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Biochimie Open 3 (2016) 49e55
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Review

Dimethyl-Benz(a)anthracene: A mammary carcinogen and a neuroendocrine

disruptor
Bernard Kerdelhue a,*,1, Claude Forest b,1, Xavier Coumoul b,1
a
CNRS UMR 8601, Chimie et Biochimie Pharmacologiques et Toxicologiques, Universite Paris Descartes, Faculte des Sciences Fondamentales et Biomedicales,
45 rue des Saints Peres, 75006 Paris, France
b
INSERM UMR-S 1124, Universite Paris Descartes, PRES Sorbonne Paris Cite, Pharmacologie Toxicologie et Signalisation Cellulaire, Faculte des Sciences
Fondamentales et Biomedicales, 45 rue des Saints Peres, 75006 Paris, France
Received 8 July 2016; accepted 30 September 2016
Available online 8 October 2016

Abstract

Polycyclic Aromatic Hydrocarbons (PAHs) are potent carcinogens. Among these, dimethylbenz(a)anthracene (DMBA) is well known for its
capacity to induce mammary carcinomas in female Sprague-Dawley (SD) rats. Ovariectomy suppresses the susceptibility of this model to
DMBA, thus suggesting that the inducible action of the carcinogen depends on ovarian hormones. The promotion of DMBA-induced adeno-
carcinoma is accompanied by a series of neuroendocrine disruptions of both Hypothalamo-Pituitary-Gonadal (HPG) and Hypothalamo-Pituitary-
Adrenal (HPA) axes and of the secretion of melatonin during the latency period of 2 months that precedes the occurrence of the first mammary
tumor. The present review analyses the various neuroendocrine disruptions that occur along the HPG and the HPA axes, and the marked
inhibitory effect of the carcinogen on melatonin secretion. The possible relationships between the neuroendocrine disruptions, which essentially
consist in an increased pre-ovulatory secretion of 17b-estradiol and prolactin, associated with a marked reduction of melatonin secretion, and the
decrease in gene expression of the receptors for aryl-hydrocarbons receptor (AhR) and 17b-estradiol (ERa; ERb) are also discussed.
© 2016 The Authors. Published by Elsevier B.V. on behalf of Société Française de Biochimie et Biologie Moléculaire (SFBBM). This is an open
access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Mammary cancer; Neuroendocrine disruption; Dimethylbenz(a)anthracene; Female rat

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2. The ovarian cycle of the female rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3. DMBA, the brain and Hypothalamo-Pituitary axes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.1. Disruption of the Hypothalamo-Pituitary-Gonadal (HPG) axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.2. Disruption of the Hypothalamo-Pituitary-Adrenal (HPA) axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.3. Disruption of the secretion of melatonin from the pineal gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4. The mechanisms of action triggered by DMBA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.1. DMBA: an estrogen-mimetic in the short-term? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2. DMBA: acting negatively on receptor function in the long-term? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Abbreviation list: ACTH, Adrenocorticotropic hormone; AhR, Aryl hydrocarbon Receptor; ARNT, AhR nuclear translocator; CRH, Corticotropin releasing
hormone; CYP, Cytochromes P450; DMBA, Dimethylbenz(a)anthracene; E2, 17b-estradiol; ERa and ERb, Estrogen receptor; FSH, Folliculo-Stimulating Hormone;
GnRH, Gonadotropin-Releasing Hormone; HPA, Hypothalamo-Pituitary-Adrenal; HPG, Hypothalamo-Pituitary-Gonadal; LH, Luteinizing hormone; PAHs, Poly-
cyclic Aromatic Hydrocarbons; PRL, Prolactin; SD, Sprague-Dawley; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XRE, Xenobiotic response elements.
* Corresponding author.
E-mail address: [email protected] (B. Kerdelhue).
1
Universite Paris Descartes, Sorbonne Paris Cite, Paris, France.

http://dx.doi.org/10.1016/j.biopen.2016.09.003
2214-0085/© 2016 The Authors. Published by Elsevier B.V. on behalf of Société Française de Biochimie et Biologie Moléculaire (SFBBM). This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
50 B. Kerdelhue et al. / Biochimie Open 3 (2016) 49e55

4.3. DMBA: a complex action on two receptors subsets? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6. Conflict of interest: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

1. Introduction genes coding for the Aryl hydrocarbon Receptor (AhR) and
for both estrogen receptors (ERa and ERb), are also discussed.
A large number of polycyclic Aromatic Hydrocarbons
(PAHs) have been identified as potent carcinogens and/or 2. The ovarian cycle of the female rat
endocrine disruptors. PAHs are widely distributed in the
environment as a result of incomplete combustion of fossil The ovarian cycle of the female rat is much shorter (4-days)
fuels and other organic molecules and are common contami- than that in the woman (28-days). However, the 2 major
nants of terrestrial and aquatic ecosystems [1,2 and therein]. events, the follicular and the luteal phases, occurring during
Numerous compounds of the PAH family have been associated the 4-days cycle in the female rat are a simple reduction in
with the development of tumors in humans and experimental time of the same events occurring during the 28-days cycle in
animals [3 and therein]. Among these, dimethylbenz(a) the woman.
anthracene (DMBA) was extensively used as a model to study In the female rat, the duration of the follicular phase is of 2
the molecular mechanisms of mammary carcinogenesis in days versus 14 days in the woman. The follicular phase is
Sprague-Dawley (SD) female rats [4]. Of particular interest is characterized by the secretion of the Folliculo-Stimulating
the observation that a single intra-gastric dose of DMBA is Hormone (FSH) in the blood, which in turn, stimulates the
sufficient to induce mammary adenocarcinoma in contrast to secretion of 17b-estradiol (E2). At the end of that phase, a pre-
the archetypal PAHs, benzo(a)pyrene or 3-methyl-cholanthrene ovulatory surge of E2 occurs which triggers pre-ovulatory
for which the administration of several doses are needed [4]. surges of hypothalamic Gonadotropin-Releasing Hormone
The induction of mammary tumors by DMBA in rats is (GnRH), pituitary Luteinizing Hormone (LH), FSH and Pro-
well documented. After metabolic activation in the mammary lactin(PRL). The surges of LH and FSH are provoked by the
gland [5], the metabolites of the carcinogen interact with surge of the hypothalamic GnRH in the Hypothalamo-
rapidly proliferating cells in the terminal end buds, forming Pituitary portal blood system. The pre-ovulatory LH surge
DNA adducts and subsequent mutations, which in turn play a provokes the ovulation of the mature follicle. The luteal phase
role in their transformation to malignant cells [6e8]. DMBA is lasts 2 days in the female rat, versus 14 days in the woman and
a highly lipophilic molecule. The breast contains significant is characterized by the secretion of progesterone in response to
amount of adipose tissue, in which DMBA can concentrate at the secretion of LH.
the epithelium contact before its metabolic activation and this Another important difference between the ovarian cycles of
property certainly correlates with the higher activity of DMBA the female rat and that of the woman is the absence of
at the mammary level. Other PAHs certainly act along the menstruation in the rodent. For that reason, the cycle of the
same line of events. female rat is named estrous and the cycle of the woman is
Sprague-Dawley (SD) rats are DMBA-sensitives, with a named menstrual. In addition, there is no pre-ovulatory PRL
peak of susceptibility at 55e60 days of age. Ovariectomy surge during the menstrual cycle of the woman.
suppresses this susceptibility, suggesting that the inducible
action of the carcinogen is related to ovarian hormones and the 3. DMBA, the brain and Hypothalamo-Pituitary axes
ovarian cycle [9].
The promotion of DMBA-induced adenocarcinoma is After one single administration (15 mg/rat by gastric intu-
accompanied by a series of neuroendocrine disruptions of the bation), DMBA is able to cross the blood-brain barrier and to
Hypothalamo-Pituitary-Gonadal (HPG) and Hypothalamo- induce the expression of cytochromes P450 (CYP) in endo-
Pituitary-Adrenal (HPA) axes and of the secretion of mela- thelial cells of blood brain interfaces [10]. Furthermore,
tonin during the latency period of 2 months that precedes the labeled [H3]-DMBA and its metabolites have been identified
occurrence of mammary tumors. The aim of this review is to in different area of the brain, including the hypothalamus, the
report on the various neuroendocrine disruptions occurring pituitary gland, the pineal gland and the frontal cortex (Can-
along the closely interconnected HPG and HPA axes, and to onico M, Lenoir V and Kerdelhue B, unpublished results). In
analyze the marked effect of the carcinogen on melatonin addition, DMBA disorganizes the neuronal plasma membranes
secretion and their influence on the carcinogenic process [10]. in rat hypothalamus [11]. This pollutant is then able to disrupt
The potential relationships between these neuroendocrine the physiological processes regulated by those brain structures,
disruptions and the observed effects on the expression of the including Hypothalamo-Pituitary axes.
 B. Kerdelhue et al. / Biochimie Open 3 (2016) 49e55 51

Fig. 1. The 17b-estradiol, Luteinizing hormone (LH) and prolactin surges in female Wistar rats, a DMBA-insensitive strain (up), and in female Sprague-Dawley
rats, a DMBA-sensitive strain (bottom) in the afternoon of the proestrus at any estrous cycle after treatment.

3.1. Disruption of the Hypothalamo-Pituitary-Gonadal contrast, in female Wistar rats, a DMBA-resistant strain, the
(HPG) axis pre-ovulatory or basal LH, FSH, PRL and E2 secretions were
not modified at any time of the estrous cycle after treatment
In female SD rats, a DMBA-sensitive strain, DMBA with the carcinogen (Fig. 1). There was also no reported dif-
significantly inhibited LH and FSH surges and stimulated the ference for any other pituitary hormone at any time of the
PRL and the E2 surges in the afternoon of the proestrus at any estrous cycle [12,13]. The pituitary gland is not the only area
estrous cycle after treatment [12,13] (Fig. 1, Table 1). No affected by DMBA exposure: ovariectomized SD rats that
difference was found for LH, FSH and PRL or E2 concen- were pre-treated with DMBA exhibited a reduced GnRH
trations in the blood at other times of the estrous cycle. In release, as measured in vitro using synaptosomes from the
addition, no difference was found for the level of any other mediobasal hypothalamus, together with a blunted in vivo LH
anterior pituitary hormone at any time of the estrous cycle. In release in response to E2 [14]. In addition, disturbances of the

Fig. 2. mRNA expression of the AhR, ERa and ERb genes in the hypothalamus and the pineal gland after one single administration of DMBA.
52 B. Kerdelhue et al. / Biochimie Open 3 (2016) 49e55

Fig. 3. The neuro-endocrine mechanisms of action triggered by DMBA on both Hypothalamo-Pituitary-Adrenal and Hypothalamo-Pituitary-Gonadal axes in the
SD female rat: DMBA alters melatonin secretion by the pineal gland, the secretion of GnRH and CRH by the hypothalamus which eventually impacts the pituitary
(secretion of FSH, LH, PRL and ACTH), adrenal gland (secretion of corticosterone) and ovary functions (secretion of E2) and the feedback loops.

expression of the GnRH and its pituitary receptor genes 3.2. Disruption of the Hypothalamo-Pituitary-Adrenal
occurred throughout the estrus cycle [15]. The early and (HPA) axis
persistent alterations in the hypothalamic centers that were
involved in the neuroendocrine cyclicity of the HPG axis very The HPA axis includes the hypothalamus that secretes
likely resulted from a direct action of DMBA. corticotropin releasing hormone (CRH) which stimulates the
In pre-menopausal women, the development of breast release of adrenocorticotropic hormone (ACTH) from the pi-
cancer is associated with reduced plasma GnRH and of FSH tuitary gland, thus allowing the secretion of glucocorticoids
secretion in post menopausal women, thus suggesting the from the adrenal cortex. Glucocorticoids control stress re-
existence of neuroendocrine disruptions in the woman that actions, the digestive and immune system, mood and emotions,
could be involved in tumor promotion and that could be sexuality, and energy storage and expenditure. Along the HPA
mimicked in a sensitive rat model with a single dose of a axis, a long-term disregulation of the circadian and E2-induced
common PAH [16]. corticosterone secretion was observed in DMBA-treated rats
[17] (Table 1). The most important disregulation observed after
administration of DMBA, is the almost complete abolition of
Table 1 the circadian rhythm of plasma corticosterone. This fact could
A summary of the DMBA effects on several neuroendocrine axes. be responsible for the change in the behavior of female rats in
Hypothalamus ANT. Pineal Ovary Adrenal preparation for mammary cancers. In an open space system,
pituitary gland gland DMBA-treated female rats exhibit a long-term reduction of
AhR
20%
20% anxiety level after treatment with the mammary carcinogen
ER-alpha
15%
20% [18]. A relationship between breast cancer and stress response
ER-beta
25%
20% has been documented in the woman [Ref. in [19]].
GnRH þ30%
CRH
20%
LH
30%
3.3. Disruption of the secretion of melatonin from the
FSH
20% pineal gland
PRL þ30%
Melatonin
70% Melatonin is a well-documented hormone secreted in a
17b-E2 þ20% circadian fashion by the pineal gland and is implicated in
Corticosterone
60%
 B. Kerdelhue et al. / Biochimie Open 3 (2016) 49e55 53

many physiological interactions [20]. The circadian synthesis estrogen receptors (ERs) and the Aryl hydrocarbon Receptor
and release of melatonin is driven by the suprachiasmatic (AhR), which have been shown to interact [29]. The AhR is a
nucleus of the hypothalamus [21]. transcription factor, expressed in the central nervous system
A single intragastric administration of DMBA to SD female (including the hypothalamus, the hippocampus & the pitui-
rats induces a long-term marked decrease in the spontaneous tary) [30]. Upon ligand binding (mostly xenobiotics), the AhR
and norepinephrine-induced melatonin secretion from the pi- translocates to the nucleus where it heterodimerizes with the
neal gland, in an in vitro perfusion system (Table 1) [22]. AhR nuclear translocator (ARNT) and the complex binds to
Melatonin secretion was completely blunted when E2 was xenobiotic response elements (XRE), regulating positively or
present in the perfusion medium, even at a low and physio- negatively the transcription of target genes, including those
logical concentration (10
10 M) [22]. In contrast, the E2 encoding detoxification enzymes [31].
antagonist tamoxifen, stimulated melatonin secretion [22].
Last but not least, melatonin had a preventive and curative 4.1. DMBA: an estrogen-mimetic in the short-term?
effect on the mammary carcinogenesis induced by DMBA
[23]. DMBA was previously shown to mimic neuroendocrine
In the woman, it can be envisioned that the long-term in- effects of E2 [11,32]. These effects were very likely provoked
hibition of melatonin secretion by ovarian steroids might play by the binding of DMBA to ERs or to the AhR (or to both
a major role during the promotion phase of the mammary receptors since an intrinsic function of the AhR was docu-
carcinogenesis. Whether or not a similar decrease in the mented as a possible key factor in female reproduction) [33].
secretion of melatonin from the pineal gland could be gener- Several studies showed that E2 down-regulated the expression
ated in the woman by non-identified carcinogenic environ- of both GnRH and GnRH-R genes [15]. We could then hy-
mental factors and therefore could constitute a promotional pothesize that DMBA acts similarly to E2 along the HPG axis,
factor for breast cancer remains to be proven. Nevertheless, a wherein it binds to ERs and subsequently delays the secretion
disruption of melatonin secretion provoked by rotating night of GnRH induced by the hypothalamus and the pituitary FSH/
shifts increases the risk of breast cancer in the woman [24]. In LH surges.
addition, low levels of urinary melatonin have been associated
with an increased risk of breast cancer in premenopausal 4.2. DMBA: acting negatively on receptor function in the
women and increased melatonin levels have been related to a long-term?
lower risk of invasive breast cancer in postmenopausal women
[25]. In this context, any modification of melatonin secretion After one single administration of DMBA and although
under the influence of breast cancer carcinogens should be this xenobiotic is not a persistent organic pollutant, a long
placed under scrutiny. lasting and similar decrease in the expression of AhR, ERa
The cellular and molecular actions of melatonin in relation and ERb genes in the hypothalamus and the pineal gland was
with breast cancer have been well documented (ref in observed (Fig. 2) [34]. No change in the expression of
[26,27]); melatonin oncostatic action is mainly due to its anti- the above-mentioned genes occurred in the hippocampus, a
estrogenic action involving the estrogen receptor alpha (ERa) non-neuroendocrine structure [34]. In view of the well-
(ref. in [28]). documented action of estrogens on both HPG and HPA axes
and of the strong interconnections between those two axes
4. The mechanisms of action triggered by DMBA [35], the decrease in expression of the ERa and ERb genes
are very likely responsible for the constant disregulations of
To summarize (Fig. 3), DMBA disrupts several key factors the HPG and HPA axes. We could then hypothesize that the
of both Hypothalamo-Pituitary axes in SD female rats: 1) along E2-induced corticosterone secretion along the HPA axis,
the HPG axis, we could hypothesize that the decreased could be impacted on the long-term by the action of DMBA
expression of GnRH (and its secretion by the hypothalamus) on the expression of ER [15].
and pituitary GnRH-R partly explains the blockade of LH and
FSH surges and the maintenance of high E2 blood concentra- 4.3. DMBA: a complex action on two receptors subsets?
tions. It is noteworthy that PRL secretion by the pituitary gland
is increased (which is consistent with the fact that PRL Since DMBA is a polycyclic aromatic hydrocarbon and
secretion does not depend on GnRH); 2) along the HPA axis, AhR ligand, we can hypothesize that, like most AhR ligands,
DMBA probably counteracts the effect of E2 on corticosterone it displays anti-estrogenic properties [36,37]. For example,
secretion and abolishes almost completely the circadian female SD rats that were treated with TCDD (2,3,7,8-
secretion of the latter hormone. 3) In the melatoninergic sys- tetrachlorodibenzo-p-dioxin), an AhR high affinity ligand,
tem, DMBA and E2 act similarly for the blunting of the display delayed incidence of mammary tumors [38]. Only a
secretion of this hormone. limited number of studies were carried out on the nervous
Overall, DMBA seems to exert pro- and anti-estrogenic system of mammals with DMBA but alternative ligands,
effects on the neuroendocrine system, that can be partly including TCDD, were sometimes used [39]. Experimental
explained by recent data and the direct/indirect effects of this studies showed that animals exposed to AhR ligands dis-
carcinogen on several key cellular receptors, including the played multiple symptoms including defects of motor
54 B. Kerdelhue et al. / Biochimie Open 3 (2016) 49e55

coordination or spatial memory, two processes that can be Acknowledgements

regulated by estrogens and ERs [40e43]. The interplay be-
tween the AhR and ERs is indeed quite complex: 1) AhR This work was funded by the Universite Paris Descartes
ligands decrease E2 blood concentration and stimulate the PRES Sorbonne Paris Cite, the Institut National de la Sante et
expression of both cytochromes P450 1A1 and 1B1 that use de la Recherche Medicale (INSERM), the Agence Nationale
E2 as a substrate [30,44,45]. 2) AhR exerts a competitive de la Recherche (ANR), ToxAhBrain, Programme CESA
action on ERs, using the same transcriptional co-activators (ANR-13-CESA-0005-01), the Centre National de la Recher-
(CBP/p300) [39]. 3) Inhibitory XREs have been character- che Scientifique (CNRS).
ized in the promoters of estrogen-responsive genes [46]. 4)
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Conflict of interest: pituitary receptor gene expression, during the promotion stage, in female
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