TPEN effects on <i>C</i>. <i>elegans</i> metal content and lifespan are zinc-specific.

<p><b>(A) TPEN effect on labile zinc levels is specific to the chelation of zinc</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM ZnSO₄ or 500μM MgSO₄ at L3 stage and collected at one-day old adult animals for analysis of relative labile zinc content. The representative micrograph shows that worms supplemented with zinc have elevated labile zinc content, while TPEN treatment decreased labile zinc. Equal molar levels of magnesium had no effect on basal or TPEN-induced fluorescence. Scale bar = 0.2mm. <b>(B) Kaplan-Meier survival curve of worm populations exposed to zinc and/or TPEN</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM ZnSO₄ at the L3 stage and monitored for effects on lifespan. Mean and maximum population lifespan were reduced with zinc supplementation and increased with TPEN treatment (p < 0.0001, log rank test). Additional 500μM ZnSO₄ attenuated the effect of TPEN on mean and maximum lifespan. <b>(C) Kaplan-Meier survival curve of worm populations exposed to magnesium and/or TPEN</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM MgSO₄ at the L3 stage and monitored for effects on lifespan. Mean and maximum population lifespan were increased with TPEN treatment (p < 0.0001, log rank test). Addition of MgSO₄ had no effect on mean and maximum lifespan.</p

Identification of genes that modulate the effects of zinc and TPEN on lifespan in <i>C</i>. <i>elegans</i>.

<p>(A) Wildtype worms, or worms with null alleles for <b>(B)</b> <i>hsf-1(sy441)</i>, <b>(C)</b> <i>daf-16(mu86)</i>, <b>(D)</b> <i>aak-2(ok524)</i>, <b>(E)</b> <i>rsks-1(ok1255)</i>, <b>(F)</b> <i>nhr-49(ok2165)</i>, <b>(G)</b> <i>skn-1(eu31)</i>, and <b>(H)</b> <i>clk-1(e2519)</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage. Mean and maximum population lifespan were reduced with zinc supplementation and increased with TPEN treatment for many mutant strains, similar to wildtype worms (p < 0.0001, log rank test). However, ZnSO₄ and TPEN-mediated changes in lifespan were attenuated in worms with mutations of <i>daf-16(mu86)</i>, <i>hsf-1(sy441)</i>, <i>skn-1(eu31)</i>.</p

Zinc Levels Modulate Lifespan through Multiple Longevity Pathways in <i>Caenorhabditis elegans</i>

<div><p>Zinc is an essential trace metal that has integral roles in numerous biological processes, including enzymatic function, protein structure, and cell signaling pathways. Both excess and deficiency of zinc can lead to detrimental effects on development and metabolism, resulting in abnormalities and disease. We altered the zinc balance within <i>Caenorhabditis elegans</i> to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. We found that increasing zinc levels <i>in vivo</i> with excess dietary zinc supplementation decreased the mean and maximum lifespan, whereas reducing zinc levels <i>in vivo</i> with a zinc-selective chelator increased the mean and maximum lifespan in <i>C</i>. <i>elegans</i>. We determined that the lifespan shortening effects of excess zinc required expression of DAF-16, HSF-1 and SKN-1 proteins, whereas the lifespan lengthening effects of the reduced zinc may be partially dependent upon this set of proteins. Furthermore, reducing zinc levels led to greater nuclear localization of DAF-16 and enhanced dauer formation compared to controls, suggesting that the lifespan effects of zinc are mediated in part by the insulin/IGF-1 pathway. Additionally, zinc status correlated with several markers of healthspan in worms, including proteostasis, locomotion and thermotolerance, with reduced zinc levels always associated with improvements in function. Taken together, these data support a role for zinc in regulating both development and lifespan in <i>C</i>. <i>elegans</i>, and that suggest that regulation of zinc homeostasis in the worm may be an example of antagonistic pleiotropy.</p></div

Zinc availability alters dauer formation, DAF-16 localization, and lifespan of insulin signaling pathway mutants in <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN</b>. Worms with null alleles for <i>daf-2</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at L4 stage animals. Mean and maximum population lifespan were reduced with zinc and increased with TPEN treatment (p < 0.0001, log rank test). <b>(B) Changes in dauer formation in worm populations exposed to zinc or TPEN as larvae</b>. Worms with null alleles for <i>daf-2</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and collected at one-day old adult animals and analyzed for dauer formation. Worms supplemented with zinc had a reduced number of worms in the dauer state, whereas worms treated with TPEN had an elevated number of worms in the dauer state (**, p<0.001, ***, p<0.0001, t-test). Data indicates the mean ± SD of 3 independent experimental replicates. <b>(C) Imaging DAF-16 localization in worm populations exposed to zinc or TPEN at 22°C</b>. DAF-16::GFP transgenic worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and measured for DAF-16 localization of one-day old adult animals. Representative micrographs showed that the subcellular localization of DAF-16::GFP was more evident in the nucleus when worms were exposed to TPEN, compared to control and zinc-supplemented worms. Scale bar = 200μm. <b>(D) Quantifying DAF-16 localization in worm populations exposed to zinc or TPEN at 22°C</b>. GFP fluorescence from DAF-16::GFP transgenice worms was imaged by fluorescence microscopy and scored for elevated nuclear DAF-16 localization. Data is represented as mean ± S.D using results of 3 experimental replicates (***, p<0.0001, log rank test). <b>(E) Imaging DAF-16 localization in worm populations exposed to zinc or TPEN at 34°C</b>. DAF-16::GFP transgenic worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and measured for DAF-16 localization of one-day old adult animals, after exposure to heat shock at 34°C for 5 min. Representative micrographs showed that the subcellular localization of DAF-16::GFP was reduced in the zinc treated group and elevated in in the TPEN group, compared to control worms. Scale bar = 200μm. <b>(F) Quantifying DAF-16 localization in worm populations exposed to zinc or TPEN at 34°C</b>. GFP fluorescence from DAF-16::GFP transgenic worms was imaged by fluorescence microscopy and scored for elevated nuclear DAF-16 localization. Data is represented as mean ± SD using results of 3 experimental replicates (***, p<0.0001, log rank test)</p

Zinc availability regulates the lifespan of <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN as larvae</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage. Mean and maximum population lifespan were reduced with zinc and increased with TPEN treatment (p < 0.0001, log rank test) <b>(B) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN as adults</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at day 5 of adulthood. Thl.ere was no significant difference in mean and maximum population lifespan due to zinc or TPEN treatment. <b>(C) Changes in total zinc content in worms exposed to zinc or TPEN as larvae</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage and collected at one-day old adult animals for analysis of total zinc content. Worms supplemented with zinc had a ~2-fold increase in total zinc content (***, p<0.0001, t-test), while worms treated with TPEN demonstrated a ~2-fold decrease in total zinc content (**, p<0.001, t-test). Data shown are the mean ± SD of 3 experimental replicates.</p

The effects of zinc availability on lifespan is dependent on HSF-1, SKN-1, and DAF-16 transcription factors in <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worms with knockdown of <i>hsf-1</i> exposed to TPEN as larvae</b>. <i>hsf-1</i> knockdown worms with or without knockdown of <i>skn-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>skn-1</i> was also knockdown (p < 0.0001, log rank test). <b>(B) Kaplan-Meier survival curve of worms with knockdown of <i>hsf-1</i> exposed to TPEN as larvae</b>. <i>skn-1</i> knockdown worms with or without knockdown of <i>hsf-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>hsf-1</i> was also knockdown (p < 0.0001, log rank test). <b>(C) Kaplan-Meier survival curve of worms with knockdown of <i>daf-16</i> exposed to TPEN as larvae</b>. <i>daf-16</i> knockdown worms with or without knockdown of <i>hsf-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>hsf-1</i> was also knockdown (p < 0.0001, log rank test). <b>(D) Kaplan-Meier survival curve of worms with knockdown of <i>daf-16</i> exposed to TPEN as larvae</b>. <i>daf-16</i> knockdown worms with or without knockdown of <i>skn-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>skn-1</i> was also knockdown (p < 0.0001, log rank test).</p

Zinc availability modulates healthspan in <i>C</i>. <i>elegans</i>.

<p><b>(A) Levels of age-related protein aggregation in worm populations exposed to zinc or TPEN</b>. L4 stage TJ1060 [spe-9(hc88)I; fer-15(b26)II] worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage for 6 days and then analyzed for SDS-insoluble protein. The representative experiment shows worms exposed to TPEN had reduced SDS-insoluble protein in comparison with control animals. (M, marker; LC, loading control of control worms; TC, loading control of TPEN treated worms; IC, SDS-insoluble protein of control animals; IT; SDS-insoluble protein of TPEN treated worms.) <b>(B) Percent paralysis of worms with knockdown for specific genes supplemented with or without TPEN</b>. HE250 worms with knockdown for <i>hsf</i>-1(RNAi), <i>skn</i>-1(RNAi), or <i>daf</i>-16(RNAi) and with or without exposure to 200μM TPEN were scored for paralysis after 48 hours. Control worms treated with 200μM TPEN showed significantly less paralysis in comparison to control animals (***, p<0.0001, log rank test). However, TPEN was less effective when used in populations with knockdown in <i>hsf</i>-1, <i>skn</i>-1, or <i>daf</i>-16 genes (NS, not significant).</p

Physiological characteristics of the dogs with urinary stones used in this study.

<p>The description of the dogs and associated data are listed. For breed, a single breed name indicates a purebred canine as reported by the owner. An “X-”preceding breed name indicates a mixed breed, with the subsequent breed name indicating the most dominant breed features of the non-purebred as reported by the owner. A listing of “mixed breed” indicates that no dominant breed characteristic pattern was observable. Age at time of stone removal is listed for most dogs, except 2 listed as “<i>NA</i>” indicating no data was available. “Status” category indicates whether the reproductive capacity of the dog was intact or spay/neutered. “Weight” category indicates weight of dog in kg at time of urinary stone collection. Composition information for both the outer surface and inner core regions of the stones is reported, with percentage of COM (% COM), COD (% COD), and apatite (% APA).</p><p>Physiological characteristics of the dogs with urinary stones used in this study.</p

Elemental Content of Calcium Oxalate Stones from a Canine Model of Urinary Stone Disease

<div><p>One of the most common types of urinary stones formed in humans and some other mammals is composed of calcium oxalate in ordered hydrated crystals. Many studies have reported a range of metals other than calcium in human stones, but few have looked at stones from animal models such as the dog. Therefore, we determined the elemental profile of canine calcium oxalate urinary stones and compared it to reported values from human stones. The content of 19 elements spanning 7-orders of magnitude was quantified in calcium oxalate stones from 53 dogs. The elemental profile of the canine stones was highly overlapping with human stones, indicating similar inorganic composition. Correlation and cluster analysis was then performed on the elemental profile from canine stones to evaluate associations between the elements and test for potential subgrouping based on elemental content. No correlations were observed with the most abundant metal calcium. However, magnesium and sulfur content correlated with the mineral hydration form, while phosphorous and zinc content correlated with the neuter status of the dog. Inter-elemental correlation analysis indicated strong associations between barium, phosphorous, and zinc content. Additionally, cluster analysis revealed subgroups within the stones that were also based primarily on barium, phosphorous, and zinc. These data support the use of the dog as a model to study the effects of trace metal homeostasis in urinary stone disease.</p></div

Association analysis of stone composition with characteristics of canines and stones.

<p>The elemental content of the canine stones was compared to physical (age, sex, and reproductive status) or dietary characteristics of the dog. The elemental content of the canine stones was also compared to mineral type of each stone. The linear regression function between element content in canine stone and dog age at time of stone collection was evaluated using the coefficient of determination (r²). The remaining categories were tested by standard t-test between element content in stone and category value. “Sex” category indicates whether dog was male or female, regardless of reproductive capacity. “Male Status” category indicates whether male dog was intact or neutered; female dogs were not compared due to few stones from intact female dogs. “Food Type” category indicates whether dog was predominantly fed commercial dry pellets or canned food. “Stone Surface” category indicates whether the surface of the canine stone was determined to be predominantly (>99%) COM or COD; stones with mixed mineral types were not included in this analysis. The mineral type at the core of the canine stone was not compared because most (>90%) stones had cores containing COM, regardless of the surface mineral type. A value of “p<0.05” indicates that the specific element significantly correlated with a specific value of the category. “<i>NA</i>” indicates that too few (n<3) points were available for comparison for the specific element and category value.</p><p>Association analysis of stone composition with characteristics of canines and stones.</p