Rapid and structure-specific cellular uptake of selected steroids

Abstract

Steroid hormones and their respective nuclear receptors are essential mediators in numerous physiologic and pathophysiologic processes, ranging from regulation of metabolism, immune function, and reproductive processes to the development of hormone-dependent cancers such as those of the breast and prostate. Because steroids must enter cells before activating nuclear receptors, understanding the mechanisms by which cellular uptake occurs is critical, yet a clear understanding of these mechanisms has been elusive. It is generally assumed that diffusion-driven uptake is similar across various steroids whereas an elevated cellular concentration is thought to reflect active uptake, but these assumptions have not been directly tested. Here we show that intact cells rapidly accumulate free steroids to markedly elevated concentrations. This effect varies widely depending on steroid structure; more lipophilic steroids reach more elevated concentrations. Strong preferences exist for 3β-OH, Δ5-steroids vs. 3-keto, Δ4-structural features and for progestogens vs. androgens. Surprisingly, steroid-structure-specific preferences do not require cell viability, implying a passive mechanism, and occur across cells derived from multiple tissue types. Physiologic relevance is suggested by structure-specific preferences in human prostate tissue compared with serum. On the other hand, the presence of serum proteins in vitro blocks much, but not all, of the passive accumulation, while still permitting a substantial amount of active accumulation for certain steroids. Our findings suggest that both passive and active uptake mechanisms make important contributions to the cellular steroid uptake process. The role of passive, lipophilicity-driven accumulation has previously been largely unappreciated, and its existence provides important context to studies on steroid transport and action both in vitro and in vivo.

Toward Realistic Dosimetry In Vitro: Determining Effective Concentrations of Test Substances in Cell Culture and Their Prediction by an In Silico Mass Balance Model

“…Several methods have been described to prepare samples for the measurement of cell-associated chemical concentrations. These include using detergents, , freezing and thawing cycles, ultrasonication, , and liquid homogenization . One of the consequences of each method is the resulting sample may or may not contain plasma membranes together with chemicals that may have bound to the outside of the cells.…”

“…10,67 The free C Medium values could be expected to be linked to a lower cellular uptake of chemicals, as binding the proteins in the medium may prevent this. McManus et al 41 reported that the use of serum-free medium resulted in higher cellular concentrations in prostate cancer cells than the serumcontaining medium. However, our results do not support this hypothesis since chemicals with high C Cell /C Medium ratios were moderately or highly bound ( f u < 35%).…”

“…Cell lysate samples underwent three thaw and freeze cycles to destroy the cellular membrane and release the cytosolic fraction from the intercellular space. 41,42 Determination of the Unbound Fraction of Test Chemicals in the Culture Medium. RED was performed as described by the manufacturer 43 to determine the fraction unbound (f u ) in culture medium.…”

“…Culture medium and cell lysate samples were stored at −20 °C until analysis. Cell lysate samples underwent three thaw and freeze cycles to destroy the cellular membrane and release the cytosolic fraction from the intercellular space. , …”

“…Cell lysate samples underwent three thaw and freeze cycles to destroy the cellular membrane and release the cytosolic fraction from the intercellular space. 41 , 42 …”