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, 102 (9), 3401-6

Identification of Mouse SLC39A8 as the Transporter Responsible for Cadmium-Induced Toxicity in the Testis

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Identification of Mouse SLC39A8 as the Transporter Responsible for Cadmium-Induced Toxicity in the Testis

Timothy P Dalton et al. Proc Natl Acad Sci U S A.

Abstract

Testicular necrosis is a sensitive endpoint for cadmium (Cd(2+), Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.

Figures

Fig. 1.
Fig. 1.
Scheme showing how the Cdm-gene-containing region was refined from >24 cM on mouse Chr 3 to 4.96 Mb, 2.37 mM, and 880 kb containing three putatively functional genes. (a) Genetic map originally generated by Taylor et al. (26). Varitint waddler (28) has now become the mucolipin 3 gene (Mcoln3) (29). (b) Phenotype-genotype association studies with the recombinant inbred line BXD14/Ty (b, B6; d, D2 allele) showing that a double crossover occurred between M12-7 and M10-26 (arrows above) and then nine recombinants derived from the (B6D2)F1 × B6 backcross further refined the Cdm locus to a region between S901 and M10-26 (arrows below). Gray circled genotypes are recombinants that ultimately define the 880-kb segment containing Cdm. (c) SNP analysis over the 880-kb segment showing “the fraction of positive SNP signatures” occurring in 40-kb intervals. “Positive” denotes those SNPs in D2 and 129S6 (Cd-sensitive strains) that differ from that in B6 and A/J (Cd-resistant strains) divided by the total number of SNPs in 40-kb intervals.
Fig. 2.
Fig. 2.
Cd toxicity in mouse fetal fibroblasts that express ZIP8 cDNA. (a) Northern blot of ZIP8 mRNA in rvZIP8 cells or rvLUC control cells. Total RNA was size-separated, and the blots were hybridized with cRNA probes for the indicated mRNA. ACTB, β-actin mRNA as a control for lane loading. (b and c) Dose–response curves for Cd-induced cell death. Cells were treated for 32 h with the indicated concentration of CdCl2 and cell death monitored by using b, the 3-(4,5-dimethlythiazol-2-yl)-2,5-diphenyl tetrasodium bromide assay, or c, the lactate dehydrogenase release assay. rvZIP8ha cells contain the HA-tagged ZIP8 protein. rvZIP8m cells contain a mutated ZIP8 cDNA. Data represent means ± SD of triplicate determinations.
Fig. 3.
Fig. 3.
Increased Cd uptake caused by membrane-localized ZIP8. (a) Time/dose-dependent 109CdCl2 uptake into rvZIP8 or rvLUC cells. (b) Western blot of ZIP8ha in microsomes (30 vs. 10 μg per lane). (c) Western blot of ZIP8ha in cytosol (C) or microsomes (M) (30 μg per lane) from rvLUC or rvZIP8ha cells. Arrow denotes band at 55 kDa. (d) Localization of the ZIP8ha protein. rvZIP8ha cells (Upper) or rvLUC cells (Lower) were fixed and incubated with a primary anti-HA antibody and a secondary goat anti-rabbit FITC-conjugated antibody. Cells were counterstained with propidium iodide (PI) to visualize nuclei. Confocal fluorescent microscopy detected FITC (Left), PI (Center), or both FITC and PI (Right).
Fig. 4.
Fig. 4.
Localization of ZIP8 mRNA from sensitive (D2 and 129S6) versus resistant (B6 and A/J) inbred mouse strains. (a) Northern analysis of testicular ZIP8 mRNA by using poly(A+) RNA. ACTB, β-actin mRNA as a control for lane loading. (b) In situ hybridization of ZIP8 mRNA in testis of the four inbred strains. Left show hematoxylin-and-eosin tissue staining (bright-field), Center show signal from in situ hybridization (dark-field), and Right show both images overlaid. Arrows show ZIP8 mRNA localized in the vascular endothelial cells of D2 and 129S6, a feature not detected in B6 or A/J mice. (c) High-magnification bright-field ZIP8 in situ image of testicular capillaries from the indicated mouse strains. (Lower) Photographic emulsion showing capillary in situ pattern (Left) was stripped and section-stained with an anti-CD31 endothelial-specific antibody that was detected by using a biotinylated secondary antibody and Alexa Fluor-488 streptavidin. Antibody reactivity is shown in green. DAPI (blue color) was used as a nuclear counterstain.

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