phorbol 12,13- dibutyrate, mezerein, and bryostatin 1, on the proliferation and lineage commitment of enriched CD34+ human myeloid progenitor cells stimulated by lL-3, GM-CSF, stem cell factor and the lL-3/GM-CSF hybrid cytokine plXY321. Coadministration of these PK-C activators with plateau concentrations of rlL-3 or rGM-CSF induced 100-150% increase in the number of day 14 CFU-GM.with a selective stimulation on neutrophil and macrophage lineages while inhibiting eosinophilic growth. plXY321 stimulated an equivalent number of CFU-GM, including a predominant eosinophilic component, when compared to the combination of saturating levels of GM-CSF and lL-3. Bryostatin 1, when coadministered with plXY321 (or with the combination of lL-3 and GM-CSF), selectively enhanced the growth of neutrophilic and monocytic lineages while inhibiting eosinophil development. The inhibition of eosinophil colonies by bryostatin 1 was not mimicked by the coadministration of rSCF, rG-CSF or rCSF-1 with plXY321. Furthermore, neutralizing antibodies to rG-CSF and rCSF-1 failed to block potentiation of neutrophil or macrophage colony formation stimulated by bryostatin 1 in conjunction with plXY321, suggesting that accessory cell effects are not solely responsible for this phenomenona. rSCF synergistically enhanced plXY321 induced colony formation by an average of 144% by selectively stimulating neutrophilic and eosinophilic growth. Coadministration of bryostatin 1 with rSCF and plXY321 further increased colony formation by an average of 81%. This combination selectively stimulated cells of the macrophage lineage, and inhibited eosinophil differentiation. However, bryostatin 1 inhibited erythroid (BFU-E) and erythroid/myeloid mixed (CFU-GEMM) colonies induced by plXY321 alone or in combination with rSCF. Together these results indicate that 1) PK-C activity is involved in the growth and lineage commitment of early and committed myeloid progenitor cells. 2) Pharmacologic manipulation of PK-C may regulate the growth and differentiation of those cells exposed to early hematopoietic growth factors. This study raises the possibility that pharmacologic intervention at PK-C,in conjunction with hematopoietic growth factors, might be useful in the ex vivo expansion of hematopoietic progenitor cells.

Previous reports have indicated a dichotomy in the actions of TCDD on humoral immunity, both in vitro and in vitro, in which enhancements and suppression have been identified. The latter effect has been correlated with induction of liver P4501A1 enzyme activity, a response which is regulated by the Ah-gene locus. Additionally, the primary alteration in suppression of antibody responses is in the differentiation of the B cell. Therefore, the current investigation was undertaken to determine the relationship between these dual actions of TCDD on humoral immunity as related to its direct actions on B lymphocyte function. Specific emphasis was placed on determining the potential role of the Ah-gene locus in the modulation of the B cell and in vitro antibody responses. Initial observations determined that: 1) the degree of suppression of in vitro antibody responses in B6C3F1 mouse (an Ah-high responder strain) splenocytes is dependent on both the lot, concentration, and type (i.e., fetal bovine versus newborn bovine) of serum used in culture; and 2) there is a similarity in the actions of TCDD and Staphylococcus aureus Cowan strain I (SAC); a polyclonal B cell activator. These observations prompted the study of the direct effects of TCDD on B cells in different stages of in vivo activation and in the presence of different serum environments for which SAC has been reported to have opposing actions. TCDD was found to increase background levels of proliferation and differentiation in resting B cells (Go); thereby suggesting that resting B cells are a primary target in enhancement of immune responses by TCDD. However, no effect was seen on either response when the cells were stimulated with lipopolysaccharide (LPS). In cycling B cells (G1), TCDD caused suppression of both background and LPS-stimulated proliferation and differentiation and demonstrated a serum dependency that paralleled its actions in whole lymphocyte antibody responses; thereby demonstrating that it is the cycling B cell that is the primary target in suppression of antibody responses by TCDD and that both proliferation and differentiation are affected. The modulatory role of serum was also determined in primary hepatocyte cultures using P4501Al enzyme induction as the endpoint. Sera which supported either no effect or enhancements in both whole lymphocyte and purified B cell responses did not support induction of P4501A1 activity above BSA, a protein control. Conversely, sera which supported a consistent and dose-related suppression of Mug antibody responses were found to enhance the induction of P4501A1 activity. More importantly, normal mouse serum was found to allow for the full expression of the Ah-dependent phenomena in vitro, where primary hepatocytes and splenocytes from Ah-high responder (B6C3F1) and Ah-low responder (DBA/2) mouse strains were affected by TCDD in a manner that parallels the effects seen in the two strains following in vivo exposures. The latter results using mouse sera are consistent with a role by the Ah-gene locus in the direct effects of TCDD on whole lymphocyte and purified B cell antibody responses. However, the results with other, more traditional sources of sera, indicate that the ultimate expression of the TCDD- induced responses can be modulated by serum factors, which are at present unidentified. Furthermore, the results of this investigation indicate that the susceptibility of the cell, as related to its stage in the cell cycle, can contribute to the complex effects that are seen in the alteration of antibody responses following TCDD exposure.

Polychlorinated biphenyls (PCBs) exert a broad range of toxicity via both parent compounds and their metabolites. Our previous study showed that hydroquinone (H2Q) metabolites of PCBs act as cosubstrates for prostaglandin H synthase (PGHS), and are oxidized by this enzyme to corresponding quinones (Q). The goal of this thesis is to illuminate the PGHS-mediated toxicity of lower chlorinated PCBs. It is hypothesized that PGHS catalyzes two sequential one-electron oxidations of PCB-H2Q to semiquinone (SQ), and Q that interact with biomolecules, such as amino acids, glutathione (GSH), protein, and DNA. In addition, the oxidation of H2Q by PGHS results in an elevation of downstream prostaglandin (PG) production in vivo. Employing 4-chlorobiphenyl-2f,5f-hydroquinone (4-CB-2f,5f-H2Q) as a model compound, I found that PGHS-2 catalyzes the one-electron oxidation of 4-CB-2f,5f-H2Q to SQ. An unusual electronically distorted SQ spectrum was observed as a result of the mixture of two different SQ species, a quartet and a doublet. Fate of 4-CB-2f,5f-SQ and/or Q in the presence of biomolecules was further investigated in the next study. 4-CB-2f,5f-SQ/Q reacts readily with the thiol-containing molecules, such as cysteine, and GSH. Oligonucleotides, and DNA did not form a covalent adduct with 4-CB-2f,5f-SQ but preferably stabilized 4-CB-2f,5f-SQ by pi-stacking interaction under the assay conditions. The in vivo study of downstream PG production in rats treated with 4-CB-2f,5f-H2Q revealed that PGE2 was significantly elevated in ratsf kidneys at 24 h post intratracheal instillation. The increased PGE2 production was correlated with an elevation of alveolar macrophages. These findings suggest two possible mechanisms of enhanced PGE2 production: i) 4-CB-2f,5f-H2Q as a cosubstrate for PGHS in kidney, and 2) release of cytokines from macrophages, leading to stimulation of PGE2 production in other tissues but released and accumulated in kidney for excretion. In summary, the toxicity of lower chlorinated PCBs metabolites is potentially mediated by PGHS. Quinones generated from the PGHS metabolic pathway covalently bind to GSH resulting in GSH depletion, and oxidative stress. The intercalation or pi-stacking of SQ in DNA may be implicated in genotoxicity as a result of the change in DNA structure.