TCDD exposure alters fecal IgA concentrations in male and female mice.

BACKGROUND: Activation of the aryl hydrocarbon receptor (AhR) can alter diurnal rhythms including those for innate lymphoid cell numbers, cytokine and hormone levels, and feeding behaviors. Because immune responses and antibody levels are modulated by exposure to AhR agonists, we hypothesized that some of the variation previously reported for the effects of AhR activation on fecal secretory immunoglobulin A (sIgA) levels could be explained by dysregulation of the diurnal sIgA rhythm. METHODS: C57Bl/6 J mice were exposed to peanut oil or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 10 or 40 µg/Kg) and fecal sIgA levels were determined in samples collected every 4 h over 4 days. RESULTS: Fecal sIgA concentrations were not significantly different between light and dark phases of the photoperiod in either male or female mice, and there were no significant circadian rhythms observed, but TCDD exposure significantly altered both fecal mesor sIgA and serum IgA concentrations, in parallel, in male (increased) and female (biphasic) mice. CONCLUSIONS: AhR activation can contribute to the regulation of steady state IgA/sIgA concentrations.

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

The growth and infectivity of Leishmania major is not altered by in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

OBJECTIVE: The numbers of Leishmania major parasites in foot lesions of C57Bl/6, BALB/c or SCID mice can be significantly reduced by pre-exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). One potential mechanism to explain this enhanced resistance to infection is that TCDD is directly toxic to L. major. This potential mechanism was addressed by exposing L. major promastigotes and amastigotes to TCDD in vitro and examining their subsequent proliferation and infectivity. RESULTS: We found no significant change in the rate of in vitro L. major proliferation (promastigotes or amastigotes) after TCDD exposure at concentrations up to 100 nM. Moreover, in vitro TCDD exposure did not significantly alter the ability of L. major to infect mice, trigger lesion formation, or survive in those lesions.

Effects of endosulfan isomers on cytokine and nitric oxide production by differentially activated RAW 264.7 cells.

Endosulfan is an organochlorine insecticide comprised of two isomers: endosulfan-α and endosulfan-ß. Endosulfan exposure has been shown to elevate some inflammatory factors, such as nitric oxide (NO) and tumor necrosis factor (TNF), in animals or cultures of animal cells. Because the two endosulfan isomers can vary in their biological activities, the goal of this study was to determine if individual endosulfan isomers differentially impact production of NO or TNF by the mouse macrophage cell RAW 264.7 at non-cytotoxic levels. We found elevated TNF with exposure to endosulfan-α (not endosulfan-ß), but only at concentrations that were cytotoxic (≥100 µM), whereas neither endosulfan isomer altered baseline levels of NO at any concentration up to 300 µM. In interferon (IFN)-γ-activated cultures, NO levels were significantly suppressed by either endosulfan isomer at 10 µM (the lowest concentration examined), whereas only endosulfan-ß significantly lowered TNF levels at non-cytotoxic concentrations. In lipopolysaccharide (LPS)-activated cultures, both endosulfan isomers significantly reduced NO, but not TNF, at non-cytotoxic concentrations. These results suggest that the endosulfan isomers have some capacity to alter inflammatory responses differentially, particularly with IFN-γ stimulation.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces Leishmania major burdens in C57BL/6 mice.

Acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can suppress adaptive immunity. In this study, pre-exposure of Leishmania major-infected mice to TCDD caused a dose-dependent and unexpected decrease in parasite burdens on day 20 after infection. In contrast, TCDD-mediated lymphoid atrophy, suppressed antibody levels, and enhanced interleukin-2 production were observed as expected. These results suggest that TCDD may enhance resistance to L. major in the face of immune suppression.

Effects of paraquat on development of preimplantation embryos in vivo and in vitro.

Paraquat can cause oxidative stress through redox cycling, and preimplantation embryos are sensitive to oxidative stress in vitro. In this study, the effects of paraquat on preimplantation embryo development were examined. Exposure of preimplantation embryos (collected on the day after ovulation) to paraquat in vitro for 24 h at concentrations as low as 8 microM caused a significant decrease in the percentage of 8-cell embryos and an increase in the percentage of compacted morulae, but the content of reduced glutathione (GSH) in embryos was not changed. Altered embryo development was most likely due to premature compaction because a 42% decrease in cell number per compacted morulae was observed in embryos exposed to paraquat at 1 mM. Exposure of preimplantation embryos to paraquat in vitro for 4 days at 200 microM or higher eliminated development beyond the blastocyst stage. Exposure of bred female mice to paraquat at 30 mg/kg on day 2 after ovulation led to a small but significant decrease in the percentage of 8-cell embryos on day 3 without a detectable increase in the percentage of compacted morulae. No detectable change in preimplantation embryo development was found following paraquat exposure on the day of ovulation (day 0), although a significant decrease in embryo GSH was found on day 1. These data indicate that paraquat can adversely impact the development of preimplantation embryos in vitro and in vivo without consistent modulation of GSH level.

Time- and dose-dependent effects of ethanol on mouse embryonic stem cells.

Ethanol is a common solvent used with mouse embryonic stem (mES) cells in protocols to test chemicals for evidence of developmental toxicity. In this study, dose-response relationships for ethanol toxicity in mES cells were examined. For cells maintained in an undifferentiated state, ethanol significantly reduced viable cell numbers with estimated half maximal inhibitory concentrations of 1.5% and 0.8% ethanol after 24 and 48h, respectively, observations which correlated with significantly increased expression of apoptotic markers. For cells cultured to induce cardiomyocyte formation, up to 0.5% ethanol during the first two days failed to alter the outcome of differentiation, whereas 0.3% ethanol for 11 days significantly reduced the fraction of cultures containing contracting areas, an observation that correlated with significantly reduced cell numbers. These results suggest that ethanol is not an inert solvent at concentrations that might be used for developmental toxicity testing.

Short report: requirement of b cells for delayed type hypersensitivity-like pathology after secondary infection with Leishmania major in resistant C57BL/6 mice.

B cell-deficient C57B1/6 (microMT) mice were resistant to Leishmania major after both primary and secondary parasite challenge. However, unlike in wild-type mice, secondary infection in microMT mice was not accompanied by a marked delayed type hypersensitivity-like response, and interferon-gamma (IFN-gamma) levels were approximately half of those in wild-type mice. These results suggest that B cells are involved in IFN-gamma production and the pathology of secondary infection.

Type 1 and type 2 responses to Leishmania major.

Leishmania major is a protozoan parasite that is transmitted to the mammalian host by its sand fly vector when the fly probes in the host's skin for a blood meal and injects the parasite within its saliva. In mice experimentally infected with L. major, outgrowth of CD4 type 1 (Th1) cells leads to resolution of the infection, but outgrowth of type 2 (Th2) cells exacerbates disease. To design an effective vaccine against the parasite (and other pathogens that induce polarized Th1 and Th2 responses), we must determine the mechanism underlying this phenomenon so that we can design the vaccine to elicit the appropriate (i.e., protective) Th cell. Recent work indicates that Th bias is influenced by a number of signals delivered by antigen-presenting cells, including cytokines and co-stimulatory molecules. Moreover, recent work also suggests that sand fly saliva influences the immune response to L. major and Th polarization. Determining the mechanisms that lead to polarized Th responses should expand our knowledge regarding immunity to L. major, and should add to our understanding of immunoregulation in general.

2,3,7,8-tetrachlorodibenzo-p-dioxin slows the progression of experimental cutaneous Leishmaniasis in susceptible BALB/c and SCID mice.

In a model of experimental cutaneous leishmaniasis, pre-exposure of Leishmania major-resistant mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor agonist, causes suppression of the protective anti-parasite T helper 1 response while paradoxically also reducing parasite burdens in those animals. In this study, we examined if TCDD exposure could also reduce parasite burdens in L. major-susceptible BALB/c mice. In the highest dose group (160 µg/Kg), TCDD treatment caused a significant reduction of parasite burdens by 10-fold after three weeks while also causing a significant lymphoid atrophy indicating suppression of the non-protective T helper 2 response. A dose-dependent delay of foot lesion progression was also observed such that lesion size in the highest dose group was less than half that of controls after 35 days of infection. Importantly, although TCDD exposure initially reduced disease severity and prolonged the course of disease by as much as three fold in some animals, this effect was transitory and TCDD did not induce resistance to L. major infection. Because TCDD exposure reduced L. major burdens in both resistant and susceptible mice, we hypothesized that TCDD reduces L. major burdens in mice by a mechanism that does not involve adaptive immunity. To test this, severe combined immunodeficient (SCID) mice were used. In mice infected with a moderate number of L. major (10,000), TCDD treatment caused a time- and dose-dependent decrease of parasite burdens by nearly 100-fold after six weeks in the highest dose group (200 µg/Kg). A significant and dose-dependent delay of foot lesion progression was also observed in these animals. These results indicate that TCDD exposure can reduce the severity of leishmanial disease in mice independent of adaptive immunity.

Resolution of an infection with Leishmania braziliensis confers complete protection to a subsequent challenge with Leishmania Major in BALB/c mice

Both Leishmania major and L. braziliensis induce cutaneous leishmaniasis in BALB/c mice. Whereas BALB/c mice die of infection with L. major, they cure an infection with L. braziliensis. We report here that after curing an infection with L. braziliensis, BALB/c mice are resistant to challenge with L. major. When challenged with L. major, L. braziliensis pre-treated BALB/c mice mounted a delayed-type hypersensitivity response to L. major and produced high amounts of interferon-gamma (IFN-gamma) but low amounts of interleukin-4. The IFN-gamma produced by the L. braziliensis pre-infected mice was involved in the protection seen against L. major challenge since treating the mice with a neutralizing anti-IFN-gamma abrogated the protection. This suggests that cross-reactive antigen epitopes exist between L. braziliensis and L. major and that pre-infection with L. braziliensis primes BALB/c mice to epitopes on L. major that can elicit a protective Th1 response to the parasite.