Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase-1 in microglial cells.

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK-3/6, MKK-1/2 and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250 µm), LPS (100 ng ml⁻¹) or Mn + LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn + LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn + LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4's phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF-α, IL-6 and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6 and MKK-4, are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect.

Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice.

The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4(+)/CD8(+) being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8(+) were increased and MHC-II(+) and CD19(+) cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic naïve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11c(high)), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4(+) lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4(+) lymphocytes, the MHC-II(+) cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.

Deliberately added and "cryptic" antioxidants in three artificial diets for insects.

Characteristics of both deliberately added and "cryptic" antioxidants were assayed from hydrophilic and lipophilic extracts from artificial diets for plant bugs, lepidopteran larvae, and green lacewings. Cryptic antioxidants are defined as substances naturally existing in diet ingredients but not deliberately added because of their antioxidant potential. Diets were tested after 1) being freshly produced, 2) stored for 48 h at 4 degrees C, or 3) held for 48 h under rearing room conditions at 27 degrees C. Tests included 1) a general assay of antioxidant capacity known as the ferric-reducing antioxidant power (FRAP) assay. 2) a cation radical-scavenging assay, 3) an ascorbic acid assay, and 4) an assay of inhibition of lipid peroxidation. In all assays, the lepidopteran diet had the highest values for protection against reactive oxygen species (ROS). The lepidopteran diet (with 0.17-0.23-mg equivalents of gallic acid equals total phenolic compounds per gram of diet) had three- to four-fold higher concentrations of phenolic compounds than did either the plant bug diet or the lacewing diet. Unexpectedly, the plant bug and the lacewing diets caused more lipid peroxidation than did the positive controls. This was attributed to the high concentrations of iron in these diets (mainly from chicken eggs), causing an ascorbate-ferric ion-induced lipid peroxidation. Diet storage, measured after 2 d at 27 or 4-6 degrees C, caused no significant declines in overall antioxidant potential. However, storage did lead to decline in ascorbic acid. The FRAP assay offered the best potential as a general, routine test of the potential of various insect diets to resist the destructive effects of ROS. The importance of addressing issues of protection against ROS in insect diets is discussed.