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Modulation of adrenal cell functions by cadmium salts: 2. Sites affected by CdCl2 during unstimulated steroid
O P Mgbonyebi1, C T Smothers, J J Mrotek
1Physiology Department, Meharry Medical College, Nashville, TN 37208.
Abstract:
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.
Insights
Cadmium chloride (CdCl2) inhibits unstimulated steroid production by affecting mitochondrial cholesterol utilization, but not cAMP-regulated pathways. This suggests CdCl2 impacts basal steroidogenesis in adrenal cells.
Area of Science:
- Endocrinology
- Cell Biology
- Toxicology
Background:
- Cadmium chloride (CdCl2) is known to inhibit steroidogenesis in Y-1 adrenal cells.
- CdCl2 affects both unstimulated and stimulated steroidogenic pathways differently.
- Mechanisms controlling unstimulated steroidogenesis remain largely unelucidated.
Purpose of the Study:
- To investigate the specific effects of CdCl2 on unstimulated steroidogenesis in Y-1 adrenal cells.
- To determine whether CdCl2 inhibits steroid precursor conversion or cholesterol transport.
- To elucidate the role of cholesterol metabolites in unstimulated steroid production.
Main Methods:
- Y-1 adrenal cells were incubated with CdCl2 and various steroid precursors (20-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, pregnenolone, progesterone) or dibutyryl cyclic AMP (dbcAMP).
- The secretion of 20-dihydroxyprogesterone (20DHP) was measured under basal and stimulated conditions.
- CdCl2 concentration was optimized to inhibit 50% of ACTH-stimulated steroid secretion.
Main Results:
- CdCl2 significantly reduced basal and 25-hydroxycholesterol-induced 20DHP secretion.
- CdCl2 did not inhibit the conversion of 20-hydroxycholesterol, 22(R)-hydroxycholesterol, pregnenolone, or progesterone to 20DHP.
- Dibutyryl cyclic AMP-stimulated 20DHP secretion was not significantly affected by CdCl2.
Conclusions:
- CdCl2 specifically inhibits the utilization of endogenous mitochondrial cholesterol and 25-hydroxycholesterol in Y-1 cells.
- The cAMP-regulated mitochondrial steps in steroidogenesis are not the primary target of CdCl2 inhibition under these conditions.
- CdCl2 toxicity may impact basal steroidogenic functions in various tissues, and 25-hydroxycholesterol is a useful tool for studying unstimulated steroid synthesis.