Cadmium Stimulates Transcription of the Cytochrome P450 Side Chain Cleavage Gene in Genetically Modified Stable Porcine Granulosa Cells1

Abstract

We investigated the effects of cadmium (Cd2+) on transcription of the cytochrome p450 side chain cleavage (p450scc) gene and on progesterone synthesis in stable granulosa cells. We used the stable porcine granulosa cell line, JC-410, genetically modified to express a luciferase genomic construct carrying 2320 base pairs (bp) of the p450scc gene promoter (p450scc-2320-LUC). A construct containing only the luciferase gene, pOLUC, was used as a promoterless control. At 1 microM, cadmium chloride (CdCl2) increased transient expression of p450scc-2320-LUC in JC-410 cells by 2.6-fold after 24-h incubation. A similar pattern of stimulation by CdCl2 was observed in cells transiently transfected with a luciferase genomic construct carrying 100 bp of the p450scc gene promoter p450scc-100-LUC, whereas no stimulation by CdCl2 was observed in cells transfected with pOLUC. At 0.6, 1, and 2 microM, CdCl2 stimulated the activity of the p450scc-2320-LUC promoter in a dose-related fashion by 1.58-, 3.19-, and 2.67-fold, respectively, after 24-h incubation. Northern blot analysis showed that CdCl2 at 0.1, 1, 2, and 3 microM increased p450scc mRNA levels by 3.13-, 1.38-, 1.61-, and 1.57-fold, respectively, after 24-h incubation. After 48-h incubation, CdCl2 at 0.6, 1, and 2 microM further increased p450scc mRNA levels by 3.43-, 2.08-, and 2.4-fold, respectively. At 1, 2, and 3 microM, CdCl2 inhibited progesterone synthesis to 0.48-, 0.38-, and 0.29-fold, respectively. After 48-h incubation, CdCl2 at 0.1 microM stimulated progesterone synthesis by 1.6-fold. We conclude that Cd2+ has a dual action in stable porcine granulosa cells: Low concentrations activate, whereas high concentrations inhibit, expression of the p450scc gene and progesterone synthesis. The stimulatory effect of Cd2+ appears to be mediated via a cis-acting element located 100 bp upstream of the p450scc gene transcription start site.

Ultrastructural Changes of Ovaries in Rabbits Following Cadmium Administration

“…In this study major alteration of endoplasmic reticulum were found in thecal and endothelial cells. Generally, there are few data describing the effect of cadmium on granulosa cells (Henson and Chendrese 2004;Smida et al 2004;Vr‰anská et al 2003;Drbohlav et al 1998) with mainly monitoring and biochemical aspects of toxicity. In luteal cells only interference of cadmium with steroid biosynthesis in rat luteal cells in vitro was studied (Paksy et al 1992).…”

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“…Both lead and cadmium caused a significant reduction in gonadotropin binding, which altered the steroidogenic enzyme activity of granulose cells (Nampoothiri & Gupta, 2006). Higher concentrations of cadmium inhibited activity of the P450scc gene (Smida, Valderrama, Agostini, Furlan & Chedrese, 2004), inhibited progesterone synthesis (Massanyi, Uhrin, Toman, Kovacik & Biro, 1999;Zhang & Jia, 2007), and facilitated changes in cell morphology and cell death (Smida,et al 2004;Kondoh, Araragi, Sato, Higashimoto, Takiguchi & Sato, 2002). Thus, polluting substances, classified as endocrine disruptors, interfere with the endogenous hormone signaling system, leading to impairment of reproductive health (Caserta et al, 2011;Gupta et al, 2010;Zhang et al, 2013).…”

“…Cd is shown to down regulate 11β-HSD by mimicking the ability of estrogen to attenuate the expression of this placental enzyme (Chowdhury & Arora, 1982). Smida et al (2004) reported the stimulatory effect of Cd on transcription of P450 side chain cleavage in porcine granulose cells resulting into increase in progesterone production.…”

Cadmium in human and animal organisms. III. Effect on reproductive system

“…У роботах M.C. Henson, A.D. Smida та інших авторів [32,68] показано, що інтенсивність синтезу прогестерону в яєчнику тварин і культи вованих клітинах зростає під впливом низьких доз, але пригнічується зі збіль шенням концентрації Кадмію.…”

“…У культивованих клітинах плаценти зумов лене Cd 2+ пригнічення синтезу прогестерону відбувається співрозмірно зі змен шенням активності P450scc [32,41]. З використанням гранульозних клітин яєчника свині встановлено, що за низьких концентрацій (0,6-3,0 мкM) Cd 2+ дозозалежно ак тивує промотор гена P450scc, а за концентрації 5 мкM -пригнічує, водночас зумов M -пригнічує, водночас зумов -пригнічує, водночас зумов люючи зниження рівня прогестерону, зменшення кількості клітин і розвиток морфо логічних змін у їхній структурі [68].…”