Available via license: CC BY 4.0
Content may be subject to copyright.
fnins-15-780698 December 2, 2021 Time: 14:17 # 1
ORIGINAL RESEARCH
published: 06 December 2021
doi: 10.3389/fnins.2021.780698
Edited by:
Vance L. Trudeau,
University of Ottawa, Canada
Reviewed by:
Stephen Joel Winters,
University of Louisville, United States
Hanna Pincas,
Mount Sinai Medical Center,
United States
*Correspondence:
Violaine Simon
violaine.simon@u-paris.fr
Joëlle Cohen-Tannoudji
joelle.cohen-tannoudji@u-paris.fr
†
†
†Present address:
Charlotte Avet,
Institute for Research in Immunology
and Cancer, Université de Montréal,
Montréal, QC, Canada
Emmanuel N. Paul,
Department of Obstetrics,
Gynecology, and Reproductive
Biology, Michigan State University
College of Human Medicine, Grand
Rapids, MI, United States
Violaine Simon,
CEA, Institut de Biosciences et
Biotechnologies de Grenoble,
Laboratoire BioSanté Inserm U1292,
Grenoble, France
‡These authors have contributed
equally to this work and share last
authorship
Specialty section:
This article was submitted to
Neuroendocrine Science,
a section of the journal
Frontiers in Neuroscience
Received: 21 September 2021
Accepted: 15 November 2021
Published: 06 December 2021
Carbon Black Nanoparticles
Selectively Alter Follicle-Stimulating
Hormone Expression in vitro and in
vivo in Female Mice
Charlotte Avet1†, Emmanuel N. Paul2†, Ghislaine Garrel1, Valérie Grange-Messent3,
David L’Hôte1, Chantal Denoyelle1, Raphaël Corre1, Jean-Marie Dupret4,
Sophie Lanone2, Jorge Boczkowski2, Violaine Simon1*†‡ and Joëlle Cohen-Tannoudji1*‡
1Université de Paris, BFA, UMR 8251, CNRS, ERL U1133, Inserm, Paris, France, 2Inserm U955, IMRB, U 955, Faculté
de Médecine, Université Paris Est (UPEC), Créteil, France, 3Sorbonne Université, CNRS, Inserm, Neuroscience Paris Seine –
Institut de Biologie Paris Seine, Paris, France, 4Université de Paris, BFA, UMR 8251, CNRS, Paris, France
Toxic effects of nanoparticles on female reproductive health have been documented
but the underlying mechanisms still need to be clarified. Here, we investigated the
effect of carbon black nanoparticles (CB NPs) on the pituitary gonadotropins, luteinizing
hormone (LH) and follicle-stimulating hormone (FSH), which are key regulators of
gonadal gametogenesis and steroidogenesis. To that purpose, we subjected adult
female mice to a weekly non-surgical intratracheal administration of CB NPs at an
occupationally relevant dose over 4 weeks. We also analyzed the effects of CB NPs
in vitro, using both primary cultures of pituitary cells and the LβT2 gonadotrope cell line.
We report here that exposure to CB NPs does not disrupt estrous cyclicity but increases
both circulating FSH levels and pituitary FSH β-subunit gene (Fshb) expression in female
mice without altering circulating LH levels. Similarly, treatment of anterior pituitary or
gonadotrope LβT2 cells with increasing concentrations of CB NPs dose-dependently
up-regulates FSH but not LH gene expression or release. Moreover, CB NPs enhance
the stimulatory effect of GnRH on Fshb expression in LβT2 cells without interfering with
LH regulation. We provide evidence that CB NPs are internalized by LβT2 cells and
rapidly activate the cAMP/PKA pathway. We further show that pharmacological inhibition
of PKA significantly attenuates the stimulatory effect of CB NPs on Fshb expression.
Altogether, our study demonstrates that exposure to CB NPs alters FSH but not LH
expression and may thus lead to gonadotropin imbalance.
Keywords: carbon black nanoparticles, pituitary, gonadotropin, GnRH, cAMP/PKA pathway, endocrine disruption
Abbreviations: FBS, fetal bovine serum; CB NPs, carbon black nanoparticles; Cga, glycoprotein hormone alpha polypeptide;
CREB, cAMP response element-binding protein; ERK1/2, extracellular signal-regulated kinase 1 and 2; FSH, follicle-
stimulating hormone; GnRH, gonadotropin-releasing hormone; HPRT, hypoxanthine phosphoribosyltransferase; IL-1β,
interleukin 1 beta; IL-6, Interleukin 6; JNK, Jun-kinase; LH, luteinizing hormone; MAPK, mitogen-activated protein kinase;
MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide; NPs, nanoparticles; PKA, protein kinase A; PKC,
protein Kinase C; TNF α, tumor necrosis factor alpha; SF3a1, splicing factor 3a subunit 1.
Frontiers in Neuroscience | www.frontiersin.org 1December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 2
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
INTRODUCTION
Reproductive processes in mammals are dependent on the
appropriate regulation of the synthesis and release of luteinizing
hormone (LH) and follicle-stimulating hormone (FSH) by
the gonadotrope cells of the anterior pituitary. These two
hormones, indeed, act in a concerted manner to regulate
gonadal hormone synthesis and gametogenesis in both males
and females. LH and FSH are glycoprotein hormones composed
of a common α-glycoprotein subunit and distinct rate-limiting
β-subunits that confer biological activity to the hormones. The
level of the three subunit transcripts (Cga,Lhb and Fshb) as
well as the release of gonadotropins are mainly controlled
by the neuropeptide, gonadotropin-releasing hormone (GnRH)
(Counis et al., 2005). GnRH is produced by hypothalamic
neurosecretory neurons and released, in a pulsatile fashion,
into the blood vessels connecting the median eminence to
the anterior pituitary. Upon binding to its receptor, which is
specifically expressed in gonadotrope cells, GnRH primarily
activates Gq/11 proteins leading to the recruitment of a wide
array of signaling pathways. GnRH notably triggers the activation
of phospholipase Cβ, the mobilization of intracellular calcium
and the increase of Protein Kinase C (PKC) activity, leading
notably to the activation of the mitogen-activated protein kinase
(MAPK) pathways (Naor and Huhtaniemi, 2013). GnRH also
stimulates, indirectly or directly by coupling to Gs-proteins, the
cAMP/Protein kinase A (PKA) pathway (Liu et al., 2002;Lariviere
et al., 2007). All PKC, PKA and MAPK pathways contribute
to the GnRH-stimulated transcription of gonadotropin genes
(Thackray et al., 2010).
The balance between LH and FSH levels is finely controlled
throughout reproductive life and disequilibrium in this balance is
associated with reproductive disorders such as polycystic ovaries
and premature ovarian failure. Polycystic ovary syndrome is
characterized by an increase specifically in LH levels, whereas
high levels of FSH are observed in patients or in animal
models of ovarian insufficiency (Guigon et al., 2005;Vandormael-
Pournin et al., 2015;Malini and Roy George, 2018). The
way in which the expression of the two gonadotropins is
finely and differentially regulated by GnRH is still poorly
understood. GnRH differentially regulates the transcription of
Lhb and Fshb via changes in pulse frequency, with increasing
GnRH pulsatility favoring LH while reduced GnRH pulsatility
favors FSH synthesis (Burger et al., 2004). In addition,
several endocrine or locally produced factors also contribute
to the fine-tuning of FSH/LH balance. Among them, is the
hormone activin, which selectively activates Fshb transcription
(Bilezikjian et al., 2012).
Reproductive fitness is extremely dependent upon
environmental signals. There is growing evidence, both
from wildlife and studies in human and animal models, that
contaminants present in the environment affect reproductive
activity (Guillette and Guillette, 1996;Danzo, 1998). Among
them, industrial compounds, such as pesticides or plasticizers
including phthalates and bisphenols have been identified
as endocrine-disrupting chemicals that alter reproductive
endocrinology and fertility. Over the past decades, a significant
increase in the production and use of nanoparticles (NPs) has
occurred. NPs are materials having at least one dimension less
than 100 nm. Among them, carbon black NPs (CB NPs) are
mainly derived from controlled incomplete combustion or
thermal decomposition of hydrocarbons. A variety of engineered
carbon NPs are used in consumer products such as car tires,
rubber, and printer toner cartridges. Furthermore, elemental
carbon-based NPs are a major part of diesel exhaust and ambient
pollution (Schauer, 2003). CB NPs are mainly taken up through
inhalation, and the translocation of intratracheally instilled CB
NPs into blood has been described in mice (Shimada et al., 2006).
Numerous experimental studies on animals have also shown
that CB NP inhalation can induce pulmonary inflammation
and cardiovascular diseases (Bachoual et al., 2007;Niwa et al.,
2008;Bourdon et al., 2012). Further underlining their toxicity,
CB NPs and their respirable aggregates/agglomerates have been
classified as possibly carcinogenic to humans [group 2B] (Iyengar
et al., 2016). The current limit for CB NPs exposure defined
by the NIOSH (National Institute of Occupational Safety and
Health) is of 3.5 mg/m3. Under occupational settings, however,
workers could be exposed to much higher concentrations of
CB NPs and levels of 79 mg/m3or even 675 mg/m3have
been reported (IARC Working Group on the Evaluation of
Carcinogenic Risks to Humans, 2010). Several recent lines of
evidence suggest that CB NPs also act as endocrine-disrupting
chemicals with detrimental effects on reproduction (Lu et al.,
2013;Hutz et al., 2014). Intratracheal administration of CB
NPs to adult male mice indeed alter testosterone production
and reduces daily sperm production (Yoshida et al., 2009).
Consistent with such an action, the in vitro exposure of a mouse
Leydig cell line to CB NPs decreases the expression of the
steroidogenic acute regulatory protein (StAR), which is the rate
limiting factor in steroid biosynthesis (Komatsu et al., 2008).
We have recently reported in human ovarian granulosa cells
that CB NPs decrease basal and FSH-stimulated expression
of the enzyme aromatase, which catalyzes the biosynthesis of
estradiol from androgens, and also decrease estradiol secretion
(Simon et al., 2017). The majority of experiments assessing
the detrimental effect of environmental contaminants, and
notably of NPs, on reproduction have been performed at the
level of the gonads (Wang et al., 2018), and although pituitary
gonadotropins are key regulators of gonadal activity, only scarce
information is available on a possible disruption of pituitary
endocrine activity.
The objective of this study was to investigate whether
CB NPs exposure could disrupt basal or GnRH-stimulated
gonadotropin secretion. To address this issue, we subjected
adult female mice to non-surgical intratracheal exposure to
CB NPs. To further explore the underlying mechanisms of
CB NPs action, we also analyzed the effects of CB NPs
in vitro, using two distinct models, i.e., the primary cultures
of pituitary cells and the LβT2 gonadotrope cell line. We
report here that CB NPs differentially alter the expression
and circulating levels of LH and FSH both in vivo in female
mice and in vitro. We further showed that CB NPs are
internalized by gonadotrope cells and that the recruitment of the
cAMP/PKA/CREB pathway by CB NPs mediates the increase in
Frontiers in Neuroscience | www.frontiersin.org 2December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 3
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FSH expression. Altogether, our study suggests that CB NPs may
act as an endocrine disruptor, leading to an imbalance of the
gonadotropins, LH and FSH.
MATERIALS AND METHODS
Exposure of Female Mice to Carbon
Black Nanoparticles
The CB NPs used were FW2 (13 nm) and obtained from Evonik
Industries/Degussa (Frankfurt, Germany). Their key physico-
chemical characteristics including diameter, surface area, zeta
potentials and hydrodynamics of the suspended particles, have
been determined previously (Sanfins et al., 2011). Stock solutions
of 20 mg/mL of CB NPs were prepared and sonicated as
previously described (Simon et al., 2017).
Studies were conducted on twelve-week-old adult female
C57BL/6 mice. Mice were maintained under controlled
conditions (12-h light/dark cycle) with food and water available
ad libitum. Once a week over four weeks, female mice received
10 µL of vehicle (NaCl group, 30 animals) or CB NPs at
10 mg/mL diluted in NaCl (CB NP group, 36 animals) that
were administered by non-surgical intratracheal instillation
performed under anesthesia [1.6 mg ketamine (Virbac, Carros,
France) and 300 mg xylazine (BayerTM, Puteaux, France)].
The protocol has been approved by our local ethic committee
(ComEth Anses/ENVA/UPEC) under the reference #12-104
(final approval #20/12/12-27). Intratracheal exposure to 100 µg
CB NPs corresponds to the mice being exposed at the current
occupational exposure limit of 3.5 mg/m3for 5,5 working days
(8 h/day) as previously reported (Bourdon et al., 2013;Husain
et al., 2015). Estrous cyclicity was monitored during the 12 days
preceding the first exposure to CB NPs or NaCl and the 12 days
preceding sacrifice. Vaginal cells from females were collected
by daily saline wash and analyzed after May-Grünwald-Giemsa
R (RAL Diagnostics) staining. Stages of the estrous cycle were
characterized, as previously described (Vandormael-Pournin
et al., 2015). Mice were sacrificed two weeks after the last
exposure. Retro-orbital blood, anterior pituitary glands, lungs
and ovaries were collected. Serum was separated from blood by
centrifuging 10 min at 1,000 ×gand stored at −20◦C. Body
and organs weights were measured. Anterior pituitaries, lungs
and ovaries were deep-frozen in liquid nitrogen and stored at
−80◦C. Four mice died during the course of the experiment
(3 NaCl- and 1 CB NP-treated mice). Moreover, because of
insufficient RNA quantity or quality, pituitary gonadotrope
function was eventually measured on 24 NaCl- and 33 CB
NP-treated mice.
Cell Culture and Exposure to Carbon
Black Nanoparticles
The pituitary gonadotrope LβT2 cell line was provided by Dr.
Pamela Mellon (University of California, San Diego) (Thomas
et al., 1996;Turgeon et al., 1996) and maintained in 12-well
plates (1 ×106per well) in DMEM (Gibco, Life Technologies)
supplemented with 10% fetal bovine serum (FBS) and 0.5%
Penicillin/Streptomycin (P/S) (Sigma-Aldrich). Primary cultures
of anterior pituitary cells were prepared from adult female
Wistar rats (225–250 g, Janvier) as previously described (Garrel
et al., 2010) and cultured in 12-well plates or 6-well plates
(1 ×106per well) for 48 h in Ham F-10 medium with 10%
FBS and 0.5% P/S. Before exposure to CB NPs, cells were
starved overnight in serum-free medium and then incubated
in serum-free medium for 24 h with increasing concentrations
of CB NPs (25–100 µg/mL corresponding to 5–20 µg/cm2,
as previously done on human luteinized granulosa cells and
on the granulosa cell line KGN (Simon et al., 2017). These
concentrations are in the range or even lower than those
classically used in several other in vitro studies (Yamawaki
and Iwai, 2006;Komatsu et al., 2008;Lee et al., 2012). At
the end of incubation, medium was collected for assaying
gonadotropin secretion. Cells were extensively washed with
culture medium and total RNAs were isolated. Shorter exposures
(30 or 60 min) to CB NPs were performed to analyze CB NP
activation of cellular signaling pathways. The potent and selective
PKA inhibitor Rp-cAMP (100 µM) was added 30 min before
CB NP addition. LβT2 cells were also stimulated during 6 h
with the GnRH agonist Triptorelin (GnRHa, 100 nM) after
having been exposed for 24 h to increasing concentrations (25–
100 µg/mL) of CB NPs.
Cell Viability
The viability of cultured anterior pituitary and LβT2 cells
exposed to CB NPs was measured using the MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Cells were serum-starved overnight and incubated for 24 h
with increasing concentrations of CB NPs (25–100 µg/mL
corresponding to 5–20 µg/cm2). Media were then aspirated
and replaced by 0.5 mL medium containing MTT (1 mg/mL).
After 2 h of incubation, cells were lysed and treated with
200 µL of dimethyl sulfoxide (DMSO). The absorbance of
the solubilized formazan crystals was read at 575 nm on a
Flexstation3 (Molecular devices).
Transmission Electron Microscopy
LβT2 cells seeded at 2 ×106cells per well in 6-well plates
in 2 mL of DMEM containing 10% FBS and 0.5% P/S and
confluent primary cultures of anterior pituitary cells cultured in
6-well plates in 2 mL of Ham F-10 medium with 10% FBS and
0.5% P/S, were used. Forty-eight hours later, cells were serum
starved overnight and incubated or not the next day with CB
NPs (LβT2: 10 or 25 µg/mL; primary cultures: 50 µg/mL) for
24 h. Cells were then extensively washed with culture medium
and washed in 0.05 M sodium cacodylate buffer (pH 7.4) before
being fixed in situ with 2.5% glutaraldehyde diluted in the same
buffer at 4◦C for 1 h. After five washes in sodium cacodylate
buffer, cell monolayers were post-fixed for 1 h using 2% osmium
tetroxide in sodium cacodylate buffer in the dark at room
temperature. Dehydration in situ was performed through an
ethanol ascending series and cell monolayers were embedded in
epoxy resin (Epoxy-Embedding Kit, cat. # 45359, Sigma Aldrich,
Switzerland). Gelatin capsules filled with resin were returned
to the cell layers and the polymerization proceeded in a 60◦C
Frontiers in Neuroscience | www.frontiersin.org 3December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 4
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
oven for 48 h. Capsules were then detached from the wells and
ultrathin sections (70 nm) of the cell monolayer were collected
on copper grids. Sections were contrasted using 2% uranyl acetate
for 5 min and Reynold’s lead citrate for 2 min. Examination was
done using a transmission electron microscope (80–120 kV 912
Omega ZEISS) equipped with a digital camera (Veleta Olympus).
Hormone Level Assays
LH and FSH were simultaneously assayed in 10 µL of serum
with the Luminex technology using the mouse pituitary
magnetic bead panel Milliplex Map kit (Merck-Millipore,
Nottingham, United Kingdom) in accordance with the
manufacturer’s instructions. Serum levels of inhibin B were
measured using a commercially available ELISA kit (Beckman
Coulter) following manufacturer’s protocol. The concentration
of estradiol and progesterone were assayed in the serum using
gas chromatography coupled with mass spectrometry (GC-MS)
procedure, as described previously (Giton et al., 2015;Francois
et al., 2017). Due to insufficient serum volume for this analysis,
serum sex steroid levels were measured in a smaller subset of
females than used for gonadotropin assays (16 NaCl- and 24 CB
NP-treated mice for progesterone and 11 NaCl- and 18 CB NP-
treated mice for estradiol). The linearity of steroid measurement
was confirmed by plotting the ratio of the respective steroid peak
response/internal standard peak response to the concentration
used for calibration standard. Lower limit of quantification
was 0.2 pg for estradiol and 5.5 pg for progesterone. In cell
culture media, LH and FSH concentrations were measured
using an ELISA method adapted from Faure et al. (2005) with
reagents supplied by Dr. Parlow (NHPP, Harbor-UCLA, CA,
United States) as previously described (Garrel et al., 2011;
Lannes et al., 2016). The minimum detectable LH and FSH
concentrations were 0.2 and 1 ng/mL, respectively and interassay
coefficients of variation were less than 10%. We checked that CB
NPs present in the cell culture medium did not interfere with the
binding between the antibody and the FSH or LH standards in
the ELISA assay (data not shown).
Reverse-Transcription and Real-Time
PCR
Total RNAs from LβT2 and cultured pituitary cells as well as
from mouse anterior pituitaries and lungs were isolated with
a RNeasy-kit (Qiagen, France). Reverse transcription (RT)
was performed using 1 µg RNA in a total volume of 20 µL
with the Reverse transcription using superscript II reverse
transcriptase (Invitrogen) and real-time PCR were carried
out in the LightCycler 480 Instrument (Roche Diagnostics)
as previously described (Simon et al., 2017). Gene expression
levels were normalized to Hprt, encoding hypoxanthine
phosphoribosyltransferase, for mouse anterior pituitaries,
to Sf3a1 encoding Splicing factor 3 subunit 1 for mouse
lungs and to Cyclophillin for LβT2 and cultured pituitary
cells. The oligonucleotide primer sequences are indicated in
Table 1. Primers were designed to target both rat and mouse
DNA sequences. Data were analyzed using the Advanced-E-
method with standard-curve derived efficiencies obtained from
TABLE 1 | Oligonucleotide primer sequences used for real-time PCR performed
on primary anterior pituitary, LβT2 cells or lung. Fshb,Lhb,Cga,Fst,Fs-288, Hprt,
Cyclophilin,Tnfa,Il1b,Il6 and Sf3a1.
Target Forward primer Reverse primer
Fshb 50TTGCATCCTACTCTGGT
GCT 30
50AGCTGGGTCCTTATACA
CCA 30
Lhb 50ATCACCTTCACCACCAG
CAT 30
50GACCCCCACAGTCAGAG
CTA 30
Cga 50GCTGTCATTCTGGTCAT
GCT 30
50GAAGCAACAGCCCATAC
ACT 30
Fst 50CAAGGTTGGCAGAGGTC
GCT 30
50CCGAGATGGAGTTGCAA
GAT 30
Fs-288 50CTCTCTCTGCGATGAGC
TGTGT 30
50GGCTCAGGTTTTACAGGC
AGAT 30
Cyclophilin 50CAAAGTTCCAAAGACAG
CAG 30
50CTGGCACATGAATCCTG
GAA 30
Hprt 50AGGACCTCTCGAA
GTGT 30
50ATTCAAATCCCTGAAGTA
CTCAT 30
Tnfa 50CCACCACGCTCTTCTGTC
TACTGAACTT 30
50GTGGGCTACAGGCTTGTC
ACTCG 30
Il1b 50TGAGAATGACCTGTTCTT
TGAAGTTG 30
50GACAGCCCAGGTCAAAG
GTTT 30
Il6 50TGAATTGGATGGTCTTGGT
CCTTAGCCAC 30
50ACAAAGCCAGAGTCCTTCA
GAGAGATACAG 30
Sf3a1 50CCACTGAGTCCAAACAGC
CAAT 30
50AGCTTCAAATTCAGGC
CCAT 30
LightCycler 480 software. The specificity of amplification was
checked by gel electrophoresis and melting curve analysis.
Protein Extraction and Immunoblotting
Membrane proteins were prepared from LβT2 cells as previously
described (Garrel et al., 2016). Equal amounts of protein
(20 µg) were separated on a 10% SDS-PAGE and transferred
to a nitrocellulose membrane. Specific antibodies and Pierce
ECL2 substrate were used to detect Phospho-CREB (P-CREB;
Cell signaling #9198; 1:1000), Phospho-Extracellular signal-
regulated kinase1/2 (P-ERK 1/2; Cell signaling #9101; 1:1000),
Phospho-p38 (P-p38; Cell signaling #4511; 1:1000), Phospho-
Jun-kinase (P-JNK; Cell signaling #4671; 1:1000), Phospho-
Smad2 (P-Smad2; Cell signaling #3108; 1:1000). Respective non-
phosphorylated proteins, including Total CREB (Cell signaling
#9197), Total ERK1/2 (Cell signaling #9102), Total p38 (Cell
signaling #8690), Total JNK (Cell signaling #4671), Total Smad2
(Cell signaling #5339) were also detected (dilution of antibodies,
1:1000) as well as vinculin (Sigma-Aldrich #V9131; 1:20 000),
used as an internal loading control. Blots were analyzed with
a Fuji LAS-4000 imager and quantified using MultiGauje
software. Full scans of the entire original gels are presented in
Supplementary Materials 1, 2.
Statistical Analyses
Data are from at least three independent experiments. The
precise number is indicated in the legends of figures. All data
were analyzed using the Prism 6 Software (GraphPad Software,
Inc). Data were first subjected to normality test and data that
did not pass the test were analyzed using non-parametric tests
Frontiers in Neuroscience | www.frontiersin.org 4December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 5
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
Mann-Whitney or Kruskal-Wallis followed by Dunnet’s multiple
comparison test. P≤0.05 was considered as significant.
RESULTS
In vivo Exposure to Carbon Black
Nanoparticles Disrupts Pituitary
Gonadotrope Activity and Selectively
Increases Follicle-Stimulating Hormone
in Female Mice
Exposure of female mice to CB NPs was carried out by non-
surgical intratracheal instillations of CB NPs performed once
weekly over four weeks. Mice did not exhibit any obvious signs of
distress (loss of activity, mobility difficulties, respiratory distress,
bristly fur or arched back) during the treatment period. Control
mice were treated with NaCl. Body weight as well as pituitary and
ovarian weights were unaffected by the treatment (Figure 1A).
As previously reported (Roulet et al., 2012), intratracheal CB
NP instillations led to pulmonary inflammation, as revealed
by a significant increase in the pulmonary expression of pro-
inflammatory cytokines such as tumor necrosis factor (TNF) α
and interleukin 6 (IL-6) in female mice (Figure 1B). Examination
of vaginal cytology showed the presence of all stages of the estrous
cycle in both groups of mice. Daily analyses of vaginal smears
over twelve days (approximately three consecutive cycles) are
illustrated for 4 CB NP and 4 control females in Figure 1C, and
revealed similar patterns of estrous cyclicity as well as similar
cycle lengths in control and CB NP-treated mice [5.19 ±0.35
in NaCI- (n= 24) versus 4.91 ±0.13 days in CB NP-treated
mice (n = 33)] (Figure 1C). To determine whether CB NPs
could disrupt pituitary gonadotrope activity, we measured serum
concentrations of gonadotropins as well as gonadotropin subunit
transcript levels in both NaCl (control)- and CB NP-treated mice.
Exposure to CB NPs significantly increased circulating FSH as
compared to controls (2.7 ±0.8-fold, p<0.05, Figure 2A).
Interestingly, no significant change in circulating LH could be
detected. The treatment of mice with CB NPs also led to a two-
fold elevation in the level of pituitary Fshb transcripts compared
to levels in control mice (2 ±0.3-fold, p<0.05, Figure 2B). In
contrast, albeit significant, the elevation was much more modest
for Lhb and Cga transcripts (Figure 2B). We then investigated
whether changes in ovarian hormones could account for the
observed changes in gonadotropin expression. Inhibin produced
by granulosa cells of growing ovarian follicles is known to
target the pituitary and antagonize activin, leading to a selective
decrease in Fshb transcription and release (Makanji et al., 2014).
However, as illustrated in Figure 2C, no difference in serum levels
of inhibin was observed between NaCl- and CB NP-treated mice.
We also measured the sex steroids, estradiol and progesterone,
by GC-MS analysis, which is the most accurate and reliable
method to measure steroids. As observed for inhibin, serum
progesterone level displayed no significant changes following
CB NP treatment. This was also the case for serum estradiol
levels, although a tendency to decrease upon CB NP treatment
could be observed.
In vitro Exposure of Anterior Pituitary
Cells to Carbon Black Nanoparticles
Alters Follicle-Stimulating Hormone but
Not Luteinizing Hormone Expression and
Secretion
To determine whether CB NPs could directly act on the pituitary
to disrupt gonadotrope activity, we first treated primary cultures
of anterior pituitary cells with increasing concentrations of CB
NPs (25–100 µg/mL) for 24 h. The potential cytotoxic effect
of CB NPs was determined by measuring cell viability with
the commonly used MTT assay. We have previously shown
that the presence of CB NPs does not interfere with this assay
(Simon et al., 2017). As indicated in Table 2, the incubation
of cells over 24 h with 50 or 100 µg/mL CB NPs did not
alter cell viability. Treatment with CB NPs induced a dose-
dependent increase in FSH secretion, as shown in Figure 3A.
The increase was significant from 50 µg/mL, with a maximum
increase of 141 ±9% as compared to untreated cells being
observed at 100 µg/mL. In contrast, CB NPs did not affect
LH secretion whatever the concentration used (Figure 3A). We
next determined the effects of CB NPs on the transcript levels
of gonadotropin subunits by real-time PCR (Figure 3B). CB
NPs significantly and dose-dependently increased Fshb transcript
levels with an increase of 137 ±8% at 100 µg/mL CB NPs.
No significant change in Lhb or Cga transcript levels could
be observed after treatment with CB NPs. Because follistatin,
produced by gonadotrope and folliculo-stellate cells of the
pituitary, binds to activin and antagonizes its action (Bilezikjian
et al., 2012), we also measured follistatin transcript levels which
were unaffected by the treatment of anterior pituitary cells with
increasing concentrations of CB NPs (Figure 3C). Alternative
splicing of follistatin mRNA can result in the formation of a
shorter 288 amino acid isoform (FS-288), which was reported to
be more active in suppressing FSH release by rat pituitary cell
cultures (Sugino et al., 1993). As observed for total follistatin, the
transcript levels of the active FS-288, determined using specific
primers (Boerboom et al., 2015;Table 1), were not affected
by the treatment with CB NPs (123 ±11% and 118 ±16%
of the levels in control cells with 50 and 100 µg/mL of
CB NPs, respectively, data not shown). Transmission electron
microscopic analyses were performed on primary cultures of
pituitary cells (Figures 4A,B). Aggregates/agglomerates of CB
NPs were visualized in all observed cells exposed to 50µg/mL of
CB NPs. They were freely dispersed in the cytoplasm or associated
with different subcellular compartments such as the smooth
endoplasmic reticulum and electron-dense vesicles. These dense-
core vesicles, of a diameter of approximately 150 nm, which were
abundantly present in the cytoplasm, correspond to secretory
vesicles (Figures 4A,B).
Carbon Black Nanoparticles Enter LβT2
Gonadotrope Cells and Selectively
Increase Fshb Transcript Levels
To further determine the effects of CB NPs on gonadotropin
expression, we used the most differentiated gonadotrope cell
Frontiers in Neuroscience | www.frontiersin.org 5December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 6
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 1 | Impact of CB NPs exposure on mice body and organs weights, pulmonary pro-inflammatory cytokine transcripts and estrous cyclicity. (A) Effect of CB
NPs on body weights and relative organ weights of female mice after an intratracheal instillation with 100 µg CB NPs/week during 4 weeks. (B) Transcript levels of
canonical pro-inflammatory cytokines in lungs of female mice exposed to CB NPs. Control saline solution (NaCl, 0.9%) or CB NPs (100 µg) were intratracheally
administered to C57BL/6 mice once a week during four weeks as described in the Materials and Methods. Mice were sacrificed and lungs were collected. Tnfa,Il6
and Il1b mRNA levels were determined by real-time quantitative PCR in NaCl- and CB NP-treated mice (11 animals in each group). (C) Effect of CB NPs on mice
estrous cyclicity. Left, Estrous cycle lengths were determined following CB NP exposure during the 12 days preceding sacrifice. No difference in cycle lengths was
observed between mice exposed to NaCl or CB NPs (5.19 ±0.35 in control mice (n= 24) versus 4.91 ±0.13 days in CB NP-treated mice (n= 33). Right, Diagrams
representing the estrous cycles at each stage (E: estrous; P: proestrous; D: diestrous; M: metestrous) for four representative controls and CB NPs mice monitored
daily during the 12 days preceding sacrifice. At the time of sacrifice, percentages of mice in each stage of the estrous cycle were roughly similar between the two
groups of mice: 16.7, 25, 29.1, and 29.2% in proestrous, estrous, diestrous and metestrous, respectively in controls vs. 18.2, 30.3, 24.2, and 27.3% in proestrous,
estrous, diestrous and metestrous, respectively in CB NP-treated mice. Data are expressed as means ±SEM. Statistical differences were determined by the
Mann-Whitney test. **p<0.01 and ***p<0.001 compared to NaCl.
line available: the LβT2 gonadotrope cell line (Pernasetti et al.,
2001). As observed with primary cultures of pituitary cells, the
incubation of LβT2 cells for 24 h with CB NPs, at concentrations
up to 100 µg/mL did not have any effect on cell viability (Table 2).
We next determined the uptake and intracellular localization
of CB NPs by transmission electron microscopy analysis using
two concentrations of CB NPs, 10 µg/mL (not illustrated)
and 25 µg/mL (Figures 4C–E). CB NPs were observed in the
cytoplasm as freely dispersed aggregates/agglomerates within or
associated with different subcellular compartments or organelles
such as the smooth endoplasmic reticulum (Figure 4C) and
mitochondrial membranes (Figure 4D). CB NPs were also
found within the numerous electron-dense vesicles (Figure 4E),
corresponding to secretory granules. In contrast and as expected,
no CB NPs could be detected in non-exposed cells (Figure 4F).
We next determined the effects of CB NPs on gonadotropin
Frontiers in Neuroscience | www.frontiersin.org 6December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 7
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 2 | Pituitary gonadotropin serum levels and subunit gene expression in female mice exposed to CB NPs. CB NPs (100 µg) or control saline solution (NaCl,
0.9%) were intratracheally administered to C57BL/6 mice once a week during four weeks as described in the Materials and Methods. Mice were sacrificed and
anterior pituitaries and blood were collected. Concentrations of FSH and LH were determined in serum by Luminex technology in 24 NaCl- and 33 CB NP-treated
mice (A).Fshb,Lhb and Cga mRNA levels were determined by real-time quantitative PCR in 24 NaCl- and 33 CB NP-treated mice (B). Serum levels of inhibin B and
sex steroids (estradiol and progesterone) were determined by ELISA and GC-MS, respectively, as described in the materials and methods (C). Assays were
performed on 23 NaCl- and 30 CB NP-treated mice for inhibin-B, 11 NaCl- and 18 CB NP-treated mice for estradiol and 16 NaCl- and 24 CB NP-treated mice for
progesterone. Data are represented as mean ±SEM. Statistical differences were determined by the Unpaired t-test (for B, Fshb mRNA) or Mann-Whitney test.
*p<0.05 and **p<0.01 compared to NaCl.
expression in the LβT2 cell line. As observed with primary
cultures of pituitary cells, treatment with CB NPs significantly
and dose-dependently increased Fshb expression (maximum
increase of 218 ±38% at 100 µg/mL). No effect could be detected
on Lhb transcript levels while a small decrease in Cga transcript
levels was observed at 100 µg/mL of CB NPs (Figure 5).
Carbon Black Nanoparticles Increase
Fshb Expression Through the cAMP
Pathway in LβT2 Cells
We next conducted experiments in LβT2 cells in order to
understand the mechanisms of action of CB NPs. Several
signaling pathways can be activated following cell exposure to
carbon NPs (Brown et al., 2000;Sydlik et al., 2006;Simon
et al., 2017;Cheng et al., 2019). Recruitment of PKA and
PKA-dependent phosphorylation of the cAMP response element-
binding protein (CREB) transcription factor have been identified
as key mechanisms mediating the preferential activation of Fshb
transcription by GnRH in LβT2 cells (Thompson et al., 2013). We
thus first sought to determine whether the PKA/CREB pathway
could be recruited by CB NPs by measuring the phosphorylation
of the PKA related transcription factor CREB. LβT2 cells
were stimulated for 30 or 60 min with 100 µg/mL of CB
NPs and CREB phosphorylation measured by immunoblotting
with antibodies recognizing the phosphorylated and total forms
Frontiers in Neuroscience | www.frontiersin.org 7December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 8
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 3 | Gonadotropin secretion and gene expression in primary cultures of anterior pituitary cells treated with CB NPs. Rat primary anterior pituitary cells were
cultured for 24 h with increasing concentrations of CB NPs (25–100 µg/mL). At the end of the incubation, FSH and LH concentrations in the culture media of primary
pituitary cells were determined by ELISA. FSH/LH ratio is also shown in panel (A). Total RNAs were extracted and Fshb,Lhb,Cga (B) and Follistatin (C) mRNA levels
were determined by real-time quantitative PCR. Data are expressed as percentage over untreated cells and expressed as means ±SEM from 4 independent
experiments. Statistical differences were determined by non-parametric Kruskal-Wallis test followed by Dunnet’s multiple comparison test. *p<0.05 and **p<0.01
compared to no CB NPs.
of CREB. Treatment of cells with CB NPs for 30 min
enhanced the phosphorylation of CREB as compared to
control conditions (Figure 6A inset), significantly increasing
the ratio of phosphorylated to total CREB (by 183 ±39%,
p<0.05; Figure 6A). To further investigate the role of the
cAMP/PKA signaling pathway in mediating CB NP-stimulated
Fshb expression, we treated LβT2 cells with CB NPs in the
presence of a selective pharmacological inhibitor of PKA, Rp-
cAMP (Figure 6B). As expected, CB NP treatment significantly
increased Fshb transcript level (by 374 ±66%, p<0.001) in
control cells. Pharmacological inhibition of PKA significantly
reduced the CB NP-dependent increase in Fshb transcript levels,
suggesting that this increase was mediated at least in part by
PKA. Because CB NPs have been reported to activate MAPK
pathways in several cell lines, including reproduction-linked
cell lines such as the ovarian granulosa cells (Sydlik et al.,
2006;Simon et al., 2017), we also examined the ability of CB
NPs to activate the three branches of the MAPK pathway:
ERK1/2, JNK and p38 (Figure 7). Detection of phosphorylated
and total forms of these kinases was carried out after 30
and 60 min of treatment with CB NPs, as done for CREB.
Immunoblotting analysis revealed that, at least during the
time period studied, CB NPs did not activate MAPK signaling
pathways as no increase of phosphorylation of ERK1/2, p38 or
JNK could be detected (P-ERK1/2/total ERK1/2: 115 ±11%
and 113 ±10% of control levels after 30 and 60 min of
CB NP treatment, respectively; P-p38/total p38: 107 ±25%
and 92 ±18% of control levels after 30 and 60 min of CB
NP treatment, respectively; P-JNK/total JNK: non-quantifiable).
In contrast, and as expected, a GnRH agonist (GnRHa:
triptorelin, 100 nM) increased their phosphorylation levels (not
shown). Because the Smad2/3 pathway strongly stimulates Fshb
transcription (Bernard, 2004;Thackray et al., 2010), we further
examined the ability of CB NPs to activate this pathway in
LβT2 cells by immunodetection of the phosphorylated form of
Smad2. As illustrated in Figure 7, CB NPs did not increase
Smad2 phosphorylation, suggesting that this pathway is not
recruited by CB NPs in LβT2 cells. Under these experimental
conditions, Smad2 phosphorylation could be induced by activin
A (data not shown).
Carbon Black Nanoparticles Increase
Gonadotropin-Releasing Hormone
Stimulation of Fshb but Not Lhb
Expression in LβT2 Cells
Since GnRH is the key regulator of gonadotrope cell activity,
we next examined whether CB NPs could alter the GnRH-
dependent-induction of gonadotropin synthesis. LβT2 cells were
pre-treated with increasing concentrations of CB NPs for
24 h followed by a 6 h-treatment with the GnRH agonist
(Figure 8A). As expected, GnRHa significantly increased Fshb
Frontiers in Neuroscience | www.frontiersin.org 8December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 9
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
and Lhb transcript levels compared to controls (182 ±19% and
130 ±6%, respectively, p<0.05). Treatment of cells with CB NPs
significantly amplified the GnRH-dependent stimulation of Fshb
transcript levels. Maximal amplification was attained at 75 µg/mL
of CB NPs (184 ±26% of levels in GnRHa-treated control cells,
p<0.05) and maintained at 100 µg/mL of CB NPs. In contrast,
CB NPs did not modify the induction of Lhb transcript levels by
GnRHa. In some experiments, GnRHa was replaced by activin
A (10 ng/mL) following the same protocol. As expected, activin
also significantly increased Fshb transcript levels (533 ±16%,
p<0.05), however, CB NP treatment did not affect activin
regulation unlike what was observed for GnRHa (Figure 8B).
DISCUSSION
Although there is now growing evidence that NPs affect the
female reproductive system, the underlying mechanisms are
still poorly understood (Iavicoli et al., 2013). Moreover, the
detrimental effects of NPs on adult reproductive activity have
been addressed mainly at the levels of the gonads and only little
information is available on their possible impact on pituitary
gonadotropins despite the crucial role of the latter in the
maintenance of normal reproductive function. Here, we report
that CB NPs increase FSH synthesis and release by pituitary
gonadotrope cells both in vivo in female mice and in vitro in
rat cultured pituitary cells, as well as in the gonadotrope cell line
LβT2. In contrast, LH synthesis was unaffected or only marginally
affected, highlighting the view that exposure to CB NPs may lead
to a gonadotropin imbalance. Because the dose of CB NPs used
in our study was occupationally relevant (Sanfins et al., 2011;
Bourdon et al., 2013), our results underline the fact that exposure
to CB NPs may be detrimental for female reproductive health.
The observed increase in FSH synthesis and release in female
mice subjected to inhaled CB NP exposure was not associated
with any significant changes in sex steroids or inhibin levels,
suggesting that FSH increase is not the consequence of CB
NP-induced alterations of the feedback control exerted by the
gonads on pituitary activity. In contrast to FSH, no variations
in circulating LH levels could be detected in CB NP-exposed
females. Since numerous studies in animals have illustrated that
pulsatile LH secretion closely reflects GnRH secretion by the
hypothalamus, this result strongly suggests that CB NPs do not
TABLE 2 | Effect of CB NPs on cellular viability of LβT2 cells or primary cultures of
anterior pituitary cells.
CB NPs (µg/mL) 0 25 50 75 100
LβT2 100 99 ±2 101 ±3 101 ±2 97 ±1
Pituitary primary culture 100 – 107 ±3 – 106 ±2
LβT2 cells and primary cultures of anterior pituitary cells were treated with
increasing concentrations of CB NPs (25–100 µg/mL) for 24 h and the MTT-
based colorimetric assay was used to quantify cell viability. Absorbance was read
at 575 nm and data are expressed as percentage over untreated cells and are
the mean ±SEM from 4 independent experiments. Statistical differences were
determined by non-parametric Kruskal-Wallis test followed by Dunnet’s multiple
comparison test. Data showed no statistical significance.
disrupt the activity of hypothalamic GnRH neurons. Altogether,
these results indicate that, among the three endocrine organs of
the reproductive axis – hypothalamus, pituitary and gonads – the
pituitary could be particularly sensitive to CB NPs. A general
effect of intranasal or intratracheal instillations reported for
distinct types of NPs, including carbon NPs, is to induce a
pulmonary inflammation at the site of deposition (Braakhuis
et al., 2014). Since cytokines are known to affect pituitary
endocrine activity (Haedo et al., 2009), such an inflammatory
response could have contributed to the observed changes in
gonadotropin secretion. LH synthesis and release have indeed
been reported in numerous studies to be altered by cytokines,
including IL-1β, IL-6 and TNFα, either in vitro (Russell et al.,
2001;Haedo et al., 2009) or in vivo (Dondi et al., 1998;Makowski
et al., 2020). Accordingly, the suppressive effect of inflammation
induced by LPS or endotoxin on GnRH secretion has been
documented in females from several species (Barabas et al., 2020).
However, we did not observe any significant alteration of LH
levels following exposure to CB NPs. Furthermore, even though
the effect of pro-inflammatory cytokines on FSH synthesis and
release in rodents has only been poorly addressed, IL-1βhas been
reported to decrease rather than increase FSH circulating levels
in female rats (Dondi et al., 1998) or activin-stimulated FSH
secretion in rat anterior pituitary cells (Bilezikjian et al., 1998).
Since the pattern of the gonadotropin changes observed in the
present study does not correspond with the changes reported to
be induced by cytokines, it is unlikely that the enhancement of
FSH secretion in female mice exposed to CB NPs is a consequence
of an associated inflammatory response.
Recent studies have shown that the deposit of CB NPs into
the lungs following NP entry into respiratory airways causes
defects into organs that are distant from the NP deposition site.
For example, inhalation of CB NPs during pregnancy damages
cerebrovascular functions in female mice (Zhang et al., 2019)
in addition to inducing neurodevelopmental changes in their
offspring (Umezawa et al., 2018). Other studies have shown
that intratracheal instillation of CB NPs causes genotoxicity in
the liver of female mice (Modrzynska et al., 2018) and alters
testosterone production and daily sperm production in testes of
adult male mice (Yoshida et al., 2009). One possible mechanism
explaining such changes would be a direct action of CB NPs on
these organs. Many NPs are indeed reported to translocate from
the lung into the blood circulation and this has been described in
particular for intratracheally instilled CB NPs in mice (Shimada
et al., 2006). Once in the blood, CB NPs can translocate into
secondary organs as recently observed in the liver of mice
(Modrzynska et al., 2018). As carbon NPs have been described
to accumulate in organs without being effectively eliminated
(Elgrabli et al., 2008;Czarny et al., 2014;Modrzynska et al., 2018),
it is tempting to speculate that CB NPs may accumulate into the
anterior pituitary gland and eventually alter pituitary endocrine
activity in vivo as we have observed here in cultured pituitary
cells. However, while it would have been interesting to address
the issue of CB NP localization and accumulation in vivo, this is
particularly difficult given the carbonaceous nature of the NPs of
interest. Indeed, without labeling (which would in turn modify
their physico-chemical characteristics and subsequent behavior),
Frontiers in Neuroscience | www.frontiersin.org 9December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 10
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 4 | Transmission electron microscopy study of uptake and intracellular localization of CB NPs in primary cultures of anterior pituitary cells and in LβT2 cells.
Primary cultures of rat anterior pituitary cells were treated for 24 h with CB NPs at a concentration of 50 µg/mL (A,B). LβT2 gonadotrope cells were incubated for
24 h with (C–E) or without (F) CB NPs at a concentration of 25 µg/mL. Cells were then extensively washed and processed for transmission electron microscopy as
described in Materials and Methods. In CB NP-treated cells (A–E), aggregates/agglomerates of CB NPs were freely dispersed within the cytoplasm or associated
with different subcellular compartments and organelles in primary pituitary cells (A,B) and in LβT2 cells (C–E). Arrows indicate mitochondria (MIT), secretory vesicles
(SV) and smooth endoplasmic reticulum (SER). Magnifications illustrate the close association of CB NPs with smooth endoplasmic reticulum (B,C), mitochondria (D)
or secretory vesicles (A,E). In contrast, no CB NPs could be detected in control untreated primary cultures (data not shown) and LβT2 cells (F). Note that almost no
CB NPs aggregates/agglomerates could be detected in the bottom of the culture dish or in intercellular spaces as compared to intracellular compartment (*). N,
nucleus. Scale bar: 2 µm.
CB NPs cannot be visualized in a biological system since it is not
possible to distinguish their chemical nature from the biological
background signal (Boczkowski and Lanone, 2012).
Our finding that CB NPs differentially regulate the two
gonadotropins in vivo is reinforced by our cell-based studies.
Indeed, in both primary cultures of pituitary cells and
gonadotrope LβT2 cells, CB NPs increase basal and GnRH-
stimulated Fshb expression while leaving Lhb unaffected.
Previous works using other types of CB NPs such as titanium
dioxide (Gao et al., 2012) or nickel (Kong et al., 2014) NPs also
reported effects on gonadotropin levels. However, in contrast to
CB NPs, exposure to nickel NPs increased both gonadotropin
Frontiers in Neuroscience | www.frontiersin.org 10 December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 11
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 5 | Gonadotropin gene expression in LβT2 gonadotrope cells treated with CB NPs. LβT2 gonadotrope cells were cultured for 24 h with increasing
concentrations of CB NPs (25–100 µg/mL). At the end of the incubation, cells were extensively washed, total RNA were extracted and Fshb,Lhb and Cga mRNA
levels were determined by real-time quantitative PCR. Data are expressed as percentage over untreated cells and expressed as means ±SEM from 5 to 8
independent experiments. Statistical differences were determined by non-parametric Kruskal-Wallis test followed by Dunnet’s multiple comparison test. *p<0.05
and **p<0.01 compared to no CB NPs.
FIGURE 6 | Role of the cAMP pathway in the effects of CB NPs on Fshb expression in LβT2 cells. (A) LβT2 cells were cultured for the indicated time with 100 µg/mL
of CB NPs. Phospho-CREB and total CREB protein levels were analyzed by immunoblotting. Phospho-CREB protein levels were normalized by total CREB signals
(histogram) and expressed as percentage over untreated cells. There was no significant alteration of total CREB content after CB NP treatment as assessed by
normalization with vinculin (total CREB/vinculin ratio was of 145 ±21% and 142 ±20% of control at 30 and 60 min, respectively, p>0.05). Data are the
mean ±SEM of 4 or 5 independent experiments. Statistical differences were determined by non-parametric Kruskal-Wallis test followed by Dunnet’s multiple
comparison test. *p<0.05 compared to no CB NPs. (B) LβT2 cells were cultured for 24 h with or without 100 µg/mL of CB NPs and PKA inhibitor, Rp-cAMP (100
µM). Total RNAs were extracted and Fshb mRNA levels were determined by real-time quantitative PCR. Data are expressed as percentage over control untreated
cells (0 CB NPs) and are the mean ±SEM of 8 independent experiments. Basal Fshb mRNA levels were not significantly affected by treatment with the PKA
inhibitors. Statistical differences were determined by the Mann-Whitney test. ***p<0.001 compared to respective untreated cells.
levels while titanium dioxide NPs administration selectively
decreased LH levels. The differences between these studies may
be related to differences in the type of NPs, the mode of
administration or the treatment duration. We have demonstrated
in the present study that the cAMP/PKA pathway, which
contributes to selective regulation of Fshb transcription by GnRH
(Thompson et al., 2013), is rapidly recruited by CB NPs in
LβT2 gonadotrope cells, as revealed by CREB phosphorylation.
NPs, including CB NPs, have been shown to activate different
signaling pathways such as MAPK, NF-kB or calcium pathways
(Brown et al., 2000;Sydlik et al., 2006;Marano et al., 2011;
Simon et al., 2017;Cheng et al., 2019) but this is the first
demonstration, to our knowledge, of their ability to recruit the
cAMP/PKA pathway. In our recent study using human granulosa
cells, incubation with CB NPs under the same conditions as
those used in this study rapidly activated the ERK1/2 signaling
Frontiers in Neuroscience | www.frontiersin.org 11 December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 12
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
FIGURE 7 | Analysis of MAPK and Smad2 signaling pathways in LβT2 cells
treated with CB NPs. LβT2 cells were cultured for the indicated times with
100 µg/mL of CB NPs. Total and phosphorylated Erk1/2, p38, JNK and
Smad2 were analyzed by immunoblotting as indicated in Materials and
Methods. Seven independent experiments were performed and a
representative immunoblot is shown for each protein.
pathway (Simon et al., 2017) whereas it fails to stimulate
the cAMP/PKA pathway (Simon V and Cohen-Tannoudji J,
unpublished observation). These discrepancies observed between
these two different cell types highlight the fact that NPs effects
may depend not only on their shape, size and physicochemical
properties but also on the cellular context. The recruitment
of the cAMP pathway by CB NPs may be explained by a
direct interaction of CB NPs with signaling entities regulating
intracellular cAMP levels, including enzymes such as adenylyl
cyclases or phosphodiesterases, or those located downstream of
cAMP, such as PKA or its related transcription factor, CREB.
Supporting this hypothesis is the demonstration, using the same
CB NPs than those used in the present study, that CB NPs
bind to the enzyme arylamine N-acetyltransferase, leading to
changes in its protein conformational and enzymatic activity
(Sanfins et al., 2011). It would be relevant in this context to
study whether direct interaction of CB NPs with signaling entities
of the cAMP pathway could occur in gonadotrope cells. Such
a direct activation would explain why, despite being broadly
distributed within gonadotrope cells, CB NPs could activate
this signaling pathway. The blockade of the CB NP-mediated
increase in Fshb expression by pharmacological inhibition of
PKA in our experiment suggests that the recruitment of this
pathway may be one of the mechanisms explaining the action
of CB NPs on the expression of FSH. We observed that CB
NPs amplified the effect of GnRH on Fshb but not Lhb gene
expression in LβT2 gonadotrope cells. It would be of interest
in future studies to assess whether GnRH regulation is also
disrupted in vivo. In contrast to their effects on GnRH regulation,
CB NPs did not affect the regulation exerted by activin, the
major regulator of FSH. This could probably be explained by the
inability of CB NPs to activate the Smad 2/3 signaling pathway,
which is the main pathway mediating the action of activin on
Fshb expression. Similarly, we observed no effect of CB NPs
on the expression of the early growth factor protein-1 (Egr-
1; not shown), a transcription factor key to controlling basal
and GnRH-dependent Lhb transcription in gonadotrope cells
(Halvorson et al., 1999;Wolfe and Call, 1999). This may explain,
at least in part, the inability of CB NPs to increase Lhb expression.
FSH plays a crucial role during ovarian folliculogenesis,
notably by promoting granulosa cell proliferation and estradiol
synthesis (McGee et al., 1997;Robker and Richards, 1998).
We did not, however, observe major alterations in the ovarian
activity of female mice exposed to CB NPs as revealed by non-
significant changes in circulating levels of sex steroids, and
inhibin, or the pattern of ovarian cyclicity. It is possible that
elevation of FSH was not maintained long enough to disrupt
ovarian activity. In vivo exposure to CB NPs, at least during the
period considered here, does not reproduce the effects observed
previously when the human granulosa cell line KGN was directly
exposed to CB NPs (Simon et al., 2017). There was, however,
a trend toward a decrease in circulating estradiol levels in CB
NP-treated mice. Analysis over longer periods of time would
FIGURE 8 | Effects of CB NPs on GnRH agonist stimulation of Fshb and Lhb expression in LβT2 cells. LβT2 cells were cultured for 24 h with increasing
concentrations (25–100 µg/mL) of CB NPs, washed and incubated or not for an additional 6 h with the GnRH agonist Triptorelin [GnRHa, 100 nM; (A)] or Activin A
[10 ng/mL; (B)]. Total RNAs were extracted, and Fshb and Lhb mRNA levels were determined by real-time quantitative PCR as indicated in Materials and Methods.
Data are expressed as percentage over GnRHa (A) or Activin A (B) stimulation in absence of CB NPs and are the mean ±SEM from 3 to 6 independent experiments.
Statistical differences were determined by non-parametric Kruskal-Wallis test followed by Dunnet’s multiple comparison test. *p<0.05 compared to no CB NPs.
Frontiers in Neuroscience | www.frontiersin.org 12 December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 13
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
be necessary to further assess the potential consequences of CB
NP-induced changes in FSH secretion on ovarian activity. In
addition, the complex and diverse effects of CB NPs at the level
of the whole organism, including the marked increase in FSH
secretion reported here, may also have obscured the effect of
CB NPs on the ovary. Interestingly, the absence of alterations
in ovarian endocrine activity contrasts with alterations observed
in testicular steroidogenesis and spermatogenesis in male mice
intratracheally exposed to very similar-sized CB NP particles (14
vs 13 nm) (Yoshida et al., 2009). Although such discrepancies
may be related the higher frequency of exposure of male mice
to CB NPs compared to the exposure regimen in the present
study, it may also reveal a differential susceptibility of gonads
to CB NPs according to sex, as already reported in rats exposed
to 2,3,7,8-tetrachlorodibenzo-pdioxin (Magre et al., 2012). Our
demonstration, based on in vitro and in vivo studies, of a selective
increase in FSH secretion in response to CB NP exposure,
potentially underlines an alteration of the reproductive function
as it is observed in patients or in animal models of ovarian
insufficiency (Guigon et al., 2005;Vandormael-Pournin et al.,
2015;Malini and Roy George, 2018). To summarize, our results
show that CB NPs, by directly and/or indirectly altering the
activity of anterior pituitary, could disrupt endocrine function
in adult females, and consequently lead to adverse health effects.
This reinforces the emerging idea that CB NPs, like other NPs,
act as endocrine disruptors (Yoshida et al., 2009;WHO, 2012;
Lu et al., 2013;Hutz et al., 2014;Simon et al., 2017). As human
exposure to CB NPs is increasing worldwide, additional studies
are needed to further assess the effects of such exposure on
female fertility.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding authors.
ETHICS STATEMENT
The animal study was reviewed and approved by ComEth
Anses/ENVA/UPEC under the reference #12-104 (final
approval #20/12/12-27).
AUTHOR CONTRIBUTIONS
CA: investigation, visualization, formal analysis, validation, data
curation, and writing – original draft. EP, CD, and DL’H:
investigation, data curation, and validation. GG: investigation,
data curation, validation, and writing – review and editing.
VG-M: investigation, visualization, validation, data curation,
and writing – review and editing. RC: investigation and
data curation. J-MD: funding acquisition and writing –
review and editing. SL and JB: funding acquisition, writing –
review and editing, and resources. VS: conceptualization,
methodology, visualization, investigation, supervision, project
administration, funding acquisition, and writing – original
draft. JC-T: conceptualization, methodology, supervision, project
administration, funding acquisition, and writing – original
draft. All authors contributed to the article and approved the
submitted version.
FUNDING
This study was supported by grants from the Agence nationale
de la sécurité sanitaire de l’alimentation, de l’environnement
et du travail (ANSES, Nanovhyp project), the Université de
Paris, CNRS and INSERM. CA was funded by an ANSES
postdoctoral fellowship.
ACKNOWLEDGMENTS
The authors are grateful to R. Wargnier (Université de
Paris, France) for its help in gene and signaling pathways
analyses and Florence Petit (Université de Paris, France) and
Philippe Caramelle (Université Paris Est, France) for their
help in collecting tissues for the in vivo study. The authors
are also grateful to Franck Giton and Mathieu Surenaud
(Hôpital Mondor, France) for determination of sex steroid
and gonadotropin serum levels, respectively. The authors thank
Pamela Mellon (University of California, San Diego, CA,
United States) and Albert F. Parlow (National Hormone and
Peptide Program, Harbor-UCLA Medical Center, Torrance, CA,
United States) for providing us with the LβT2 cell line and rat
LH reagents, respectively. The authors also thank S. Rasika for
English editing.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnins.
2021.780698/full#supplementary-material
REFERENCES
Bachoual, R., Boczkowski, J., Goven, D., Amara, N., Tabet, L., On, D., et al. (2007).
Biological effects of particles from the paris subway system. Chem. Res. Toxicol.
20, 1426–1433. doi: 10.1021/tx700093j
Barabas, K., Szabo-Meleg, E., and Abraham, I. M. (2020). Effect of inflammation
on female gonadotropin-releasing hormone (GnRH) neurons: mechanisms and
consequences. Int. J. Mol. Sci. 21:529. doi: 10.3390/ijms21020529
Bernard, D. J. (2004). Both SMAD2 and SMAD3 mediate activin-stimulated
expression of the follicle-stimulating hormone beta subunit in mouse
gonadotrope cells. Mol. Endocrinol. 18, 606–623. doi: 10.1210/me.2003-0264
Bilezikjian, L. M., Justice, N. J., Blackler, A. N., Wiater, E., and Vale, W. W. (2012).
Cell-type specific modulation of pituitary cells by activin, inhibin and follistatin.
Mol. Cell Endocrinol. 359, 43–52. doi: 10.1016/j.mce.2012.01.025
Bilezikjian, L. M., Turnbull, A. V., Corrigan, A. Z., Blount, A. L., Rivier, C. L.,
and Vale, W. W. (1998). Interleukin-1beta regulates pituitary follistatin and
Frontiers in Neuroscience | www.frontiersin.org 13 December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 14
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
inhibin/activin betaB mRNA levels and attenuates FSH secretion in response
to activin-A. Endocrinology 139, 3361–3364. doi: 10.1210/endo.139.7.6190
Boczkowski, J., and Lanone, S. (2012). Respiratory toxicities of nanomaterials – a
focus on carbon nanotubes. Adv. Drug Deliv. Rev. 64, 1694–1699. doi: 10.1016/
j.addr.2012.05.011
Boerboom, D., Kumar, V., Boyer, A., Wang, Y., Lambrot, R., Zhou, X., et al. (2015).
beta-catenin stabilization in gonadotropes impairs FSH synthesis in male mice
in vivo.Endocrinology 156, 323–333. doi: 10.1210/en.2014-1296
Bourdon, J. A., Saber, A. T., Jacobsen, N. R., Jensen, K. A., Madsen, A. M., Lamson,
J. S., et al. (2012). Carbon black nanoparticle instillation induces sustained
inflammation and genotoxicity in mouse lung and liver. Part. Fibre Toxicol. 9:5.
doi: 10.1186/1743-8977- 9-5
Bourdon, J. A., Saber, A. T., Jacobsen, N. R., Williams, A., Vogel, U., Wallin, H.,
et al. (2013). Carbon black nanoparticle intratracheal instillation does not alter
cardiac gene expression. Cardiovasc. Toxicol. 13, 406–412. doi: 10.1007/s12012-
013-9223- 1
Braakhuis, H. M., Park, M. V., Gosens, I., De Jong, W. H., and Cassee, F. R.
(2014). Physicochemical characteristics of nanomaterials that affect pulmonary
inflammation. Part. Fibre Toxicol. 11:18. doi: 10.1186/1743-8977-11-18
Brown, D. M., Stone, V., Findlay, P., MacNee, W., and Donaldson, K. (2000).
Increased inflammation and intracellular calcium caused by ultrafine carbon
black is independent of transition metals or other soluble components. Occup.
Environ. Med. 57, 685–691. doi: 10.1136/oem.57.10.685
Burger, L. L., Haisenleder, D. J., Dalkin, A. C., and Marshall, J. C. (2004). Regulation
of gonadotropin subunit gene transcription. J. Mol. Endocrinol. 33, 559–584.
doi: 10.1677/jme.1.01600
Cheng, Z., Chu, H., Wang, S., Huang, Y., Hou, X., Zhang, Q., et al. (2019). TAK1
knock-down in macrophage alleviate lung inflammation induced by black
carbon and aged black carbon. Environ. Pollut. 253, 507–515. doi: 10.1016/j.
envpol.2019.06.096
Counis, R., Laverriere, J. N., Garrel, G., Bleux, C., Cohen-Tannoudji, J., Lerrant, Y.,
et al. (2005). Gonadotropin-releasing hormone and the control of gonadotrope
function. Reprod. Nutr. Dev. 45, 243–254. doi: 10.1051/rnd:2005017
Czarny, B., Georgin, D., Berthon, F., Plastow, G., Pinault, M., Patriarche, G.,
et al. (2014). Carbon nanotube translocation to distant organs after pulmonary
exposure: insights from in situ (14)C-radiolabeling and tissue radioimaging.
ACS Nano 8, 5715–5724. doi: 10.1021/nn500475u
Danzo, B. J. (1998). The effects of environmental hormones on reproduction. Cell
Mol. Life Sci. 54, 1249–1264. doi: 10.1007/s000180050251
Dondi, D., Limonta, P., Montagnani Marelli, M., and Piva, F. (1998). Mechanism
of action of interleukin-1 in modulating gonadotropin secretion. In vivo and
in vitro studies. Biol. Signals Recept. 7, 55–60. doi: 10.1159/000014528
Elgrabli, D., Floriani, M., Abella-Gallart, S., Meunier, L., Gamez, C., Delalain, P.,
et al. (2008). Biodistribution and clearance of instilled carbon nanotubes in rat
lung. Part. Fibre Toxicol. 5:20. doi: 10.1186/1743-8977-5-20
Faure, M. O., Nicol, L., Fabre, S., Fontaine, J., Mohoric, N., McNeilly, A., et al.
(2005). BMP-4 inhibits follicle-stimulating hormone secretion in ewe pituitary.
J. Endocrinol. 186, 109–121. doi: 10.1677/joe.1.05988
Francois, C. M., Petit, F., Giton, F., Gougeon, A., Ravel, C., Magre, S., et al. (2017).
A novel action of follicle-stimulating hormone in the ovary promotes estradiol
production without inducing excessive follicular growth before puberty. Sci.
Rep. 7:46222. doi: 10.1038/srep46222
Gao, G., Ze, Y., Li, B., Zhao, X., Zhang, T., Sheng, L., et al. (2012). Ovarian
dysfunction and gene-expressed characteristics of female mice caused by long-
term exposure to titanium dioxide nanoparticles. J. Hazard Mater. 243, 19–27.
doi: 10.1016/j.jhazmat.2012.08.049
Garrel, G., Racine, C., L’Hote, D., Denoyelle, C., Guigon, C. J., di Clemente, N., et al.
(2016). Anti-Mullerian hormone: a new actor of sexual dimorphism in pituitary
gonadotrope activity before puberty. Sci. Rep. 6:23790. doi: 10.1038/srep23790
Garrel, G., Simon, V., Denoyelle, C., Cruciani-Guglielmacci, C., Migrenne, S.,
Counis, R., et al. (2011). Unsaturated fatty acids stimulate LH secretion via
novel PKCepsilon and -theta in gonadotrope cells and inhibit GnRH-induced
LH release. Endocrinology 152, 3905–3916. doi: 10.1210/en.2011-1167
Garrel, G., Simon, V., Thieulant, M. L., Cayla, X., Garcia, A., Counis, R., et al.
(2010). Sustained gonadotropin-releasing hormone stimulation mobilizes the
cAMP/PKA pathway to induce nitric oxide synthase type 1 expression in rat
pituitary cells in vitro and in vivo at proestrus. Biol. Reprod. 82, 1170–1179.
doi: 10.1095/biolreprod.109.082925
Giton, F., Sirab, N., Franck, G., Gervais, M., Schmidlin, F., Ali, T., et al. (2015).
Evidence of estrone-sulfate uptake modification in young and middle-aged rat
prostate. J. Steroid Biochem. Mol. Biol. 152, 89–100. doi: 10.1016/j.jsbmb.2015.
05.002
Guigon, C. J., Coudouel, N., Mazaud-Guittot, S., Forest, M. G., and Magre, S.
(2005). Follicular cells acquire sertoli cell characteristics after oocyte loss.
Endocrinology 146, 2992–3004. doi: 10.1210/en.2005-0045
Guillette, L. J. Jr., and Guillette, E. A. (1996). Environmental contaminants and
reproductive abnormalities in wildlife: implications for public health? Toxicol.
Ind. Health 12, 537–550. doi: 10.1177/074823379601200325
Haedo, M. R., Gerez, J., Fuertes, M., Giacomini, D., Paez-Pereda, M., Labeur, M.,
et al. (2009). Regulation of pituitary function by cytokines. Horm. Res. 72,
266–274. doi: 10.1159/000245928
Halvorson, L. M., Kaiser, U. B., and Chin, W. W. (1999). The protein kinase C
system acts through the early growth response protein 1 to increase LHbeta gene
expression in synergy with steroidogenic factor-1. Mol. Endocrinol. 13, 106–116.
doi: 10.1210/mend.13.1.0216
Husain, M., Kyjovska, Z. O., Bourdon-Lacombe, J., Saber, A. T., Jensen, K. A.,
Jacobsen, N. R., et al. (2015). Carbon black nanoparticles induce biphasic
gene expression changes associated with inflammatory responses in the lungs
of C57BL/6 mice following a single intratracheal instillation. Toxicol. Appl.
Pharmacol. 289, 573–588. doi: 10.1016/j.taap.2015.11.003
Hutz, R. J., Carvan, M. J. III, Larson, J. K., Liu, Q., Stelzer, R. V., King-Heiden,
T. C., et al. (2014). Familiar and novel reproductive endocrine disruptors:
xenoestrogens, dioxins and nanoparticles. Curr. Trends Endocinol. 7, 111–122.
IARC Working Group on the Evaluation of Carcinogenic Risks to Humans (2010).
Carbon Black, Titanium Dioxide, and Talc, Vol. 93. Lyon: International agency
for research on cancer.
Iavicoli, I., Fontana, L., Leso, V., and Bergamaschi, A. (2013). The effects of
nanomaterials as endocrine disruptors. Int. J. Mol. Sci. 14, 16732–16801. doi:
10.3390/ijms140816732
Iyengar, P., Lau, S., Donington, J. S., and Suh, R. D. (2016). Local therapy for
limited metastatic non-small cell lung cancer: what are the options and is there
a benefit? Am. Soc. Clin. Oncol. Educ. Book 35, e460–e467.
Komatsu, T., Tabata, M., Kubo-Irie, M., Shimizu, T., Suzuki, K., Nihei, Y., et al.
(2008). The effects of nanoparticles on mouse testis Leydig cells in vitro.Toxicol.
In Vitro 22, 1825–1831. doi: 10.1016/j.tiv.2008.08.009
Kong, L., Tang, M., Zhang, T., Wang, D., Hu, K., Lu, W., et al. (2014). Nickel
nanoparticles exposure and reproductive toxicity in healthy adult rats. Int. J.
Mol. Sci. 15, 21253–21269. doi: 10.3390/ijms151121253
Lannes, J., L’Hote, D., Fernandez-Vega, A., Garrel, G., Laverriere, J. N., Cohen-
Tannoudji, J., et al. (2016). A regulatory loop between miR-132 and miR-125b
involved in gonadotrope cells desensitization to GnRH. Sci. Rep. 6:31563. doi:
10.1038/srep31563
Lariviere, S., Garrel, G., Simon, V., Soh, J. W., Laverriere, J. N., Counis, R., et al.
(2007). Gonadotropin-releasing hormone couples to 3’,5’-cyclic adenosine-5’-
monophosphate pathway through novel protein kinase Cdelta and -epsilon
in LbetaT2 gonadotrope cells. Endocrinology 148, 1099–1107. doi: 10.1210/en.
2006-1473
Lee, J. K., Sayers, B. C., Chun, K. S., Lao, H. C., Shipley-Phillips, J. K., Bonner,
J. C., et al. (2012). Multi-walled carbon nanotubes induce COX-2 and iNOS
expression via MAP kinase-dependent and -independent mechanisms in mouse
RAW264.7 macrophages. Part. Fibre Toxicol. 9:14. doi: 10.1186/1743-8977-9-
14
Liu, F., Usui, I., Evans, L. G., Austin, D. A., Mellon, P. L., Olefsky, J. M., et al. (2002).
Involvement of both G(q/11) and G(s) proteins in gonadotropin-releasing
hormone receptor-mediated signaling in L beta T2 cells. J. Biol. Chem. 277,
32099–32108. doi: 10.1074/jbc.M203639200
Lu, X., Liu, Y., Kong, X., Lobie, P. E., Chen, C., and Zhu, T. (2013). Nanotoxicity:
a growing need for study in the endocrine system. Small 9, 1654–1671. doi:
10.1002/smll.201201517
Magre, S., Rebourcet, D., Ishaq, M., Wargnier, R., Debard, C., Meugnier, E.,
et al. (2012). Gender differences in transcriptional signature of developing rat
testes and ovaries following embryonic exposure to 2,3,7,8-TCDD. PLoS One
7:e40306. doi: 10.1371/journal.pone.0040306
Makanji, Y., Zhu, J., Mishra, R., Holmquist, C., Wong, W. P., Schwartz, N. B., et al.
(2014). Inhibin at 90: from discovery to clinical application, a historical review.
Endocr. Rev. 35, 747–794. doi: 10.1210/er.2014-1003
Frontiers in Neuroscience | www.frontiersin.org 14 December 2021 | Volume 15 | Article 780698
fnins-15-780698 December 2, 2021 Time: 14:17 # 15
Avet et al. Nanoparticles Lead to Gonadotropin Imbalance
Makowski, K. N., Kreisman, M. J., McCosh, R. B., Raad, A. A., and Breen, K. M.
(2020). Peripheral interleukin-1beta inhibits arcuate kiss1 cells and LH pulses
in female mice. J. Endocrinol. 246, 149–160. doi: 10.1530/JOE-20- 0165
Malini, N. A., and Roy George, K. (2018). Evaluation of different ranges of LH:FSH
ratios in polycystic ovarian syndrome (PCOS) – clinical based case control
study. Gen. Comp. Endocrinol. 260, 51–57. doi: 10.1016/j.ygcen.2017.12.007
Marano, F., Hussain, S., Rodrigues-Lima, F., Baeza-Squiban, A., and Boland, S.
(2011). Nanoparticles: molecular targets and cell signalling. Arch. Toxicol. 85,
733–741. doi: 10.1007/s00204-010- 0546-4
McGee, E. A., Perlas, E., LaPolt, P. S., Tsafriri, A., and Hsueh, A. J. (1997). Follicle-
stimulating hormone enhances the development of preantral follicles in juvenile
rats. Biol. Reprod. 57, 990–998. doi: 10.1095/biolreprod57.5.990
Modrzynska, J., Berthing, T., Ravn-Haren, G., Jacobsen, N. R., Weydahl, I. K.,
Loeschner, K., et al. (2018). Primary genotoxicity in the liver following
pulmonary exposure to carbon black nanoparticles in mice. Part. Fibre Toxicol.
15:2. doi: 10.1186/s12989-017- 0238-9
Naor, Z., and Huhtaniemi, I. (2013). Interactions of the GnRH receptor with
heterotrimeric G proteins. Front. Neuroendocrinol. 34:88–94. doi: 10.1016/j.
yfrne.2012.11.001
Niwa, Y., Hiura, Y., Sawamura, H., and Iwai, N. (2008). Inhalation exposure to
carbon black induces inflammatory response in rats. Circ. J. 72, 144–149. doi:
10.1253/circj.72.144
Pernasetti, F., Vasilyev, V. V., Rosenberg, S. B., Bailey, J. S., Huang, H. J.,
Miller, W. L., et al. (2001). Cell-specific transcriptional regulation of follicle-
stimulating hormone-beta by activin and gonadotropin-releasing hormone in
the LbetaT2 pituitary gonadotrope cell model. Endocrinology 142, 2284–2295.
doi: 10.1210/endo.142.6.8185
Robker, R. L., and Richards, J. S. (1998). Hormone-induced proliferation and
differentiation of granulosa cells: a coordinated balance of the cell cycle
regulators cyclin D2 and p27Kip1. Mol. Endocrinol. 12, 924–940. doi: 10.1210/
mend.12.7.0138
Roulet, A., Armand, L., Dagouassat, M., Rogerieux, F., Simon-Deckers, A., Belade,
E., et al. (2012). Intratracheally administered titanium dioxide or carbon black
nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats.
BMC Pulm. Med. 12:38. doi: 10.1186/1471-2466-12-38
Russell, S. H., Small, C. J., Stanley, S. A., Franks, S., Ghatei, M. A., and Bloom,
S. R. (2001). The in vitro role of tumour necrosis factor-alpha and interleukin-
6 in the hypothalamic-pituitary gonadal axis. J. Neuroendocrinol. 13, 296–301.
doi: 10.1046/j.1365-2826.2001.00632.x
Sanfins, E., Dairou, J., Hussain, S., Busi, F., Chaffotte, A. F., Rodrigues-Lima, F.,
et al. (2011). Carbon black nanoparticles impair acetylation of aromatic amine
carcinogens through inactivation of arylamine N-acetyltransferase enzymes.
ACS Nano 5, 4504–4511. doi: 10.1021/nn103534d
Schauer, J. J. (2003). Evaluation of elemental carbon as a marker for diesel
particulate matter. J. Expo. Anal. Environ. Epidemiol. 13, 443–453. doi: 10.1038/
sj.jea.7500298
Shimada, A., Kawamura, N., Okajima, M., Kaewamatawong, T., Inoue, H.,
and Morita, T. (2006). Translocation pathway of the intratracheally instilled
ultrafine particles from the lung into the blood circulation in the mouse. Toxicol.
Pathol. 34, 949–957. doi: 10.1080/01926230601080502
Simon, V., Avet, C., Grange-Messent, V., Wargnier, R., Denoyelle, C., Pierre, A.,
et al. (2017). Carbon black nanoparticles inhibit aromatase expression and
estradiol secretion in human granulosa cells through the ERK1/2 pathway.
Endocrinology 158, 3200–3211. doi: 10.1210/en.2017-00374
Sugino, K., Kurosawa, N., Nakamura, T., Takio, K., Shimasaki, S., Ling, N.,
et al. (1993). Molecular heterogeneity of follistatin, an activin-binding protein.
Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate
proteoglycans on the ovarian granulosa cell. J. Biol. Chem. 268, 15579–15587.
Sydlik, U., Bierhals, K., Soufi, M., Abel, J., Schins, R. P., and Unfried, K. (2006).
Ultrafine carbon particles induce apoptosis and proliferation in rat lung
epithelial cells via specific signaling pathways both using EGF-R. Am. J. Physiol.
Lung Cell Mol. Physiol. 291, L725–L733. doi: 10.1152/ajplung.00131.2006
Thackray, V. G., Mellon, P. L., and Coss, D. (2010). Hormones in synergy:
regulation of the pituitary gonadotropin genes. Mol. Cell. Endocrinol. 314,
192–203. doi: 10.1016/j.mce.2009.09.003
Thomas, P., Mellon, P. L., Turgeon, J., and Waring, D. W. (1996). The L beta T2
clonal gonadotrope: a model for single cell studies of endocrine cell secretion.
Endocrinology 137, 2979–2989. doi: 10.1210/endo.137.7.8770922
Thompson, I. R., Ciccone, N. A., Xu, S., Zaytseva, S., Carroll, R. S., and Kaiser, U. B.
(2013). GnRH pulse frequency-dependent stimulation of FSHbeta transcription
is mediated via activation of PKA and CREB. Mol. Endocrinol. 27, 606–618.
doi: 10.1210/me.2012-1281
Turgeon, J. L., Kimura, Y., Waring, D. W., and Mellon, P. L. (1996). Steroid and
pulsatile gonadotropin-releasing hormone (GnRH) regulation of luteinizing
hormone and GnRH receptor in a novel gonadotrope cell line. Mol. Endocrinol.
10, 439–450. doi: 10.1210/mend.10.4.8721988
Umezawa, M., Onoda, A., Korshunova, I., Jensen, A. C. O., Koponen, I. K., Jensen,
K. A., et al. (2018). Maternal inhalation of carbon black nanoparticles induces
neurodevelopmental changes in mouse offspring. Part. Fibre Toxicol. 15:36.
doi: 10.1186/s12989-018- 0272-2
Vandormael-Pournin, S., Guigon, C. J., Ishaq, M., Coudouel, N., Ave, P., Huerre,
M., et al. (2015). Oocyte-specific inactivation of Omcg1 leads to DNA damage
and c-Abl/TAp63-dependent oocyte death associated with dramatic remodeling
of ovarian somatic cells. Cell Death Differ. 22, 108–117. doi: 10.1038/cdd.2014.
122
Wang, R., Song, B., Wu, J., Zhang, Y., Chen, A., and Shao, L. (2018). Potential
adverse effects of nanoparticles on the reproductive system. Int. J. Nanomed.
13, 8487–8506. doi: 10.2147/IJN.S170723
WHO (2012). State of the Science of Endocrine Disrupting Chemicals. WHO.
Available online at: http://www.who.int/ceh/publications/endocrine/en/
(accessed April 14, 2021)
Wolfe, M. W., and Call, G. B. (1999). Early growth response protein 1 binds to
the luteinizing hormone-beta promoter and mediates gonadotropin-releasing
hormone-stimulated gene expression. Mol. Endocrinol. 13, 752–763. doi: 10.
1210/mend.13.5.0276
Yamawaki, H., and Iwai, N. (2006). Mechanisms underlying nano-sized air-
pollution-mediated progression of atherosclerosis: carbon black causes
cytotoxic injury/inflammation and inhibits cell growth in vascular endothelial
cells. Circ. J. 70, 129–140. doi: 10.1253/circj.70.129
Yoshida, S., Hiyoshi, K., Ichinose, T., Takano, H., Oshio, S., Sugawara, I., et al.
(2009). Effect of nanoparticles on the male reproductive system of mice. Int.
J. Androl. 32, 337–342. doi: 10.1111/j.1365-2605.2007.00865.x
Zhang, Y., Tu, B., Jiang, X., Xu, G., Liu, X., Tang, Q., et al. (2019). Exposure
to carbon black nanoparticles during pregnancy persistently damages the
cerebrovascular function in female mice. Toxicology 422, 44–52. doi: 10.1016/
j.tox.2019.04.014
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Publisher’s Note: All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations, or those of
the publisher, the editors and the reviewers. Any product that may be evaluated in
this article, or claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Citation: Avet C, Paul EN, Garrel G, Grange-Messent V, L’Hôte D, Denoyelle C,
Corre R, Dupret J-M, Lanone S, Boczkowski J, Simon V and Cohen-Tannoudji J
(2021) Carbon Black Nanoparticles Selectively Alter Follicle-Stimulating Hormone
Expression in vitro and in vivo in Female Mice. Front. Neurosci. 15:780698.
doi: 10.3389/fnins.2021.780698
Copyright © 2021 Avet, Paul, Garrel, Grange-Messent, L’Hôte, Denoyelle, Corre,
Dupret, Lanone, Boczkowski, Simon and Cohen-Tannoudji. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academicpractice. No
use, distribution or reproduction is permitted which does not comply with theseterms.
Frontiers in Neuroscience | www.frontiersin.org 15 December 2021 | Volume 15 | Article 780698
