Báo cáo khoa học: Nanoparticles can induce changes in the intracellular metabolism of lipids without compromising cellular viability docx

We provide evidence that both uncoated and ZnS-coated quantum dots can induce the accumulation of lipids increase in cytoplasmic lipid droplet formation in two cell culture models: glial

metabolism of lipids without compromising cellular

viability

Ewa Przybytkowski, Maik Behrendt, David Dubois and Dusica Maysinger

Department of Pharmacology and Therapeutics, McGill University, Montre´al, Canada

Introduction

Quantum dots (QDs) are colloidal semiconductor

nanoparticles (NPs) with unique luminescence

charac-teristics and wide biological and industrial applications

[1,2] They could become attractive tools for imaging

in basic research and, eventually, in medicine [3] How-ever, some QDs can be harmful to cells, particularly if

Keywords

fat oxidation; hypoxia; lipid droplets;

nanoparticles; quantum dots

Correspondence

D Maysinger, Department of Pharmacology

and Therapeutics, McGill University, 3655

Promenade Sir-William-Osler, Montre´al, QC,

Canada, H3G 1Y6

Fax: (514) 398 6690

Tel: (514) 398 1264

E-mail: dusica.maysinger@mcgill.ca

(Received 5 June 2009, revised 17 August

2009, accepted 24 August 2009)

doi:10.1111/j.1742-4658.2009.07324.x

There is growing concern about the safety of engineered nanoparticles, which are produced for various industrial applications Quantum dots are colloidal semiconductor nanoparticles that have unique luminescence char-acteristics and the potential to become attractive tools for medical imaging However, some of these particles can cause oxidative stress and induce cell death The objective of this study was to explore quantum dot-induced metabolic changes, which could occur without any apparent cellular dam-age We provide evidence that both uncoated and ZnS-coated quantum dots can induce the accumulation of lipids (increase in cytoplasmic lipid droplet formation) in two cell culture models: glial cells in primary mouse hypothalamic cultures and rat pheochromocytoma PC12 cells Glial cells treated with CdTe quantum dots accumulated newly synthesized lipids in a phosphoinositide 3-kinase-dependent manner, which was consistent with the growth factor-dependent accumulation of lipids in PC12 cells treated with CdTe and CdSe⁄ ZnS quantum dots In PC12 cells, quantum dots, as well as the hypoxia mimetic CoCl2, induced the up-regulation of hypoxia-inducible transcription factor-1a and the down-regulation of the b-oxida-tion of fatty acids, both of which could contribute to the accumulab-oxida-tion of lipids On the basis of our results, we propose a model illustrating how nanoparticles, such as quantum dots, could trigger the formation of intra-cellular lipid droplets, and we suggest that metabolic measurements, such

as the determination of fat oxidation in tissues, which are known sites of nanoparticle accumulation, could provide useful measures of nanoparticle safety Such assays would expand the current platform of tests for the determination of the biocompatibility of nanomaterials

Abbreviations

DIV 8, day (in vitro) 8; FAS, fatty acid synthase; FFA, free fatty acid; HIF-1a, hypoxia-inducible factor-1a; HIFs, hypoxia-inducible transcription factors; LD, lipid droplet; NP, nanoparticle; PEG, polyethylene glycol; PI3K, phosphoinositide 3-kinase; PSN, penicillin ⁄ streptomycin ⁄ neomycin; QD, quantum dot; ROS, reactive oxygen species; SCD-1, stearoyl-coenzyme A desaturase-1; SREBP-1, sterol regulatory element binding protein-1.

their surface is not fully protected or if they degrade

within the biological environment We have studied

the effects of QDs on living cells and have reported

their internalization, the intracellular production of

reactive oxygen species (ROS) and the damage to

mul-tiple cellular sites induced by various QDs [4–6] We,

as well as others, have shown that the degree of

inter-nalization of QDs and various other NPs is dependent

on their size, surface charge, concentration in the

med-ium and duration of exposure [4,7–11] We have also

shown the release of free cadmium from QDs

contain-ing a CdTe core [6] However, this could not explain

fully their harmful effects We have postulated that

intracellular ROS formation or interactions with

cellu-lar structures (mitochondria in particucellu-lar) could

con-tribute to the observed cytotoxicity [4–7] Although

coating of NPs with ZnS or polyethylene glycol (PEG)

commonly prevents some undesirable effects [6,12], the

long-term stability of these materials in the biological

milieu is not well understood [13]

The purpose of this study was to investigate the

more subtle effects of QDs which could occur without

any evident morphological cellular damage or cell

death In particular, we explored QD-induced changes

in lipid metabolism Using well-characterized in vitro

cell model systems [primary mouse hypothalamic

cultures and pheochromocytoma cells (PC12)], we have

provided evidence that poorly fluorescent CdTe NPs,

without ZnS capping but with cysteamine coating, as

well as highly fluorescent CdSe⁄ ZnS NPs, capped with

ZnS and coated with cysteamine on the surface, can

induce the accumulation of lipids in cytoplasmic lipid

droplets (LDs)

LDs are macromolecular lipid assemblies consisting

of neutral lipids, such as triacylglycerols,

diacylglyce-rols, cholesterol esters and cholesterol, surrounded by

a monolayer of phospholipids [14] Many cell types

are able to store excess fat as cytoplasmic LDs

How-ever, under physiological conditions, LDs are found

mostly in tissues involved directly in energy

meta-bolism, such as adipocytes, liver and muscles [14]

LDs are much more than simply blobs of fat

segre-gated from the hydrophilic milieu of the cytoplasm

They are organelles with a particular structure and

organization [15] They are formed when free

(uneste-rified) fatty acids (FFAs) from exogenous or

endoge-nous sources are available inside the cells Such FFAs

are either esterified to form complex lipids, which are

then stored in droplets or become part of the cellular

membrane, or are oxidized in mitochondria for

energy production The formation and maintenance

of LDs are complex, dynamic and highly regulated

processes [15,16]

The formation of LDs induced by NPs could be par-tially explained by a reduction in fat oxidation, which occurs in parallel with an increase in LD number As the measurement of fat oxidation is relatively simple to perform and gives clear objective values, it has the potential for broad application in the assessment of the metabolic effect described in this study Given that high levels of cytoplasmic LDs present in nonadipose tissues are considered to be harmful, such assays would expand the current platform of tests for the determination of nanomaterial biocompatibility Excess fat in nonadipose cells may be involved in several human pathologies, such as fatty liver, obesity, athero-sclerosis and type 2 diabetes, and may contribute to the development of insulin resistance and lipotoxic tis-sue damage [17] The accumulation of neutral lipids in cytoplasmic LDs occurs following exposure to mito-chondrial toxins [18], during chronic viral infections [19,20], in response to protease inhibitors [21] and during hypoxia [22–24]

In this study, we also showed that exposure to colloidal semiconductor NPs leads to an increased expression of hypoxia-inducible transcription factor-1a (HIF-factor-1a) The family of hypoxia-inducible tran-scription factors (HIFs) regulates the adaptation to hypoxic conditions, which is critical for cell survival during decreased availability of oxygen in tissues [25,26] Hypoxia and hypoxia-related signaling have been associated with major pathologies, such as car-diovascular disease, stroke and cancer [27] The sig-naling for hypoxia was of interest in this study because QDs, which are redox-active NPs, can release

Cd2+ and induce the intracellular formation of ROS, and, as such, make good candidates for hypoxia mimetics In addition, signaling induced by hypoxia promotes alterations in cellular metabolism HIFs bind to DNA, forming heterodimers composed of one oxygen-regulated a subunit (HIF-1a, HIF-2a and HIF-3a) and one stable b subunit [28] In normoxia, the oxygen-dependent hydroxylation of critical proline within the a subunit promotes its degradation by the ubiquitin–proteosome system At low oxygen concen-trations, HIF-a subunits become more stable and thus can participate in the transcription of target genes, initiating the hypoxic response [25,26,28] It is well accepted that HIF-a subunits can also be stabi-lized at normoxia by nonhypoxic stimuli, such as ROS, divalent metals and some mitochondrial meta-bolites [29,30] In this study, we used CoCl2 as a con-trol for the induction of signaling for hypoxia [31,32] QDs were much poorer inducers of HIF-1a than was CoCl2 In addition, the induction of HIF-1a by QDs was not correlated with the accumulation of lipids,

thus suggesting that the two phenomena are most

probably independent

In the present study, we used hypothalamic glial

cells as a model system because the hypothalamus is a

brain structure that is not completely protected by the

blood–brain barrier and thus can be accessed by

xeno-biotics and NPs [33] Various other tissues, such as

liver, kidney, spleen and bone marrow, are known sites

of NP accumulation in vivo [34,35] The evaluation of

fat oxidation in these tissues could complement current

toxicological assays for the safety screening of NPs

and other nanomaterials

Results

Short- and long-term effects of uncoated CdTe

QDs on mouse primary hypothalamic cultures

The effects of green, positively charged CdTe QDs with

cysteamine surfaces [4,6,7] were investigated in primary

mouse hypothalamic cultures Mixed neural cultures

were obtained from 5-day-old animals, and experiments

were initiated at day (in vitro) 8 (DIV 8), when neural

cells were fully differentiated (Fig 1A) A few neurons with small cell bodies were visible on top of supporting glia In this study, we focused on glial cells

To examine the short-term effects, the cultures were exposed to QDs (0–20 lgÆmL)1) for a period of 24 h

in serum-free Neurobasal A medium with supplements Cells responded to QD treatment by forming multiple cytoplasmic LDs (Fig 1B–D) The number of LDs increased with increasing concentration of QDs (Fig 1E) Within this time period, cells exposed to relatively low concentrations of QDs (5 lgÆmL)1) were viable, and only the highest concentration (20 lgÆmL)1) induced cell detachment and cell death (data not shown) To examine the effects of long-term exposure, primary mouse hypothalamic cultures were exposed to 5 lgÆmL)1of QDs for 4 days The cultures were viable and contained multiple cytoplasmic LDs (Fig 1G) The number of LDs in control, untreated glial cells increased gradually with aging of the cultures However, cells treated with QDs for 4 days contained more and much larger droplets than those found in the corresponding controls (Fig 1F versus Fig 1G)

E

Fig 1 CdTe QDs trigger the formation of lipid droplets in glial cells from primary mouse hypothalamic cultures (A–D, F, G) Representative photomicrographs of primary mouse hypothalamic cultures (A) Phase contrast photomicrograph taken at DIV 9 (B–D) Photomicrographs of cultures stained with Oil Red O to visualize neutral lipids at DIV 9 (B) Control untreated cultures (C, D) Cultures treated for 24 h (DIV 8 to DIV 9) with 5 lgÆmL)1(C) and 10 lgÆmL)1 (D) of CdTe QDs (E) The mean number of lipid droplets per microscopic field evaluated as described in Materials and methods The data represent the mean and SEM from two independent experiments (n = 10) (F, G) Photomicro-graphs of cultures stained with Oil Red O at DIV 12 (F) Control untreated cultures (G) Cultures treated with 5 lgÆmL)1of CdTe QDs for

4 days (DIV 8 to DIV 12) The scale bars correspond to 50 lm.

LD formation triggered by CdTe QDs in glial cells

depends on the de novo synthesis of lipids and

phosphoinositide 3-kinase (PI3K) signaling

Cytoplasmic LDs contain neutral lipids, including

triacylglycerols, diacylglycerols and cholesterol esters

To verify whether de novo fat synthesis is involved in

LD formation during treatment with QDs,

hypotha-lamic cultures were exposed to CdTe QDs (5 lgÆmL)1)

in the presence of the fatty acid synthase (FAS)

inhibi-tor cerulenin (5 lgÆmL)1) FAS is responsible for the

synthesis of FFAs, which can then be esterified to

form components of cell membranes or lipids stored in

LDs Cerulenin, an antifungal antibiotic isolated from

Cephalosporium caerulens, irreversibly binds to FAS,

thereby inhibiting its activity [36] Treatment with

5 lgÆmL)1 of cerulenin for 24 h had little effect on

glial cell viability and resulted in the disappearance

of LDs (Fig 2), suggesting that these cells carried

out lipogenesis (de novo synthesis of lipids) in control cultures Interestingly, cerulenin also inhibited LD formation in cultures treated with 5 lgÆmL)1 CdTe QDs for 24 h (Fig 2C–E) These results suggest that increased LD formation triggered by low dose (5 lgÆmL)1) CdTe QDs in glial cells involves de novo lipid synthesis

The PI3K⁄ Akt signaling pathway is best known for its role in the maintenance of cell survival, but is also responsible for the up-regulation of glucose metabo-lism and the induction of lipogenesis [37–40] We hypothesized that signaling via the PI3K⁄ Akt pathway was involved in the promotion of the formation of LDs in cells treated with QDs To test this hypothesis, primary mouse hypothalamic cultures were exposed to the PI3K inhibitor LY294002 (50 lm), alone or in combination with CdTe QDs (5 lgÆmL)1) Treatment with LY294002 completely inhibited LD formation in glial cells under control conditions (Fig 3A, B, E), and

E

Fig 2 Formation of LDs induced by QDs in

glial cells from primary mouse hypothalamic

cultures depends on the de novo synthesis

of lipids (A–D) Representative

photomicro-graphs of primary mouse hypothalamic

cultures stained with Oil Red O (A) Control

cultures at DIV 9 Cultures treated for 24 h

with 5 lgÆmL)1of cerulenin (B), 5 lgÆmL)1

of CdTe QDs (C) and both 5 lgÆmL)1of

CdTe QDs and 5 lgÆmL)1of cerulenin (D).

The scale bars correspond to 50 lm (E) The

quantification of the lipid droplet number

from (A) to (D) The data represent the

mean and SD from three independent fields.

***P < 0.001.

inhibited the formation of LDs in QD-treated cells

(Fig 3C–E), indicating that the formation of LDs was

dependent on signaling via PI3K Glial cell survival

was not markedly compromised by the inhibition of

PI3K signaling with LY294002 in a fully supplemented

medium and within the tested time period (Fig 3B, D)

QDs induce the formation of LDs in PC12 cells

in a growth factor-dependent manner without

compromising cellular viability

Rat pheochromocytoma PC12 cells are commonly used

as a model cell line to study trophic factor signaling,

cell death by trophic factor deprivation [41–43] and

effects of NPs [4,6] The cells were treated with two

different types of QD in two different culture

condi-tions: in full medium and in serum-free medium

(buffered with 10 mm Hepes) We used CdTe QDs,

which contain a CdTe core and have no protective shell on the surface, as well as CdSe⁄ ZnS QDs (i.e CdSe core and ZnS shell) [44] The latter are much less harmful to cells [6,12] Indeed, CdSe⁄ ZnS QDs were not toxic to PC12 cells during 24 h of exposure

in the presence or absence of serum, but unprotected CdTe QDs were nontoxic only in the presence of serum (Fig 4A, B) Serum albumin can reduce QD entry into the nucleus [7], and serum trophic factors can support cell survival by signaling via the PI3K⁄ Akt pathway [38,45] PC12 cells treated in the presence of serum with both CdTe and CdSe⁄ ZnS QDs contained more LDs than did untreated con-trols (Fig 4C–E, K) The effect was dose dependent and was much more pronounced with uncoated CdTe QDs (Fig 4K) The activation of PI3K⁄ Akt was necessary for LD formation in PC12 cells (Fig 4F–H)

E

Fig 3 Formation of LDs induced by CdTe QDs in glial cells from primary mouse hypo-thalamic cultures depends on the PI3K signaling pathway (A–D) Representative photomicrographs of primary mouse hypo-thalamic cultures stained with Oil Red O (A) Control cultures at DIV 11 (B) Cultures treated with 50 l M LY294002 for 2 days (DIV 9 to DIV 11) (C) Cultures treated with

5 lgÆmL)1of CdTe QDs for 3 days (DIV 8 to DIV 11) (D) Cultures treated with 5 lgÆmL)1

of CdTe QDs and 50 l M LY294002 for

3 days (DIV 8 to DIV 11) The scale bars correspond to 50 lm (E) The quantification

of the lipid droplet number from (A) to (D) The data represent the mean and SD from three independent fields **P < 0.01.

As QDs are redox-active NPs (effective electron

donors and acceptors) which can release Cd2+and are

able to induce intracellular formation of ROS in PC12

and MCF 7 cells [4,6], they make good candidates for

hypoxia mimetics [27] Thus, we used the known

hypoxia mimetic, CoCl2, and tested whether it also

induced the formation of LDs in PC12 cells in a

growth factor-dependent manner PC12 cells treated with 100 lm CoCl2for 24 h contained more and larger LDs than the control (Fig 4I versus Fig 4C, K) Cells treated with CoCl2 in the absence of serum did not contain LDs, suggesting that the activation of PI3K⁄ Akt by trophic factors is also necessary for LD formation induced by this hypoxia mimetic (Fig 4J)

C

D

G

Fig 4 Uncoated and coated QDs trigger the formation of LDs in PC12 cells in a growth factor-dependent manner without compromising cellular viability Pheochromocytoma PC12 cells were treated with QDs in fully supplemented medium (A, C–E, I and K) and in serum-free medium (B, F–H, J) (A, B) Percentage cell survival relative to control (determined with Alamar blue assay) after exposure to two different concentrations of QDs in fully supplemented medium (A) and in serum-free medium (B) Data represent the mean and SEM from two inde-pendent experiments Representative photomicrographs of cells stained with Oil Red O (C, F) Control untreated cells (D, G) Cells treated with 20 lgÆmL)1of CdTe QDs for 24 h Cells treated with 20 lgÆmL)1of CdSe ⁄ ZnS QDs (E, H) and with 100 l M CoCl 2 (I, J) for 24 h The scale bars correspond to 50 lm ***P < 0.001; *P < 0.05 (K) Number of lipid droplets found in PC12 cells treated with two different con-centrations of QDs in fully supplemented medium The data represent the mean and SEM from two independent experiments.

QDs can induce signaling implicated in the

response to hypoxia and can reduce the rate of

fat oxidation in PC12 cells

We hypothesized that QDs and CoCl2induce the

accu-mulation of lipids in cytoplasmic LDs by the

activa-tion of HIF-1a Transcripactiva-tion factors involved in

signaling for hypoxia are known to stimulate glucose

metabolism [26] and to promote the accumulation of

lipids [23,24] PC12 cells were incubated with QDs or

CoCl2 for 24 h, and the expression of HIF-1a protein

was analyzed by western blotting QDs caused the

up-regulation of HIF-1a, but only when added at

higher concentration (20 lgÆmL)1) and to serum-free

medium (Fig 5A, B) Thus, it is unlikely that HIF-1a

is involved directly in the accumulation of lipids

trig-gered by QDs under the conditions of uncompromised

cell survival in the presence of serum

On the basis of the results obtained in this study

showing that glial cells treated with QDs accumulate

newly synthesized lipids, we further hypothesized that

NP-induced lipid accumulation could be a result of the

down-regulation of b-oxidation of FFAs Saturated

fatty acid palmitate (C16:0), synthesized de novo, is

elongated, desaturated and esterified to form other

FFAs and eventually more complex lipids (including

triacylglycerols stored in LDs), or can be transported

to mitochondria where it is oxidized The

down-regula-tion of the b-oxidadown-regula-tion of palmitate in mitochondria

would provide more FFAs available for esterification

and storage in LDs PC12 cells were treated with QDs for 24 h and the rate of fat oxidation was measured The oxidation of exogenous palmitate was decreased

in cells treated with QDs or CoCl2in a dose-dependent manner in both the presence and absence of serum (Fig 6A, B) In the presence of serum, fat oxidation decreased by 25–40% after treatment with CdTe QDs and by 20% after treatment with 20 lgÆmL)1 of CdSe⁄ ZnS QDs In the absence of serum, the effect of QDs was even more pronounced, as fat oxidation decreased by 40–50% after treatment with CdTe QDs and by 19–36% after treatment with CdSe⁄ ZnS QDs (Fig 6A) These results strongly suggest that: (a) QDs can induce changes in cellular lipid metabolism with-out affecting cellular viability; and (b) QD-induced

A

B

Fig 5 Uncoated and coated QDs increase the expression of

HIF-1a in PC12 cells Pheochromocytoma PC12 cells were treated with

QDs for 24 h in fully supplemented medium (A) and in serum-free

medium (B) After treatment, HIF-1a protein levels were analyzed

by western blot.

A

B

Fig 6 Uncoated and coated QDs decrease the rate of b-oxidation

of fatty acids in PC12 cells Pheochromocytoma PC12 cells were treated with QDs for 24 h in fully supplemented medium (A) and in serum-free medium (B) After treatment, fatty acid oxidation was measured using [1-14C]palmitate as a substrate The data represent the mean and SEM from two independent experiments *P < 0.05;

**P < 0.01; ***P < 0.001.

accumulation of lipids in cytoplasmic LDs could be

explained, in part, by the down-regulation of fat

oxida-tion triggered by these NPs

Discussion

This study shows that exposure of cells to CdTe and

CdSe NPs can affect the intracellular metabolism of

lipids and induce HIF-1a-mediated signaling The

results suggest that QDs, together with trophic factors,

promote the accumulation of lipids in cytoplasmic

LDs, in part by down-regulating the oxidation of

de novo-synthesized fatty acids (Fig 7)

PC12 cells, as most cell types, require trophic factors

for survival and differentiation [41–43] When placed

into culture, they will not grow or survive for extended

periods of time without trophic factors in the cellular medium Serum, a mixture of proteins isolated from the blood (in this study, from the blood of bovine fetus), is a source of trophic factors The PI3K⁄ Akt signaling pathway is activated on the cytoplasmic side

of the plasma membrane when various trophic factors involved in the regulation of cell growth, survival and proliferation bind to their receptors on the cell surface [38,42,43,45,46] Stimulation of this signaling pathway also enhances metabolism, resulting in an increase in glucose uptake and an up-regulation of glycolysis [37,38] In addition, PI3K signaling has been shown to

be involved in the up-regulation of lipogenic enzymes, such as FAS, most probably via sterol regulatory element binding protein-1 (SREBP-1) transcription factor [39,40] Thus, in many cell types, signaling via PI3K⁄ Akt ensures the supply of substrate for lipid synthesis and enhances the activities of lipogenic enzymes, setting the stage for the accumulation of lipids Consistent with this, we have observed a small number of LDs in glial cells and in PC12 cells grown

in a fully supplemented medium (control conditions) Interestingly, when glial cells from primary mouse hypothalamic cultures were exposed to QDs, the num-ber and⁄ or size of LDs increased markedly LDs have been recognized recently as ubiquitous dynamic organ-elles, which communicate with other cellular compart-ments and participate in important functions, such as transport and communication between different vesi-cles and compartments inside the cell [47] Some of these functions are probably dependent on the pres-ence of specific proteins on the surface of droplets [48]

It has been shown previously that QDs can cause distortion and⁄ or damage to cellular membranes, which are composed mostly of complex lipids [49] This could result in the release of FFAs, which then would be available for esterification and the formation

of triacylglycerols (the main components of LDs) We considered such a possibility; however, the inhibition

of PI3K with LY294002 and the inhibition of FAS with cerulenin caused the disappearance of droplets and prevented the formation of new droplets during exposure to NPs when trophic factors were present in the medium These findings indicate that glial cells (from primary mouse hypothalamic cultures) accumu-lated newly synthesized lipids when exposed to NPs This was also true for PC12 cells treated with NPs, as they accumulated lipids mainly in the presence of serum Thus, our results suggest that the fatty acids necessary for LD formation during exposure to NPs are synthesized de novo by the cells, rather than being released from the damaged membranes However, NP interaction with particular membrane domains, such as

Trophic factors QDs/CoCl 2

PI3K/AKT

Serum withdrawal

SREBP-1

FAS (Lipogenesis) Cerulenin

HIF-1α

FFA

?

LD Esterification

FFA oxidation in mitochondrion

(Fat utilization)

products of FFA (Fat storage)

Fig 7 A model illustrating how colloidal semiconductor

nanoparti-cles, such as QDs, could trigger the formation of intracellular lipid

droplets Activation of the PI3K ⁄ Akt pathway by trophic ⁄ growth

factors creates the metabolic state, in which cells are able to

syn-thesize FFAs These newly synsyn-thesized FFAs are stored in LDs in

the form of triacylgycerols or are oxidized in mitochondria We

hypothesize that QDs interfere with this processes by

down-regu-lating fat oxidation As a result, more FFAs become available for

esterification and storage in LDs The PI3K ⁄ Akt signaling pathway

stimulates lipogenesis via SREBP-1 FAS is the enzyme responsible

for the synthesis of palmitate, the precursor of FFAs The inhibition

of signaling by trophic factors, removal of trophic factors or

inhibi-tion of fat synthesis result in the down-regulainhibi-tion of lipogenesis

and the disappearance of LDs QDs can also induce the

up-regula-tion of HIF-1a, most probably via the producup-regula-tion of ROS QDs and

the hypoxia mimetic CoCl2 down-regulate the oxidation of FFAs

and induce the accumulation of lipids Further studies are needed

to clarify the relationship between HIF-1a-mediated signaling and

the metabolism of lipids in cells exposed to nanoparticles.

caveolae, which consist of small invaginations in

plasma membranes, containing the protein caveolin

and a particular lipid content, could contribute to the

accumulation of lipids It has been shown recently that

caveolae can act as regulatory sites for the synthesis

and trafficking of triacylglycerols [50] Moreover, it is

well documented that NGF signaling via the trk

recep-tor in PC12 cells involves caveolin [51], and that

cave-olin associates with LDs [52] Therefore, NPs may

induce the accumulation of lipids in PC12 cells by

disturbing the function of cellular membranes on the

cell surface, as well as by disturbing cellular functions

inside the cells

Several mechanisms could explain our results: (a)

NPs could up-regulate de novo lipogenesis; (b) NPs

could increase the esterification of FFAs; (c) NPs

could down-regulate the b-oxidation of FFAs; and⁄ or

(d) NPs could modulate the expression of proteins

involved in the retention of lipids in LDs, such as

lip-ases or other LD-associated proteins We have shown

that at least one of these mechanisms is implicated in

lipid accumulation triggered by NPs in PC12 cells

Exposure to ‘degenerated’ (uncoated and uncapped)

CdTe NPs, as well as fluorescent CdSe⁄ ZnS NPs,

significantly down-regulated the b-oxidation of FFAs,

making them available for esterification and storage in

LDs The rate of esterification itself was not altered by

treatment with QDs (data not shown) However, the

mechanisms involved in the down-regulation of fat

oxidation by NPs require further investigation

Fat accumulation and the down-regulation of the

b-oxidation of FFAs were induced in PC12 cells by

exposure to both NPs and the hypoxia mimetic CoCl2

Several studies have shown that the accumulation of

lipids may occur during hypoxia [22–24] Intermittent

hypoxia induced hyperlipidemia in mouse liver via

signaling through SREBP-1 and via up-regulation of

stearoyl-coenzyme A desaturase-1 (SCD-1) [22]

Hyp-oxic conditions also enhanced the synthesis of neutral

lipids in human macrophages via the up-regulation of

lipogenesis (increase in SCD-1 activity) and also via

the down-regulation of the b-oxidation of fatty acids

[23] Hypoxia has also been shown to induce the

for-mation of LDs in various tumors [24] Both NP and

CoCl2 treatment induced the up-regulation of HIF-1a

in PC12 cells However, the up-regulation of HIF-1a

by NP exposure was detectable only in serum-free

con-ditions, whereas LDs were produced mainly in the

presence of serum These findings suggest that the

up-regulation of HIF-1a may not be necessary for

the accumulation of lipids induced by NPs in trophic

factor-supplemented medium, or that changes in its

levels were too subtle to be detected by western

blot-ting [53] Further studies are needed to clarify the rela-tionship between HIF-1a-mediated signaling and the metabolism of lipids in cells exposed to NPs HIF-mediated signaling not only induces the expression of genes involved in cellular adaptation to low oxygen [26], but also alters the cell’s response to various stres-sors [27] In this regard, it could be predicted that exposure to NPs could also modify the cellular responses to various xenobiotics

The lipid accumulation induced in PC12 cells by two types of NP was concentration dependent and largely preceded the manifestation of cell death QDs are redox-active NPs (effective electron donors and accep-tors), which can induce the formation of ROS [4,35] Several studies have shown that QDs and other NPs can generate ROS, particularly if they are exposed to light [54–58] However, we and others have shown that exposure to QDs causes the intracellular production of ROS with and without illumination [4,6,59,60] Both exogenously and endogenously produced ROS cause

an imbalance in the cellular redox state, resulting in oxidative stress It is possible that the accumulation of fat in cytoplasmic LDs in nonadipose cells, such as glia from the hypothalamus, may be an early sign of dam-age and⁄ or oxidative stress induced by certain types of

NP There is evidence that NPs, such as fullerenes and carbon nanotubes, may also produce ROS in vitro [54] and in vivo [57] If so, these NPs could probably induce changes in lipid metabolism and⁄ or the up-regulation

of HIFs We have also tested NPs which are consid-ered to be safe and which do not induce oxidative stress in cells (gold NPs and latex beads) for their abil-ity to induce LD formation We did not observe any increase in LD formation in PC12 cells exposed to these NPs These findings corroborate results from studies with biological NPs and latex beads in macro-phages, reviewed by D’Avila et al [61] Thus, it appears that biological and artificial NPs which cause oxidative stress and⁄ or the release of specific cytokines are potentially hazardous and are the prime candidates for LD induction

The majority of in vitro tests designed for the assess-ment of NP safety are based on viability assays, the peroxidation of membrane lipids, the depletion of cellular glutathione or the secretion of inflammatory mediators [35] The results from the present study sug-gest that metabolic measurements, such as the determi-nation of changes in fat oxidation, could be used as an additional sensitive test for the evaluation of NP safety⁄ biocompatibility Metabolic measurements, especially those related to mitochondrial function and nonhypoxic induction of metabolic effects normally observed with hypoxia, such as changes in oxygen

consumption, are already being considered as

impor-tant criteria for the evaluation of drug-induced toxicity

[62–64]

The accumulation of lipids in cytoplasmic LDs in

glial cells from primary mouse hypothalamic cultures

has not been reported previously Changes in lipid

metabolism induced by metallic ions and NPs in

hypo-thalamus, liver, kidney, spleen or bone marrow could

compromise organismal homeostasis Thus, metabolic

measurements, such as the determination of the rate of

fat oxidation, in these organs or tissues, which are also

known sites of NP accumulation in vivo [34,35,65],

could provide useful measures of NP biocompatibility

and safety

Materials and methods

Materials

The sources of the reagents were as follows: phenol red-free

RPMI, DMEM and Neurobasal A media, fetal bovine

serum, B27 supplement, l-glutamine, penicillin⁄ streptomycin ⁄

neomycin (PSN) cocktail, Ca2+⁄ Mg2+-free NaCl⁄ Pi,

0.25% trypsin⁄ EDTA, Hepes buffer, trypan blue solution

and 545 amino (PEG) QDs from Invitrogen (Burlington,

Canada); Oil Red O, Harris hematoxylin, polyornithine

and laminine from Sigma (Oakville, Canada); formalin

from Fisher Scientific (Nepean, Canada); Alamar blue

reagent from Biosource (Montreal, Canada);

paraformal-dehyde (PFA) from BDH Laboratories (Poole, UK);

glia-specific rabbit GFAP antibody (Z0334) from Doko

Cytomation (Glostrup, Denmark); anti-HIF-2a IgG

(NB100-122SS) from Novus Biologicals (Littleton, CO,

USA); Texas red goat anti-rabbit IgG-conjugated

second-ary antibody (111-075-045) from Jackson ImmunoResearch

(West Grove, PA, USA); Hoechst Dye 33342 (H1399) from

Molecular Probes (Carlsbad, CA, USA); Aqueous Mount

mounting medium from ScyTec (Hornby, Canada); and

VECTASHIELD medium from VECTOR Laboratories

Inc (Burlingame, CA, USA)

Nanoparticles

CdTe and CdSe⁄ ZnS NPs were prepared and characterized

as originally described by Gaponik et al [66] and modified

by Lovric et al [7] CdTe NPs were prepared as described

by Lovric et al [7]; they contain a CdTe core, their

dia-meter is 2.8 nm and they have an emission maximum at

535 nm There was no ZnS cap on the CdTe core and

cysteamine was attached directly to the surface [7]

CdSe⁄ ZnS NPs were prepared as described by Hoshino et al

[44]; they contain a CdSe core, their diameter is 2.4 nm and

they have an emission maximum of 518 nm The CdSe core

was capped with one layer of ZnS to which cysteamine was

attached, and thus the particle size, measured by the dynamic light scattering method, was about 10 nm [44] QDs were added to the cellular media in different amounts and for different lengths of time, as indicated in the figures

Primary mouse hypothalamic cultures

All experiments were conducted with the approval of the McGill University Animal Care Committee Mouse (strain 129T2⁄ SV) hypothalamus was obtained by dissection at postnatal day 5 and freed from the meninges Tissue pooled from at least six animals was stored in ice-cold sterile

Ca2+⁄ Mg2+NaCl⁄ Pi The tissues were dissociated mechan-ically by gentle pipetting using sterile 1 mL pipette tips, and digested with 0.25% trypsin⁄ EDTA at 37 C for

10 min Dissociated cells were resuspended in DMEM med-ium containing 10% fetal bovine serum and PSN cocktail Cells in suspension were seeded onto polyornithine- and laminine-coated 12 mm2 glass microscope slide coverslips and incubated in a 95% air⁄ 5% CO2 atmosphere at 37C for 1 h The coverslips with attached cells were placed in DMEM medium containing 10% fetal bovine serum and PSN for 24 h in 24-well plates (Corning, Nepean, Canada) The next day, the cells were washed twice with pre-warmed Neurobasal A medium and, finally, Neurobasal A medium supplemented with B27, l-glutamine and PSN was added

to the cultures The cultures were maintained at 37C in a 95% air⁄ 5% CO2 atmosphere Experiments were initiated

at DIV 8

Cell culture

Rat pheochromocytoma (PC12, ATCC # CRL-1721) cells were cultured at 37C in phenol red-free RPMI medium containing 2 mm glutamine and 10% fetal bovine serum For survival experiments, cells were seeded onto 24-well plates (25 000 cells per well) and, for LD staining, onto

12 mm2 glass coverslips (13 000 cells per coverslip) Treat-ments with NPs were performed in serum-containing and serum-free medium buffered with 10 mm Hepes (pH 7.4)

LD staining and quantification

LDs were stained with Oil Red O, as described in detail by Przybytkowski et al [67] Briefly, cells were washed with NaCl⁄ Piand incubated with Oil Red O working solution for

15 min at room temperature The cells were then washed once with NaCl⁄ Pi, and fixed with 10% formalin for 25 min Subsequently, the cells were washed again with NaCl⁄ Pi, stained for 2 min with Harris hematoxylin, washed with distilled water and mounted on microscopic slides using Aqueous Mount mounting medium Photomicrographs were taken from representative fields using an Olympus BX2 microscope (Olympus America Inc., Center Valley, PA,