Libia Vega

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells

Abstract. Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.

Metabolism and Toxicity of Arsenicals in Cultured Cells

Publisher Summary The metabolism and toxicities of arsenite, arsenate, and trivalent and pentavalent methylated arsenicals have been examined in primary rat hepatocytes and in cells derived from human liver, skin, urinary bladder, and cervix. Among the cell lines examined, primary rat hepatocytes exhibit the greatest capacity for methylation of arsenicals. Trivalent arsenicals, arsenite, diiodomethylarsine, and methylarsine oxide are better substrates for the methylation reactions than are pentavalent arsenate and methylarsenate. Compared to primary rat hepatocytes, the capacity for methylation of arsenicals is significantly lower in primary human hepatocytes. Even lower capacity for As methylation was found in HeLa and normal human epidermal keratinocytes. The Urotsa cell line, an SV-40 transformed human urinary bladder cell line, did not methylate any arsenical tested. In primary rat hepatocytes incubated with 0.1 to 1 μM arsenite, dimethylarsenics (DMAs) were the major methylated metabolite and were found mainly in culture media. Small amounts of monomethylarsenics (MAs) were detected in cells. Incubation of primary rat hepatocytes with 4 to 20 μM arsenite resulted in partial inhibition of the methylation reactions, a decreased DMAs/MAs ratio, and the release of significant amounts of MAs from cells. In cell lines with low capacities for As methylation, Asi and/or MAs accumulated in the cells, suggesting that complete methylation (dimethylation) is a prerequisite for clearance of As from cells. Addition of glutathione, glutathione ethyl ester, or N-acetylcysteine to culture media stimulated the efflux of MAs from cells decreasing the DMAs/MAs ratio. For all cell lines examined, trivalent mono- and dimethylated arsenicals are more toxic than is arsenite. There is no correlation between methylation capacity of cell lines and resistance to the cytotoxicity of trivalent arsenicals.

The unexpected role for the aryl hydrocarbon receptor on susceptibility to experimental toxoplasmosis.

The aryl hydrocarbon receptor (AhR) is part of a signaling system that is mainly triggered by xenobiotic agents. Increasing evidence suggests that AhR may regulate immunity to infections. To determine the role of AhR in the outcome of toxoplasmosis, we used AhR−/− and wild-type (WT) mice. Following an intraperitoneal infection with Toxoplasma gondii (T. gondii), AhR−/− mice succumbed significantly faster than WT mice and displayed greater liver damage as well as higher serum levels of tumor necrosis factor (TNF)-α, nitric oxide (NO), and IgE but lower IL-10 secretion. Interestingly, lower numbers of cysts were found in their brains. Increased mortality was associated with reduced expression of GATA-3, IL-10, and 5-LOX mRNA in spleen cells but higher expression of IFN-γ mRNA. Additionally, peritoneal exudate cells from AhR−/− mice produced higher levels of IL-12 and IFN-γ but lower TLR2 expression than WT mice. These findings suggest a role for AhR in limiting the inflammatory response during toxoplasmosis.

Synergistic effect of influenza A virus on endotoxin-induced mortality in rat pups: A potential model for sudden infant death syndrome

Sudden infant death syndrome is the most common cause of postneonatal infant mortality in the developed world. It is a diagnosis of exclusion with peak age of incidence between 2 and 6 mo. Fifty to 63% of these infants have a preexisting upper respiratory tract infection before death. We hypothesized that the immature immune system may be altered by a primary infection, preventing a protective response after secondary challenge. To mimic dual infection, we used a nonlethal strain of a rat-adapted influenza A virus and a sublethal dose of endotoxin to establish a model that results in pathology and death in 12-d-old rat pups similar to that seen in infants dying of sudden infant death syndrome. Mortality only occurred when specific criteria such as timing between infectious insults and developmental age of the pup were met. Results suggest that mortality is caused by a rapid systemic shock event rather than lung-specific damage. Gross pathologic findings such as lung petechiae and liquid blood around the heart on necropsy were consistent with those seen in infants dying of sudden infant death syndrome. Histopathologic lesions including subendocardial hemorrhage and mild cortical thymocyte necrosis were found with greater severity and frequency in dually challenged animals. Macrophage subpopulation in rat-adapted influenza A virus-inoculated animals was significantly elevated in the spleen at the time of death. Our model suggests that the developing immune system can be primed to respond in an exaggerated way to a second immune challenge resulting in unexpected death.

Levocetirizine inhibits migration of immune cells to lymph nodes and induces treg cells in a murine type I allergic conjunctivitis model.

Background & Purpose: Levocetirizine is a histamine H(1) receptor antagonist. Here, we utilised DO11.10TCR transgenic mice to establish an antigen-specific T cell-dependent allergic conjunctivitis (AC) model to determine the effect of the topical application of an ophthalmic formulation of Levoceritizine as a treatment for AC. Experimental Approach: DO11.10 mice (n=6/each) were exposed to ovalbumin (OVA, 50 µg) and treated with a Levocetirizine ophthalmic formulation (0.001–0.02% v/w) or placebo (vehicle) for 24–72 h. Serum, aqueous/vitreous humour and conjunctiva were obtained. Immunoglobulin (Ig)-E, interleukin (IL)-10 and lipoxin (LX)A4 were determined by ELISA. Levels of tumour necrosis factor (TNF)-α, transforming growth factor (TGF)-β, interferon (IFN)-γ and 18rS expression were measured by RT-PCR. Proportions of total and activated antigen-presenting cells (APC), recruited T lymphocytes (CD4+), activated T lymphocytes (CD25+) and T regulatory cells (Treg) were measured by flow cytometry. Key Results: OVA exposure induced AC in the animal model indicated by increased expression of LXA4, TNF-α and TGF-β. Levocetirizine treatment (0.01–0.02% v/w) reduced LXA4 in the eye humours. This treatment approach increased systemic IL-10 secretion and reduced TNF-α and TGF-β expression in conjunctiva without changing IFN-γ expression. Levocetirizine reduced APC levels in draining lymph nodes but increased the proportion of total lymphocytes recruited and their differentiation to Treg cells. Conclusions & Implications: Levocetirizine effectively reduces the activation and migration of APC to local draining lymph nodes and induces differentiation of Treg cells as one possible mechanism of its anti-inflammatory action.