No abstract

Cultured Y-1 mouse adrenal tumor cells, which secrete 20-alpha-hydroxy-4-pregnen-3-one (20-DHP), were used to investigate the acute nonlethal effects of incremental cadmium chloride (CdCl2) concentrations on basal and maximally stimulated steroid secretion. In addition, cumulative CdCl2 effects during 4-hr incubations, effect reversibility, and viability were determined. Cells were incubated in 1 ml serum-free Eagle's Minimal Essential Medium (FMEM) with or without 0.5 IU (ca. 1.5 microM) adrenocorticotropin (ACTH) in the presence or absence of CdCl2. Following incubation, cell viability was quantitated using trypan blue exclusion. The 20-DHP secreted into the experimental incubation medium was measured by radioimmunoassay. CdCl2 levels of 10.0 micrograms/ml or greater significantly inhibited basal 30 min steroid secretion in a dose-dependent manner; ACTH-stimulated steroid secretion was significantly inhibited by levels 5.0 micrograms/ml or greater. At least 80% of all control and stimulated cells in the presence or absence of cadmium ions excluded trypan blue. The reduction in ACTH-stimulated steroid secretion was greater than the reduction in basal steroid secretion at any cadmium concentration level. The CdCl2 concentration that reduced stimulated steroid hormone secretion by 50% (IC50) was 45.0 micrograms/ml. Exposing Y-1 cells to either 5.0, 10.0, 45.0 or 500.0 micrograms CdCl2/ml FMEM for periods ranging from 0.5 to 4 hr inhibited ACTH-stimulated steroid secretion in a time-dependent manner. After 30 min exposure to 10.0, 45.0 or 500.0 micrograms CdCl2/ml FMEM with or without ACTH, cadmium inhibition was irreversible. When 5.0 micrograms CdCl2/ml was used, basal and stimulated inhibition was reversible by reincubating in medium containing ACTH alone. The relatively greater cadmium effects on ACTH stimulated steroidogenesis might suggest that cadmium modulated the rate-limited transducing system between the ACTH plasma membrane receptor complex and cholesterol side-chain cleaving mitochondrial enzymes. However, cadmium influences on basal secretion indicated effects on the non-rate-limited steroidogenic pathway.

In previous studies cadmium chloride (CdCl2) nonlethally inhibited Y-1 mouse adrenal tumor cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. In addition, dibutyryl cAMP-stimulated 20DHP secretion was unaffected by CdCl2, while the site of the unstimulated effect was indirectly shown to involve steps between endogenous cholesterol utilization and 20-hydroxycholesterol association with mitochondrial cytochrome P450 side-chain cleavage enzyme. In the present study we determined CdCl2 effects on plasma membrane sites preceding pre-dbcAMP-stimulation of 20DHP secretion. Y-1 cells were incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited adrenocorticotropin- (ACTH)-stimulated steroid secretion by 50%) together with exogenously added maximally stimulating concentrations of ACTH, cholera toxin, forskolin, or adenosine triphosphate. Cholera toxin, forskolin and ATP bypass specific plasma membrane sites involved in the synthesis of intracellular cAMP and activate the steroid hormone biosynthetic pathway. Cadmium effects on ACTH-stimulated endogenous cAMP secretion were also examined. CdCl2 significantly reduced Y-1 cell 20DHP secretion following exposure to ACTH, cholera toxin, forskolin, and ATP; it also significantly decreased endogenous cAMP secretion into culture medium. These data may be interpreted to suggest that CdCl2 altered Y-1 cell regulation of adenyl cyclase activity, which reduced cAMP-activated cholesterol uptake by mitochondria as a consequence.

In previous studies cadmium chloride (CdCl2) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl2 effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22(R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl2 might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl2 could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl2. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22(R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl2, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl2 specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl2 toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesis.

In previous studies, nonlethal CdCl2 concentrations apparently inhibited basal Y-1 mouse adrenal tumor cell endogenous mitochondrial cholesterol conversion to pregnenolone. In addition, CdCl2 inhibited all agents stimulating both plasma membrane-dependent cAMP synthesis and 20 alpha-hydroxy-4-pregnen-3-one (20DHP) secretion. Bypassing the plasma membrane using dibutyryl-cAMP (dbcAMP) stimulated cytoplasmic cholesterol metabolism and 20DHP secretion in the presence of CdCl2. Since CdCl2 competed at metabolic steps requiring Ca2+ in other tissues, experiments were designed to examine Cd2+ competition with Ca2+ during steroidogenesis. Sets of cells incubated with either medium or adrenocorticotropin (ACTH) with or without CdCl2 were also treated with 0, 1.0, 5.0 or 10.0 mmol/L CaCl2 in the presence or absence of EGTA, a relatively specific Ca2+, but not Cd2+, chelating agent. Another experimental cell set incubated with either medium or ACTH, with or without CdCl2, was treated with or without 1 mmol/L A23187, an ionophore specifically facilitating extracellular Ca2+ transfer across plasma membranes. Besides determining Ca2+ involvement in steroidogenesis using steroid secretion as an endpoint, we directly measured Ca2+ concentrations using intracellular fura-2 fluorescence. Following loading with 2 mumol/L fura-2, cells remained untreated or medium was infused with CdCl2, ACTH, ACTH/CdCl2 or ACTH followed after 50 s by CdCl2. Using Ca(2+)-supplemented media, we observed that Cd2+ inhibition of ACTH-stimulated 20DHP secretion was completely reversed. Standard Ca(2+)-containing medium supplemented with Ca2+ also enhanced maximally stimulated 20DHP secretion by ACTH. 20DHP secretion by ACTH-treated and ACTH/Cd(2+)-treated cells was only reduced by EGTA, when Ca2+ was not supplemented. The ionophore A23187 increased basal and ACTH-stimulated 20DHP secretion by Cd(2+)-treated cells, suggesting that extracellular Ca2+ resources may compete against Cd2+ effects on plasma membrane cAMP synthesis and on basal cholesterol metabolism by mitochondria. No time-dependent change in Ca2+ concentrations occurred within untreated cell suspensions. ACTH stimulation caused a 25 s burst in Ca2+ concentrations before returning to basal, steady-state levels. Cd2+ also stimulated intracellular fura-2 fluorescence. Untreated cell suspensions infused with Cd2+ exhibited a continuous rise in intracellular fluorescence. ACTH/CdCl2-treated cells exhibited a hyperbolic rise in intracellular fluorescence over the 300 s study period. Cells treated with Cd2+ 50 s after ACTH treatment initially exhibited the 25 s fluorescence burst followed by a Cd(2+)-induced hyperbolic rise in intracellular Cd2+. These fluorescence measurements suggested that cytoplasmic Ca2+ changes do not appear to be necessary for basal 20DHP synthesis and secretion; only a 25 s burst in intracellular Ca2+ is necessary to a slightly higher plateau level for stimulated 20DHP synthesis and secretion. Cd2+ freely enters the cell under basal conditions and Cd2+ entry is accelerated...

If 1 mM supplemental Ca++, or 1.6 mg hemoglobin (Hb)/ml medium (23.5 uM, a concentration comparable to that in 1 ml mouse blood), was added to Eagle's minimum essential medium (MEM) (which contains 1 mM Ca++), basal and maximally stimulated 20-dihydroprogesterone (20-DHP) secretion by cultured Y-1 mouse adrenal tumor cells [49-65th passages] could be measured by radioimmunoassay after a 0.5 hr incubation. ACTH-stimulated, but not basal, 20-DHP secretion increased after 1 mM Ca++ treatment. 5 mM EGTA significantly reduced basal and stimulated 20-DHP secretion, although significant ACTH stimulation still remained. Basal and stimulated secretion significantly increased when Hb was present. Although O2 involvement in the Hb effect was tested by bubbling medium with 100% O2 for 10 minutes, basal and stimulated secretion was unaffected. Since proteins, such as Hb, non-specifically bind free steroids, enhancing secretion, albumin (Al) was compared to Hb. Al enhanced unstimulated, but not ACTH-stimulated, 20-DHP secretion. The Hb effect may not be due to non-specific protein-steroid binding. Ca++ supplementation and chelation studies suggest the necessity to optimize co-factors in steroidogenic tissue incubation media to maximize basal and stimulated steroid synthesis and secretion. Since physiologically relevant Hb levels enhanced basal and stimulated Y-1 cell steroid secretion by mechanisms other than protein-steroid binding and soluble O2 had little effect, Hb may more efficiently transport O2 to cultured cells than soluble O2 diffusion through medium does.