Identification and Characterization of Cellular Manganese Efflux Mechanisms

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2011.Manganese (Mn) is a ubiquitous trace element that is required for life; however, exposures to high levels of Mn can have severe adverse consequences, including the development of a Parkinson’s-like disease known as manganism. The mechanisms by which Mn is transported across cell membranes to reach its target sites are now beginning to be elucidated, although much remains to be learned about these processes, and in particular about cellular export mechanisms. The overall goal of this project was to identify and characterize cellular mechanisms of Mn efflux. Initial studies examined the hypothesis that the iron exporter ferroportin (FPN1) also mediates cellular Mn export, by studying the ability of FPN1 to transport 54Mn in Xenopus laevis oocytes co-expressing the human divalent metal transporter-1 (DMT1/NRAMP2/SLC11A2). The results demonstrated that Mn is indeed a substrate for FPN1, and that FPN1 may be a multi-specific metal efflux carrier that is both pH and membrane potential-sensitive. When compared to oocytes expressing only DMT1, 54Mn accumulation was lower in oocytes also expressing FPN1. In addition, FPN1-expressing oocytes exhibited a four-fold increase in 54Mn export when compared to control oocytes. Export was concentration-dependent and could be partially cis-inhibited by Fe, Co, and Ni. In addition, transport ability was significantly reduced at pH 5.5, but not at pH 8.5, while incubation in high K media lowered Mn efflux, indicating that Mn export on FPN1 is sensitive to pH and membrane potential. To search for additional Mn transporters, the human genome was searched for proteins with predicted amino acid homology to known or suspected Mn transporters from bacteria, yeast and plants. This in silico homology screen identified twenty-two proteins as candidate Mn transporters, and four of these were screened for their ability to transport 54Mn in Xenopus laevis oocytes co-expressing DMT1. Although the injection of the cRNA for the three ATP proteins failed to alter 54Mn accumulation in Xenopus oocytes, oocytes expressing the zinc transporter-2 (ZNT2/SLC30A2) accumulated much less 54Mn, suggesting that ZNT2 may modulate Mn uptake or efflux. ZNT2 expressing oocytes exported twice as much 54Mn when compared to control oocytes, whereas 54Mn uptake was unaffected. Export from the oocytes could be partially cis-inhibited by 100 M Cd, but not by the other metals tested. In addition, incubation in high K media had no effect on 54Mn efflux in ZNT2 expressing oocytes, indicating that this process is not dependent on the membrane potential. Additional studies examined whether the putative yeast vacuolar metal transporter Ypk9p contributes to Mn transport. Under the conditions tested, a role in Mn transport for Ypk9p was not supported, as accumulation of 54Mn was similar in the parental and ypk9- strains. Overall, these findings provide novel insight into mechanisms of Mn transport and a starting point for future research not only into Mn transport, but also to metal transport in general

Temporal changes in rat liver gene expression after acute cadmium and chromium exposure.

U.S. Service Members and civilians are at risk of exposure to a variety of environmental health hazards throughout their normal duty activities and in industrial occupations. Metals are widely used in large quantities in a number of industrial processes and are a common environmental toxicant, which increases the possibility of being exposed at toxic levels. While metal toxicity has been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify candidate biomarkers, rats were exposed via a single intraperitoneal injection to three concentrations of CdCl2 and Na(2)Cr(2)O(7), with livers harvested at 1, 3, or 7 days after exposure. Cd and Cr accumulated in the liver at 1 day post exposure. Cd levels remained elevated over the length of the experiment, while Cr levels declined. Metal exposures induced ROS, including hydroxyl radical (•OH), resulting in DNA strand breaks and lipid peroxidation. Interestingly, ROS and cellular damage appeared to increase with time post-exposure in both metals, despite declines in Cr levels. Differentially expressed genes were identified via microarray analysis. Both metals perturbed gene expression in pathways related to oxidative stress, metabolism, DNA damage, cell cycle, and inflammatory response. This work provides insight into the temporal effects and mechanistic pathways involved in acute metal intoxication, leading to the identification of candidate biomarkers

Exposure to toxic metals triggers unique responses from the rat gut microbiota.

Our understanding of the interaction between the gut microbiota and host health has recently improved dramatically. However, the effects of toxic metal exposure on the gut microbiota remain poorly characterized. As this microbiota creates a critical interface between the external environment and the host's cells, it may play an important role in host outcomes during exposure. We therefore used 16S ribosomal RNA (rRNA) gene sequencing to track changes in the gut microbiota composition of rats exposed to heavy metals. Rats were exposed daily for five days to arsenic, cadmium, cobalt, chromium, nickel, or a vehicle control. Significant changes to microbiota composition were observed in response to high doses of chromium and cobalt, and significant dose-dependent changes were observed in response to arsenic, cadmium and nickel. Many of these perturbations were not uniform across metals. However, bacteria with higher numbers of iron-importing gene orthologs were overly represented after exposure to arsenic and nickel, suggesting some possibility of a shared response. These findings support the utility of the microbiota as a pre-clinical tool for identifying exposures to specific heavy metals. It is also clear that characterizing changes to the functional capabilities of microbiota is critical to understanding responses to metal exposure

Exposure to toxic metals triggers unique responses from the rat gut microbiota.

Our understanding of the interaction between the gut microbiota and host health has recently improved dramatically. However, the effects of toxic metal exposure on the gut microbiota remain poorly characterized. As this microbiota creates a critical interface between the external environment and the host's cells, it may play an important role in host outcomes during exposure. We therefore used 16S ribosomal RNA (rRNA) gene sequencing to track changes in the gut microbiota composition of rats exposed to heavy metals. Rats were exposed daily for five days to arsenic, cadmium, cobalt, chromium, nickel, or a vehicle control. Significant changes to microbiota composition were observed in response to high doses of chromium and cobalt, and significant dose-dependent changes were observed in response to arsenic, cadmium and nickel. Many of these perturbations were not uniform across metals. However, bacteria with higher numbers of iron-importing gene orthologs were overly represented after exposure to arsenic and nickel, suggesting some possibility of a shared response. These findings support the utility of the microbiota as a pre-clinical tool for identifying exposures to specific heavy metals. It is also clear that characterizing changes to the functional capabilities of microbiota is critical to understanding responses to metal exposure

Cr, but not Cd exposure induced DNA damage in rat liver.

<p>A comet assay was performed to assess DNA strand breaks in hepatocytes from Cd (A) or Cr (B) treated animals. Cd exposure had no effect on Comet tail length. However, Cr induced DNA strand breaks at the medium and high doses, and this was sustained at all days examined. Data are expressed as mean ±SE, n = 2–7 animals per group. ^†Only two comet assays were available for analysis on days 1 and 3. *Significantly different from control on each day, p<0.05.</p

Time-dependent changes in candidate biomarker gene expression.

<p>The figure shows hierarchical clustering of the top 5 up-regulated DEGs on each day for each metal. When the same gene was one of the top genes for a different day of the same metal the next highest up-regulated gene was included for that day. The values shown in the heat map are the log_<b>2</b> ratio of change of the genes compared to the unexposed controls.</p