Raymond Derk

Estimating the effective density of engineered nanomaterials for in vitro dosimetry

The need for accurate in vitro dosimetry remains a major obstacle to the development of cost-effective toxicological screening methods for engineered nanomaterials. An important key to accurate in vitro dosimetry is the characterization of sedimentation and diffusion rates of nanoparticles suspended in culture media, which largely depend upon the effective density and diameter of formed agglomerates in suspension. Here we present a rapid and inexpensive method for accurately measuring the effective density of nano-agglomerates in suspension. This novel method is based on the volume of the pellet obtained by bench-top centrifugation of nanomaterial suspensions in a packed cell volume tube, and is validated against gold-standard analytical ultracentrifugation data. This simple and cost-effective method allows nanotoxicologists to correctly model nanoparticle transport, and thus attain accurate dosimetry in cell culture systems, which will greatly advance the development of reliable and efficient methods for toxicological testing and investigation of nano-bio interactions in vitro.

Effect of Fiber Length on Carbon Nanotube-Induced Fibrogenesis

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-β) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-β release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-β activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

Tracking translocation of industrially relevant engineered nanomaterials (ENMs) across alveolar epithelial monolayers in vitro

Abstract Relatively little is known about the fate of industrially relevant engineered nanomaterials (ENMs) in the lungs that can be used to convert administered doses to delivered doses. Inhalation exposure and subsequent translocation of ENMs across the epithelial lining layer of the lung might contribute to clearance, toxic effects or both. To allow precise quantitation of translocation across lung epithelial cells, we developed a method for tracking industrially relevant metal oxide ENMs in vitro using neutron activation. The versatility and sensitivity of the proposed in vitro epithelial translocation (INVET) system was demonstrated using a variety of industry relevant ENMs including CeO2 of various primary particle diameter, ZnO, and SiO2-coated CeO2 and ZnO particles. ENMs were neutron activated, forming gamma emitting isotopes 141Ce and 65Zn, respectively. Calu-3 lung epithelial cells cultured to confluency on transwell inserts were exposed to neutron-activated ENM dispersions at sub-lethal doses to investigate the link between ENM properties and translocation potential. The effects of ENM exposure on monolayer integrity was monitored by various methods. ENM translocation across the cellular monolayer was assessed by gamma spectrometry following 2, 4 and 24 h of exposure. Our results demonstrate that ENMs translocated in small amounts (e.g. <0.01% of the delivered dose at 24 h), predominantly via transcellular pathways without compromising monolayer integrity or disrupting tight junctions. It was also demonstrated that the delivery of particles in suspension to cells in culture is proportional to translocation, emphasizing the importance of accurate dosimetry when comparing ENM–cellular interactions for large panels of materials. The reported INVET system for tracking industrially relevant ENMs while accounting for dosimetry can be a valuable tool for investigating nano–bio interactions in the future.

Differential effects of octylphenol, 17β-estradiol, endosulfan, or bisphenol A on the steroidogenic competence of cultured adult rat Leydig cells

In the current studies, we evaluated the effects of 4-tert-octylphenol (OP), endosulfan, bisphenol A (BPA), and 17beta-estradiol on basal or hCG-stimulated testosterone formation by cultured Leydig cells from young adult male rats. Exposure of Leydig cells to increasing concentrations of OP (1 to 2000 nM), 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM) or BPA (1 to 1000 nM), alone or with 10 mIU/mL hCG for 4 or 24 h, did not lower ambient testosterone levels, although cells exposed to higher OP concentrations + hCG for 24 h often had modest declines in testosterone (10 to 20%). Of interest, exposure to the highest concentration OP (2000 nM) alone for 4 or 24 h increased testosterone levels (approximately 2-fold in 4-h exposed cells). Whether prior exposure to OP + hCG for 24 h affects the subsequent conversion of steroid substrates to testosterone over 4 h was evaluated. Progressive declines in 1 microM 22(R) hydroxycholesterol, 1 microM pregnenolone, or 1 microM progesterone conversion to testosterone was observed beginning at 100 to 500 nM OP exposure (maximal declines of 40 to 12% of controls were observed); however, the conversion of 1 microM androstenedione to testosterone was not affected by OP. These results suggested that 24-h exposure to OP + hCG has no effect on 17beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, but that it inhibits the 17alpha-hydroxylase/C17-20 lyase step, which converts progesterone to androstenedione. In addition, potentially, OP could inhibit cholesterol side/chain cleavage activity, which converts cholesterol to pregnenolone, and/or 3beta-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone. Of interest, exposure to increasing concentrations of 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM), or BPA (1 to 1000 nM) + hCG for 24 h had no effect on subsequent conversion of 22(R)hydroxycholesterol to testosterone. Furthermore, the inhibiting effects of OP + hCG exposure on subsequent conversion of progesterone to testosterone was unaffected by concomitant exposure to the pure estrogen antagonist, ICI 182,780, or the antioxidants, ascorbate or dimethyl sulfoxide, suggesting that the actions of OP are not mediated through binding to estrogen receptor alpha or beta or by free radical induced damage to steroidogenic enzymes, respectively. These results demonstrate that direct exposure of adult Leydig cells to OP may have subtle effects on their ability to produce testosterone, which may not be detected by measuring ambient androgen levels. In addition, the effects of OP on Leydig cell testosterone formation appear to be different from those of the native estrogen, 17beta-estradiol, and from other reported weak xenoestrogens such as endosulfan and BPA.

Neoplastic-like transformation effect of single-walled and multi-walled carbon nanotubes compared to asbestos on human lung small airway epithelial cells

Abstract Accumulating evidence indicates that carbon nanotubes (CNTs) are biopersistent and can cause lung damage. With similar fibrous morphology and mode of exposure to asbestos, a known human carcinogen, growing concern has arisen for elevated risk of CNT-induced lung carcinogenesis; however, relatively little is known about the long-term carcinogenic effect of CNT. Neoplastic transformation is a key early event leading to carcinogenesis. We studied the ability of single- and multi-walled CNTs to induce neoplastic transformation of human lung epithelial cells compared to asbestos. Long-term (6-month) exposure of the cells to occupationally relevant concentrations of CNT in culture caused a neoplastic-like transformation phenotype as demonstrated by increased cell proliferation, anchorage-independent growth, invasion and angiogenesis. Whole-genome expression signature and protein expression analyses showed that single- and multi-walled CNTs shared similar signaling signatures which were distinct from asbestos. These results provide novel toxicogenomic information and suggest distinct particle-associated mechanisms of neoplasia promotion induced by CNTs and asbestos.

Biphasic effects of octylphenol on testosterone biosynthesis by cultured Leydig cells from neonatal rats

The present studies evaluated the suitability of using cultured dispersed testicular cells from neonatal rats as a source for fetal Leydig cells and the use of these cells to examine direct toxic effects of environmental/occupational chemicals on androgen biosynthesis. For the current studies, the direct actions of octylphenol (OP), a surfactant additive widely used in the manufacture of various detergents, on testosterone biosynthesis by cultured rat neonatal Leydig cells were examined. Octylphenol is considered a xenoestrogen and has been reported to mimic the actions of estrogen in many cellular systems. Following exposure of cultured cells for 24 h to varying concentrations of OP (1 to 2000 nM) together with 10 mlU/mL human chorionic gonadotropin (hCG), the lower concentrations of OP (1 and 10 nM) consistently enhanced testosterone levels (approximately 10 to 70% above control), whereas higher OP concentrations (100 to 2000 nM) progressively decreased testosterone from peak levels to approximately 40 to 80% below control at the highest OP concentration. Interestingly, increasing concentrations of 17beta-estradiol (1 to 1000 nM) were without effect on testosterone biosynthesis under the same conditions, and the biphasic pattern of testosterone biosynthesis elicited by increasing OP concentrations was unaffected by concomitant treatment with 10 or 100 nM ICI 182,780, which is considered a pure estrogen antagonist. Therefore, the actions of OP on testosterone biosynthesis by cultured neonatal Leydig cells do not appear to be mediated through the classic estrogen receptor alpha or beta pathway. Although the increase in testosterone levels after exposure to lower OP concentrations and to 0.1 and 1.0 mM 8-Br-cAMP was attenuated, suggesting that lower OP concentrations may alter cellular cAMP levels, because hCG-stimulated cAMP levels were unaffected by any of the OP concentrations evaluated, it appears that its main site(s) of action occurs after the generation of cAMP. In addition, because pretreatment of cells with increasing OP concentrations and hCG had no effect on the conversion of steroid precursors (22(R)-hydroxycholesterol, pregnenolone, progesterone, or androstenedione) to testosterone, it seems that the main actions of OP under the present conditions occur before the mitochondrial cholesterol side-chain cleavage step. Furthermore, because concomitant treatment of cells with various antioxidants (alpha-tocopherol, butylated hydroxyanisole, or ascorbic acid) did not alter the biphasic pattern of testosterone response to increasing concentrations of OP and hCG, it seems that OP is not acting as an anti- or pro-oxidant in producing these effects. It will be important to determine whether this dose-sensitive response to OP is observed in vivo, and whether the maturational status of Leydig cells influences their pattern of response to OP and similar chemicals.

Octylphenol inhibits testosterone biosynthesis by cultured precursor and immature Leydig cells from rat testes.

4-tert-octyphenol (OP) is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to mimic the actions of estrogen in many cellular systems. The present studies evaluated the direct effects of OP on human chorionic gonadotropin (hCG)-stimulated testosterone biosynthesis by cultured precursor and immature Leydig cells from 23-day old (prepubertal) rats. Exposure to increasing OP concentrations (1 to 2000 nM) progressively decreased hCG-stimulated testosterone formation in both precursor and immature Leydig cells at higher OP concentrations (100 or 500 to 2000 nM). Testosterone levels were reduced approximately 30 to 70% below control at the highest concentration in both cell types. Similar reductions in testosterone associated with OP exposure were observed in cells stimulated with 1 mM 8-Br-cAMP, suggesting that the main actions of OP occur after the generation of cAMP. Increasing concentrations of 17beta-estradiol (1 to 1000 nM) had no effect on hCG-stimulated testosterone formation in both precursor and immature Leydig cells and the inclusion of 100 nM ICI 182,780, a pure estrogen antagonist, in precursor and immature Leydig cells exposed to OP and hCG, did not alter the inhibition by higher OP concentrations of testosterone formation in both cell types. These results suggest that OP is a hormonally active agent, but that some of its actions are distinct from those of 17beta-estradiol and are not mediated through the estrogen receptor alpha or beta pathway. To further localize the potential site(s) of action of OP, cultured precursor and immature Leydig cells were exposed to increasing concentrations of OP and hCG for 24 h. Next, fresh media containing 1 microM 22(R)-hydroxycholesterol, 1 microM pregnenolone, 1 microM progesterone, or 1 microM androstenedione was added, and the conversion of each substrate to testosterone was determined after incubation for 4 h. The conversion of androstenedione to testosterone was unaffected by exposure to OP, suggesting that the 17beta-hydroxysteroid dehydrogenase step is not inhibited. However, the conversion of 22(R)-hydroxycholesterol, pregnenolone and progesterone all were inhibited by prior exposure to OP and hCG. This finding suggests that the 17alpha-hydroxylase/c17-20-lyase step, which converts progesterone to androstenedione, is inhibited by OP, and that the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase -isomerase steps, which convert cholesterol to pregnenolone and pregnenolone to progesterone, respectively, are other potential sites of OP action. Because concomitant exposure to the antioxidants alpha-tocopherol or ascorbate did not alter the inhibition of testosterone formation by higher OP concentrations, it does not appear that OP is acting as a pseudosubstrate for the generation of free radicals, which can damage P450 enzymes.

Identification of TGF-β receptor-1 as a key regulator of carbon nanotube-induced fibrogenesis

Carbon nanotubes (CNTs) induce rapid interstitial lung fibrosis, but the underlying mechanisms are unclear. Previous studies indicated that the ability of CNTs to penetrate lung epithelium, enter interstitial tissue, and stimulate fibroblasts to produce collagen matrix is important to lung fibrosis. In this study, we investigated the activation of transforming growth factor-β receptor-1 [TGF-β R1; i.e., activin receptor-like kinase 5 (ALK5) receptor] and TGF-β/Smad signaling pathway in CNT-induced collagen production in human lung fibroblasts. Human lung fibroblasts and epithelial cells were exposed to low, physiologically relevant concentrations (0.02-0.6 μg/cm(2)) of single-walled CNTs (SWCNT) and multiwalled CNTs (MWCNT) in culture and analyzed for collagen, TGF-β1, TGF-β R1, and SMAD proteins by Western blotting and immunofluorescence. Chemical inhibition of ALK5 and short-hairpin (sh) RNA targeting of TGF-β R1 and Smad2 were used to probe the fibrogenic mechanism of CNTs. Both SWCNT and MWCNT induced an overexpression of TGF-β1, TGF-β R1 and Smad2/3 proteins in lung fibroblasts compared with vehicle or ultrafine carbon black-exposed controls. SWCNT- and MWCNT-induced collagen production was blocked by ALK5 inhibitor or shRNA knockdown of TGF-β R1 and Smad2. Our results indicate the critical role of TGF-β R1/Smad2/3 signaling in CNT-induced fibrogenesis by upregulating collagen production in lung fibroblasts. This novel finding may aid in the design of mechanism-based risk assessment and development of rapid screening tests for nanomaterial fibrogenicity.

Exposure to octylphenol increases basal testosterone formation by cultured adult rat Leydig cells

4-Tert-octylphenol (OP) is a breakdown product of 4-tert-octylphenol ethoxylate, which is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to exhibit weak estrogenic activity in many assay systems. The studies described herein examined an unusual effect of OP in increasing constitutive testosterone levels of cultured Leydig cells from young adult rats. The increase in testosterone was both dose and time sensitive, and this response was observed in medium lacking both calcium and magnesium and containing a membrane-permeable calcium chelator, suggesting that the increase in testosterone was not mediated by an increase in the permeability of extracellular calcium into cells or the redistribution/release of calcium from intracellular stores, respectively. Cellular cAMP levels also were unaffected by OP alone in cultured Leydig cells. Furthermore, initial exposure to 2000nM OP alone for 4h did not alter the subsequent conversion of endogenous cholesterol or exogenously added 22 (R)hydroxycholesterol to testosterone, suggesting that the increase in testosterone was not due to the enhanced availability of endogenous cholesterol or an increase in cholesterol side-chain cleavage activity, respectively. The increase in testosterone also was observed in the presence of the pure estrogen antagonist, ICI 182,780, or a 5alpha-reductase inhibitor, suggesting that this effect of OP was not mediated through the estrogen receptor alpha or beta pathway or by inhibition of Leydig cell testosterone metabolism, respectively. In addition, exposure of cells to comparable concentrations of two different detergents, Triton X-100 or sodium cholate, did not increase testosterone levels, suggesting that this effect of OP was not due to its potential detergent qualities. Although these studies did not identify specific mechanism(s) that increase constitutive testosterone levels by OP, they identify specific pathways that appear not to be involved. The physiological relevance of this observation is not known; nevertheless, they illustrate potential diverse actions of OP in modulating the level of androgen secreted by Leydig cells, and they emphasize that some actions of OP do not appear to be mediated through the estrogen receptor alpha or beta pathway.

The methoxychlor metabolite, HPTE, inhibits rat luteal cell progesterone production

UNLABELLED The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum. RESULTS Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to <22% of control at 500 nM HPTE. Similarly, HPTE progressively inhibited progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors.