The role of zinc (Zn) in lead (Pb)-induced reproductive toxicity

Abstract

Objective: We here seek initial testicular gene expression changes conferring sensitivity/resistance to Pb-induced decreases in spermatogenesis. In 96 occupationally unexposed men, 15/21 post-IVF pregnancy failures were attributed to elevated Pb in the male tract, but 9/24 men with elevated Pb were successful, possibly due to normal Zn levels. Here, seminal plasma Pb and Zn levels correlated inversely (Spearman correlation [SC], n=67, r = -0.4, P < 0.001). Pb and Zn had opposing effects on sperm count (SC, respectively, r = -0.3, P < 0.02 and r = 0.3, P < 0.01). In obstructive azoopsermia, testis biopsy (TB) Pb was negatively related to sperm count (SC, n=33, r = -0.5, P < 0.002), apparently related to the correlation between Pb and % apoptosis (SC, n=17, r = 0.4, P < 0.08 [trend]). Expression of L-type voltage-dependent calcium channel (LVDCC) α1C subunit mRNA deletions in exons 6–9 (regulating heavy metal transport; Fisher’s exact test, n=13, P < 0.01) apparently protected against Pb. Consistently, in rodents, Pb (a calcium competitor) accumulates within testicular cell nuclei; while Zn co-administration with Pb prevents Pb accumulation and Pb-induced gene expression changes. Design: Adult (100 day) Pb-resistant Sprague-Dawley (SD) and Pb-sensitive Wistar male rats were given deionized water containing 0.0%, 0.025%, 0.05%, 0.15% or 0.3% lead acetate (PbAc) for 10 days. Materials and Methods: [1] Zn and Pb levels by atomic absorption; [2] total gene expression by microarrays (Rat Genome U34 Set; Affymetrix) with data mining via GeneSpring (Silicon Genetics); [3] LVDCC splicing variations in exons 6–9 by RT-PCR and amplicon sequence analysis; [4] percentage testicular apoptosis by TUNEL. Results: Highest-level PbAc-treated animals had blood leads >40 μg/dL, with <1 μg/dL in controls. The increase in testicular Pb was higher in Wistar rats. Testicular Zn increased in SD, but was unchanged in Wistars. In SD testes: [1] transcription factor modulators: CaMK IV increased and calcineurin decreased, altering CRE-containing (Ca regulated) gene expression gene dose-dependently [2] growth factors, xenobiotic detoxification genes (e.g. metallothioneins [MT]), DNA repair, and protein synthesis were all down-regulated; [3] undeleted LDCC α1C mRNA decreased, aberrant splicing increased, and [4] caspases were up-regulated. Apoptosis was increased. Only ∼10% of gene expression changes occured in Wistar testes, including both increased (transcription factors, ion channels) and decreased expression (ribosomal proteins). All magnitudes were greater in Wistar testes. Wistar-unique were increased expression (heat shock factors, cell cycle control proteins) and unchanged expression (e.g., MT). Undeleted LDCC α1C mRNA increased and aberrant splicing decreased. Conclusion: Levels of Pb that altered testicular gene expression are well below that causing widespread organ failure. In SD rats, increased testicular Zn associated with induction of MT, sequestering Pb. Altered Zn levels in Wistar testes with MT induction failure contributes to Pb sensitivity. Expression of deleted LVDCC α1C, reducing Pb transport, also contributes to Pb resistance. The data emphasize the importance of metal interactions to normal reproductive function. Supported by: NIH Grant Nos. ES06100 and ES10496 to SB and ES03749 to RZS.