Van Vugt-Lussenburg , Rosan B

Home , Hexestrol

Reproductive Toxicology 75 (2018) 40–48

Contents lists available at ScienceDirect

Reproductive Toxicology

j ournal homepage: www.elsevier.com/locate/reprotox

Incorporation of metabolic enzymes to improve predictivity of

reporter gene assay results for estrogenic and anti-androgenic activity

a,∗ a a

Barbara M.A. van Vugt-Lussenburg , Rosan B. van der Lee , Hai-Yen Man ,

a a,b a a

Irene Middelhof , Abraham Brouwer , Harrie Besselink , Bart van der Burg

BioDetection Systems bv, Science Park 406, 1098XH Amsterdam, The Netherlands

VU University, Department of Animal Ecology, 1081HV Amsterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Identiﬁcation and monitoring of so-called endocrine-disrupting compounds has received ample atten-

Received 28 July 2017

tion; both the OECD and the United States Environmental Protection Agency (US EPA) have designed tiered

Received in revised form 12 October 2017

testing approaches, involving in vitro bioassays to prioritize and partly replace traditional animal exper-

Accepted 14 November 2017

iments. Since the estrogen (ER) and androgen (AR) receptor are frequent targets of endocrine disrupting

Available online 21 November 2017

chemicals, bioassays detecting interaction with these receptors have a high potential to be of use in risk

assessment of endocrine active compounds. However, in many bioassays in vivo hepatic metabolism is

Keywords:

not accounted for, which hampers extrapolation to the in vivo situation. In the present study, we have

Endocrine disruption

developed a metabolic module using rat liver S9 as an add-on to human cell-based reporter gene assays.

Reporter gene assay

Metabolism The method was applied to reporter gene assays for detection of (anti-) estrogens and (anti-) andro-

Bioactivation gens, but can be extended to cell-based reporter gene assays covering a variety of endpoints related to

endocrine disruption.

1. Introduction ples include certain pesticides, polychlorinated biphenyls, and

brominated ﬂame retardants [4,6]. Other compounds are reaching

Over the last two decades, evidence has accumulated that expo- signiﬁcant levels because of their high production volumes, such as

sure to a group of chemicals with a potential to alter the normal certain phthalates, bisphenol A and alkylphenols.

functioning of the endocrine system has been associated with Although the potency of most EDCs is orders of magnitude lower

adverse health effects in both wildlife and humans. These endocrine than that of natural hormones, and the exposure concentrations

disrupting compounds (EDCs) are deﬁned as exogenous substances often relatively low, this does not mean that they pose no health

or mixtures that alter function(s) of the endocrine system, and con- hazard. Because it has been shown that the activity of many EDCs is

sequently cause adverse health effects in an intact organism, its additive, effects of low concentrations of environmental EDCs can

progeny, or subpopulations [1]. add up to pass a threshold of adversity [3,7–9].

Exposure to EDCs has been associated with a wide range of Endocrine active substances can act via various modes of action

adverse events, including disruption of hormone function, early [5], such as activation or inactivation of nuclear receptor-mediated

puberty, effects on reproduction and fertility, diabetes, obesity, and transcription, induction or inhibition of receptor expression, or

the induction of several hormone-related cancers (breast, testis, metabolism of endogenous hormones e.g. through modulation of

prostate) [2–5]. steroidogenic enzyme expression and function. The Organisation

Man-made EDCs have been found in many different areas; they for Economic Co-operation and Development (OECD) has designed

can enter the environment during production, use or disposal. a conceptual framework covering several of these steps in a draft

Several EDCs are persistent and tend to bioaccumulate; exam- testing battery consisting of different information levels [10]. The

ﬁrst level involves gathering of existing data and non-test informa-

tion, the second level involves data obtained from in vitro assays,

∗ and higher levels involve in vivo experimentation. Likewise, the US

Corresponding author.

EPA has designated a tiered testing system in their Endocrine Dis-

E-mail addresses: [email protected] (B.M.A. van Vugt-Lussenburg),

[email protected] (R.B. van der Lee), [email protected] (H.-Y. Man), ruptor Screening Program (EDSP) [11]. In both testing schedules

[email protected] (I. Middelhof), [email protected] (A. Brouwer), interaction with the estrogen, androgen, and thyroid hormone sys-

[email protected] (H. Besselink), [email protected] (B. van der Burg).

https://doi.org/10.1016/j.reprotox.2017.11.005

B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48 41

tem are key elements. In particular, the estrogen receptor (ER) and ative (no receptor activation) compounds were used. Posi-

androgen receptor (AR) have been found to be frequent targets of tives: 17␣-estradiol; 17␣-ethinylestradiol; 17␤-estradiol; 19-

endocrine disrupting chemicals. nortestosterone; 4-cumylphenol; 4-tert-octylphenol; bisphenol

This paper focuses on these two major EDC targets, using CALUX A; butylbenzyl phthalate; coumestrol; diethylstilbestrol; ethyl

reporter gene assays [12]. The ER␣ CALUX assay has been vali- paraben; genistein; kaempferol; kepone; meso-hexestrol; p,p’-

dated successfully and is included in an OECD guideline for stably methoxychlor; norethynodrel. Negatives: atrazine; corticosterone;

transfected transactivation assays to detect estrogen agonists and ketoconazole; linuron; reserpine; spironolactone.

antagonists (TG455) [13,14]. More recently a related OECD guide- In OECD TG457 [27], the VM7Luc4E2 estrogen receptor trans-

line has been drafted to assess chemical interactions with the activation test method for identifying estrogen receptor agonists

androgen receptor, and the AR CALUX assay is currently under- and antagonists, a very similar, largely overlapping list of 27

going validation for The European Union Reference Laboratory for positive and nine negative compounds is described. Positives:

alternatives to animal testing (EURL-ECVAM) to be incorporated in 17-methyltestosterone; 17␣-estradiol; 17␣-ethinylestradiol; 17ß-

this guideline [15]. Currently, in the context of the US-EPA Toxicity estradiol; 4-cumylphenol; 4-nonylphenol; 4-tert-octylphenol;

Forecaster (ToxCast) program a battery of tests has been proposed 5␣-dihydrotestosterone; apigenin; bisphenol A; butylbenzyl

to predict both of these endpoints [16]. Since application of the Tox- phthalate; chrysin; daidzein; p,p’-DDE; o,p’-DDT; di(2-ethylhexyl)

Cast battery of tests would lead to a signiﬁcant higher demand on phthalate; dicofol; dibutyl phthalate; diethylstilbestrol; estrone;

resources than running a single assay for each endpoint, we com- ethylparaben; fenarimol; genistein; kaempferol; kepone; meso-

pared the published results from the EPA battery with experimental hexestrol; p,p’-methoxychlor. Negatives: atrazine; corticosterone;

results of the CALUX assays. ﬂutamide; haloperidol; ketoconazole; linuron; procymidone;

Several review papers have been published indicating that reserpine; spironolactone.

metabolism is particularly important when testing EDCs, since Finally, in the OECD Validation Management Group for Non-

several hormonally active chemicals are known to be subject Animal testing (VMG-NA) paper [17], several preliminary reference

to metabolism [5,17,18]. Because compounds can be converted compounds are proposed for validation of metabolising systems for

into either more active or less active metabolites, neglecting to ER- and AR- assays: 17␤-estradiol; 4-tert-octylphenol; bisphenol

determine metabolism can lead to both false negatives and false A; ﬂutamide; genistein; p,p’-methoxychlor; tamoxifen; testos-

positives. terone; vinclozolin.

In the current study we show that the CALUX system has very

low basal metabolic competence. Therefore we have developed a 2.3. Chemicals

metabolic module using rat liver S9 as an add-on to the CALUX

assays; a proof-of-principle of this method was published recently Aroclor-induced Sprague-Dawley rat liver S9 fraction was

[19]. In genotoxicity testing, mimicking in vivo hepatic metabolism obtained from MolTox (11-101). Phenobarbital/␤-naphtoﬂavone

using S9 fractions is common practice [20–22]. This approach has (PB-BNF) induced Sprague-Dawley rat liver S9 mix was obtained

also been explored for EDCs [23–25]. from MolTox (11-105). NADPH-tetrasodium salt was from

The current paper describes the ﬁrst step towards a validated Applichem.

␣ method to incorporate metabolism in CALUX reporter gene assays 17 -estradiol, genistein, glucose-6-phosphate and testosterone

for detection of (anti-)estrogens and (anti-)androgens [26]. This were supplied by Wako. 3 -Phosphoadenosine-5 -Phosphosulfate

provides important data on the role of metabolism in EDC activ- (PAPS) was from Santa Cruz Biotechnology. Apigenin, chrysin, cor-

ity, which supports better options for in vitro/in vivo extrapolation. ticosterone, coumestrol, cumylphenol, daidzein, dibutyl phthalate,

For efﬁciency, the methods were made compatible with automated dicofol, diethylstilbestrol, estradiol, estrone, 17␣-ethinylestradiol,

high-throughput screening. ethyl paraben, ﬂutamide, glucose-6-phosphate-dehydrogenase,

glutathione (GSH), haloperidol, kaempferol, linuron, meso-

hexestrol, 4-nonylphenol, p,p’- Dichlorodiphenyldichloroethylene

2. Materials and methods

(p,p’- DDE), p,p’-methoxychlor, norethynodrel, 19-nortestosteron,

reserpine, spironolactone, tamoxifen, 4-tert-octylphenol, procymi-

2.1. Cell lines and cell culture

done, rifampicin, stanozolol, Uridine 5 -diphospho-glucuronic Acid

(UDPGA) and vinclozolin were obtained from Sigma. Atrazine,

The ER␣ CALUX and AR CALUX cell lines as described by Sonn-

butylbenzyl phthalate, bis-(2-ethylhexyl)phthalate, fenarimol, 17-

eveld et al., [12] are human U2-OS cells stably transfected with an

methyltestosterone and ketoconazole were obtained from Fluka.

expression construct for the full-length human ER␣- or AR recep-

Bisphenol A was obtained from Aldrich. Kepone was obtained

tor, and a reporter construct consisting of multimerized responsive

from Supelco. Dihydrotestosterone was obtained from Steraloids.

elements for the cognate receptor coupled to a minimal promoter

o,p’- Dichlorodiphenyltrichloroethane (o,p’- DDT) was obtained

element (TATA) and a luciferase gene. Cells were maintained as

from Chem Service. Tributyltinacetate was obtained from Merck.

described previously [12]. The Cytotox CALUX, used as a control

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was obtained

line for non-speciﬁc effects, consists of human U2-OS cells stably

from Cerilliant.

transfected with an expression construct constitutively expressing

the luciferase gene, and is described in [20]. Wild-type U2-OS cells

2.4. CALUX assay procedure

(HTB-96) and wild-type HepG2 cells (HB-8065; used as control cell

line to assess metabolic capacity) were obtained from ATCC.

Testing was performed in non-blinded fashion. The automated

CALUX assays were carried out as described earlier [28]. In brief,

2.2. Compound selection the assay was performed in assay medium, consisting of DMEM

without phenol red indicator (Gibco) supplemented with 5%

The 42 compounds selected for this study were based charcoal-stripped fetal calf serum (DCC), 1 × non-essential amino

on several OECD-guidelines. For validation according to the acids (Gibco) and 10 U/ml penicillin and 10 ␮g/ml streptomycin.

OECD TG455 (Test Guideline for Stably Transfected Transac- A cell suspension in assay medium was made of 1 × 10 cells/ml,

tivation In Vitro Assays to Detect Estrogen Receptor Ago- and white 384-wells plates were seeded with 30 ␮l cell suspen-

nists and Antagonists), 17 positive (agonist) and six neg- sion/well. After 24 h, exposure medium was prepared. A dilution

42 B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48

series in 0.5log unit increments of each test compound (in DMSO) 2.7. Assessment of metabolic capacity of U2-OS cells

was added to a 96-wells plate containing assay medium. Of this

exposure mixture, 30 ␮l was added to the assay plates contain- To determine whether phase I and phase II metabolic genes

ing the CALUX cells, resulting in a ﬁnal DMSO concentration of are expressed in U2-OS cells, RT-qPCR experiments were per-

0.1%. Additionally, DMSO blanks and a full dose response curve formed. HepG2 cells were used as positive control. Cells were

of the reference compounds 17␤-estradiol (E2, ER␣ CALUX), dihy- seeded in 6 cm petri dishes (U2-OS: 3.5E5 cells/dish; HepG2: 3E6

drotestosterone (DHT, AR CALUX) or tributyltinacetate (cytotox cells/dish) in culture medium. After 24 h, cells were exposed to

CALUX) were included on each plate. All samples were tested in cytochrome P450-inducers, or DMSO (vehicle control). Inducers

triplicate. The preparation of the compound dilution series as well used were: 2,3,7,8-TCDD 1E–8 M (CYP1A1/2, CYP1B1, UGT) or

as the exposure of the cells were performed on a Hamilton Star- Rifampicin 1E–4 M (CYP2A6, CYP3A4 and GST). After 16 h expo-

let liquid handling robot coupled to a Cytomat incubator. After sure, cells were harvested and mRNA was isolated using an mRNA

24 h exposure the exposure medium was removed using an EL406 isolation kit (Machery Nagel). Of each sample, 1 ␮g of mRNA was

washer-dispenser (BioTek) and 10 ␮l/well triton lysisbuffer was subsequently converted to cDNA using an iScript cDNA Synthe-

added by the EL406. Subsequently, the luciferase signal was mea- sis Kit (BioRad). Finally, an RT-qPCR was performed in an MyiQ

sured in a luminometer (InﬁnitePro coupled to a Connect Stacker, iCycler (BioRad) using SYBR green. Each 25 ␮l reaction contained

both TECAN). In order to be able to detect receptor antagonism, 12.5 ␮l 2 x iQ SYBR Green Supermix (BioRad), 0.2 ␮M sense/anti-

the assays were also performed in antagonistic mode. The assay sense primers, and cDNA from 25 ng mRNA. Cycling parameters

◦ ◦

procedure was as described above, with the only exception that were: 3 at 95 C; 40 cycles of 10 at 95 C followed by 40 at

◦

the EC50 concentration of the reference agonist was present during ; 1 at 95 C; 1 at

−12 −09 ◦

the exposure (6 × 10 M 17␤-estradiol, or 5 × 10 M stanozolol, ture>. The annealing temperature was 54 C for CYP2A6, 2E1, 3A4,

◦

respectively). The choice for stanozolol instead of the biological GSTA1, NAT1, UGT1A1 and SULT1A1, and 56 C for CYP1A1, 1A2,

agonist DHT was governed by the fact that DHT is metabolised by rat 1B1 and 36B4 (housekeeping). For primer sequences see supple-

liver S9, while stanozolol is metabolically stable [19]. The reference mentary table S1.

compounds for the antagonist assays were tamoxifen (anti-ER␣) To ascertain whether any cytochrome P450 activity could be

and ﬂutamide (anti-AR). detected in U2-OS cells, P450-Glo substrates were used (Promega).

Rat liver S9 was used as a positive control. The substrates used were

Luciferin-2B6, Luciferin-1A2, Luciferin-H (CYP2C9) and Luciferin-

IPA (CYP3A4). Luciferin-2B6 and Luciferin-1A2 reactions were

2.5. Incorporation of phase I and phase II metabolism: the rat carried out in PBS + salicylamide (3 mM), and Luciferin-H and

liver S9 procedure Luciferin-IPA reactions were carried out in medium (DMEM, no

additives). Substrate concentrations were 3 ␮M (Luciferin-2B6),

␮

When applicable, 6 l/well of a 10 x concentrated S9 mix was 4 ␮M (Luciferin-1A2), 100 ␮M (Luciferin-H) and 3 ␮M (Luciferin-

added after the addition of the exposure medium. 10 x S9 mix for IPA). Of this reaction mix, 30 ␮l was added to a 96-wells plate

phase I metabolism consisted of: 0.1 mg/ml Aroclor-induced or PB- containing either 13.000 cells/well U2-OS cells, or 1% ﬁnal v/v rat

BNF induced Sprague-Dawley rat liver S9, 2 mM NADPH, 30 mM liver S9 (aroclor-induced) + 2 mM NADPH. The plates were incu-

◦

glucose-6-phosphate, 50 mM MgCl2, and 3 units/ml glucose-6- bated for 1.5 h at 37 C to allow the P450 enzymes to convert the

phosphate dehydrogenase. In case of phase I + II metabolism, the substrates into luciferin; then, 25 ␮l supernatant was transferred to

10 x S9 mix additionally contained 20 mM GSH, 5 mM UDPGA and a clean, white 96-wells plate, and 25 ␮l detection reagent contain-

␮

20 M PAPS. Negative control incubations, where only the S9 frac- ing luciferase protein (Promega) was added. After 20 incubation at

tion was added but no cofactors, were also performed to investigate room temperature, the resulting luminescent signal was measured

whether shifts in potency could be explained by protein bind- in a luminometer for 4 s/well.

ing of the test compounds to the S9 fraction proteins, rather than metabolism.

3. Results

3.1. Analysis of 42 reference compounds on the ER˛ CALUX

2.6. Data analysis

Recently, the ER␣ CALUX has been validated for inclusion in

The luminometer data was analysed as follows; the average of

the OECD test guideline 455 [14], following an earlier successful

the triplicate wells was determined, and the average blank (DMSO)

prevalidation [13]. The reference chemicals used to assess accuracy

value was subtracted. The maximum response elicited by the ref-

and reliability of the assay were adapted from the OECD Perfor-

erence compound was set to 100% (full receptor activation), and

mance Standards document [14]. The list was composed of 17 ER

the other values were scaled accordingly. GraphPad Prism was

agonists and six negatives, selected to represent different chemical

used to ﬁt a sigmoidal curve through the data (four parameters,

classes and to span a range of potencies. In the present study, the

variable slope). The PC10 concentration was deﬁned as the concen-

list was expanded to include a total of 42 compounds. The results

tration where the response elicited by the test compound equals

of the ER␣ CALUX analysis (Table 1) show 100% concordance with

10% of the maximum response of the reference compound. For

consensus classiﬁcation published by The Interagency Coordinat-

antagonist- and cytotoxicity experiments, PC80 values were deter-

ing Committee on the Validation of Alternative Methods (ICCVAM)

mined instead, which was deﬁned as the concentration where the [29].

test compound causes a 20% decrease in the basal signal, which

In the context of the US-EPA ToxCast program a battery of 18

was set to 100%. For typical reference graphs, see supplemen-

tests has been proposed to predict ER-agonism in order to com-

tary Fig. S1. Concentrations where the compounds were cytotoxic

pensate for assay interference [16]. Fig. 1 shows the ER␣ CALUX

according to the Cytotox CALUX were excluded from the analysis.

data (1A/B) versus the published ToxCast data (1B) for the 36 com-

For metabolism studies, compounds were considered to be signiﬁ-

pounds that were present in both datasets, arranged by potency

cantly activated or inactivated if their PC10/PC80 value shifted 0.5

class. It can be observed that, for this set of reference compounds,

log units.

the predictions made by the single ER␣ CALUX assay are at least

B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48 43

Table 1

ER␣ CALUX results for 30 known ER agonists, 9 known negatives, one ER antagonist and two additional compounds. Results in PC10 (M). PC10 = concentration where the

signal elicited by the compound equals 10% of the maximum signal elicited by the reference compound 17␤-estradiol. NA (> concentration) = no activity observed up to the

highest concentration tested. N/A: not applicable, compound is not on the ICCVAM list. [*: only tamoxifen was tested as ER␣-antagonist, while all other compounds were

tested as agonists].

Compound CAS no. ER␣ CALUX PC10 (M) ICCVAM Classiﬁcation

17␣-ethinyl estradiol 57-63-6 6.3E-13 Pos

17␤-estradiol 50−28-2 6.2E-12 Pos

diethylstilbestrol 56−53-1 6.0E-12 Pos

meso-hexestrol 84−16-2 2.1E-11 Pos − norethynodrel 68 23-5 2.0E-10 Pos

␣

17 -estradiol 57-91-0 2.5E-10 Pos

estrone 53−17-7 3.2E-10 Pos

coumestrol 479−13-0 2.1E-09 Pos

bisphenol A 80−05-7 2.0E-08 Pos

5␣-dihydrotestosterone 521−18-6 3.2E-08 Pos −

genistein 446 72-0 3.4E-08 Pos

4-tert-octylphenol 140−66-9 4.3E-08 Pos

19-nortestosterone 434−22-0 6.1E-08 Pos

kepone 143−50-0 8.4E-08 Pos −

o,p’-DDT 789 02-6 1.3E-07 Pos

daidzein 486−68-8 1.3E-07 Pos

−

4-cumylphenol 599 64-4 1.8E-07 Pos

apigenin 520−36-5 2.5E-07 Pos

butylbenzyl phthalate 85−68-7 5.0E-07 Pos

p,p’-methoxychlor 72−43-5 6.3E-07 Pos

testosterone 58−22-0 6.3E-07 N/A −

kaempferol 520 18-3 7.6E-07 Pos

chrysin 480−40-0 1.0E-06 Pos

−

17-methyltestosterone 58 18-4 1.0E-06 Pos

fenarimol 60168−88-9 2.0E-06 Pos

dicofol 115−32-2 5.0E-06 Pos −

p,p’-DDE 72 55-9 5.0E-06 Pos

ethyl paraben 120−47-8 5.5E-06 Pos

−

dibutylphthalate 84 74-2 6.3E-06 Pos

4-nonylphenol 104−40-5 7.9E-06 Pos

di(2-ethylhexyl) phthalate 117−81-7 1.0E-04 Pos

atrazine 1912−24-9 NA (>3E-05) Neg

corticosterone 50−22-6 NA (>1E-04) Neg

−

ﬂutamide 13311 84-7 NA (>1E-04) Neg

procymidone 32809−16-8 NA (>1E-04) Neg

haloperidol 52-86-8 NA (>1E-04) Neg

linuron 330−55-2 NA (>1E-04) Neg

spironolactone 52−01-7 NA (>1E-04) Neg

ketoconazole 65277−42-1 NA (>1E-04) Neg

reserpine 50-55-5 NA (>1E-04) Neg

−

vinclozolin 50471 44-8 NA (>3E-05) N/A

tamoxifen 10540−29-1 1.6E-08* Antagonist

as accurate as the predictions based on multiple ER assays as pub- any of the 10 enzymes, while in HepG2 cells all enzymes were sig-

lished by ToxCast. niﬁcantly induced, except for CYP2E1, and NAT1 and SULT1A1 (but

the latter two enzymes were already expressed signiﬁcantly in the

3.2. Metabolic capacity of U2-OS cells absence of inducers).

The RT-qPCR results indicate that U2-OS shows no detectable

The performance of the ER␣ CALUX assay with the 42 ref- expression of any of the metabolic enzymes tested, except for

erence compounds was very good. However, several hormonally NAT1. In order to conﬁrm that no cytochrome p450 activity can

active chemicals are known to be subject to metabolism. The pos- be detected in U2-OS cells, luminescent P450-Glo substrates were

sibility to include hepatic metabolism in the assay, will increase used for four major p450 isoforms. Aroclor-induced rat liver S9 was

the predictivity and with that the applicability of the results used as positive control. Fig. 2C shows that no activity could be

for hazard assessment. It was therefore investigated whether detected in U2-OS cells, while S9 activity was detected for all 4

the CALUX-host cell line U2-OS has the ability to express substrates used.

metabolic enzymes. RT-qPCR was used to detect mRNA of six Overall, it can be concluded that the basic U2-OS cell line does

cytochromes p450 relevant for hepatic xenobiotic metabolism, not express the Phase II enzymes UGT, GST and SULT based on RT-

and the Phase II enzymes glutathione-S-transferase A1 (GSTA1), qPCR results, and that they do not express Phase I cytochrome p450

N-acetyltransferase-1 (NAT1), UDP-Glucuronosyltransferase 1A1 enzymes, based on both RT-qPCR results and P450-Glo activity

(UGT1A1) and sulfotransferase 1A1 (SULT1A1). HepG2 cells, known assays.

to be able to express these hepatic enzymes, were used as a positive

control. Because some enzymes are expressed only after xenobiotic 3.3. Phase I and phase II metabolism of 27 reference compounds

induction, cells were cultured in the presence of several inducers.

Fig. 2A shows that in the absence of inducers, only NAT1 seems To introduce metabolic capacity in the U2-OS-based CALUX

to be expressed in U2-OS. In HepG2, CYP3A4, NAT1 and SULT1A1 assays, we have developed a metabolic module that enables the

are expressed constitutively. Fig. 2B shows the mRNA increase com- incorporation of Phase I metabolism (cytochromes p450) and Phase

pared to non-induced cells; in U2-OS, no increase was observed for II metabolism (UGTs, SULTs and GSTs), consisting of rat liver S9

44 B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48

Fig. 1. A: ER␣ CALUX results for 27 known positives and 9 known negatives, in PC10 −LOG[(M)]. PC10 = concentration where the signal elicited by the compound equals 10%

of the maximum signal elicited by the reference compound 17␤-estradiol. The 36 compounds are grouped by potency class. Fig. 1B: ER␣ CALUX PC10 values versus ToxCast

AUC values (adapted from [Judson (2015)]) on the same 36 compounds. A colour gradient was applied on the values, ranging from green (inactive) to red (most potent). (For

interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Fig. 2. A: RT-qPCR results of non-induced U2-OS cells (white bars) and HepG2 cells (grey bars); values are expressed as% of h36B4 expression (housekeeping). Fig. 2B:

RT-qPCR results of induced U2-OS cells (white bars) and HepG2 cells (grey bars); values are expressed as fold induction relative to non-induced cells. Fig. 2C: P450-Glo results

for U2-OS cells (white bars) and rat liver S9 (grey bars), expressed as fold-induction relative to incubations without cells or S9 fraction.

fraction supplemented with the required cofactors. To investigate of S9 recommended by the OECD [18]; the results for both types of

whether shifts in potency could be explained by protein bind- S9 were very comparable (not shown).

ing of the test compounds to the S9 fraction proteins rather than Experiments performed in the absence of S9 fraction showed

metabolism, control incubations were performed with S9 fraction identical results to control experiments with S9 fraction but with-

but in the absence of any cofactors. The Phase I and Phase II cofac- out any cofactors. This indicates that the observed potency shifts

tors can be added either independently or simultaneously to the can indeed be attributed to metabolism, rather than to binding of

CALUX assays. To demonstrate the performance of this modular the test compounds to the S9 proteins. One exception was ethyl

␣

set-up, 27 compounds were analysed on the ER CALUX assay in paraben, which was always converted into a less potent estrogen

the absence of metabolism, in the presence of Phase I metabolism, in the presence of S9 fraction, irrespective of the presence of any

and in the presence of Phase I + II metabolism. Furthermore, since cofactors. This can be explained by the fact that ethyl paraben is

estrogenic compounds are often also able to act as anti-androgens, metabolised by esterases, which are present in S9 fraction and do

the experiment was also performed on the anti-AR CALUX assay not require any cofactors [31].

[28,30]. As a control for cytotoxicity and aspeciﬁc effects, the Cyto- Of the 27 compounds, 19 were active on the ER␣ CALUX when

tox CALUX was used. Cytotox CALUX results showed that the cell tested in absence of S9. The addition of Phase I enzymes had

viability was 100% for the incubations with S9 mix, indicating that mainly an activating effect: 12 compounds were more active in

the S9 mix is not toxic to the cells at the concentration used. The the presence of Phase I enzymes, including p,p’-methoxychlor

data is summarised in Table 2, and several representative graphs (Fig. 3D) which became 30-fold more potent. It is especially note-

are shown in Fig. 3. For this experiment hepatic S9 derived from worthy that of the eight compounds that showed no activity on the

␣

Aroclor-induced rats was used. The experiment was also carried ER CALUX without metabolism, seven were activated by Phase

out using hepatic S9 from PB-BNF induced rats, which is the type I enzymes (although corticosterone still did not reach the PC10-

threshold even after activation). One example is linuron, shown in

B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48 45

Table 2

PC10 (agonism) or PC80 (antagonism) values for ER␣-CALUX, anti-AR-CALUX and Cytotox-CALUX for 27 reference compounds in the absence and presence of metabolic

enzymes (phase I only, or phase I + II) are displayed. Red shading: PC10/PC80 increases >3 x upon metabolism; Green shading: PC10/PC80 decreases >3 x upon metabolism.

Orange shading: no change is observed upon metabolism. NA (>concentration) = no activity observed up to the highest concentration tested. [*: tamoxifen was tested as

ER␣-antagonist instead of −agonist; testosterone was tested as AR-agonist instead of −antagonist].

CompoundERα CALUX PC10 (M) Anti-AR CALUX PC80 (M) Cytotox CALUX PC80 (M)

No S9 S9 Phase I S9 Phase I+II No S9 S9 Phase I S9 Phase I+II No S9 S9 Phase I S9 Phase I+II 17α-ethinylestradiol 6.3E-13 1.0E-12 1.0E-12 2.0E-08 3.2E-08 3.2E-08 NA (>1E-04) NA (>1E-04) NA (>1E-04)

17β-estradiol 6.2E-12 1.0E-11 3.7E-11 1.0E-08 NA (>1E-08) NA (>1E-08)NA (>1E-08) NA (>1E-08) NA (>1E-08)

diethylstilbestrol 6.0E-12 3.2E-11 3.2E-11 2.0E-06 1.6E-06 2.0E-06 1.6E-05 2.5E-05 3.2E-05

meso-hexestrol 2.1E-11 3.9E-11 1.1E-10 1.6E-06 1.3E-06 2.0E-06 3.2E-05 7.9E-05 7.9E-05

norethynodrel 2.0E-10 4.0E-11 1.6E-10 NA (>1E-05) NA (>1E-05) NA (>1E-05)NA (>1E-05) NA (>1E-05) NA (>1E-05)

17α-estradiol 2.5E-10 5.6E-10 6.4E-10 2.4E-08 1.0E-07 1.0E-07 NA (>1E-07) NA (>1E-07) NA (>1E-07)

coumestrol 2.1E-09 2.1E-09 3.2E-09 6.4E-06 7.9E-06 5.0E-06 NA (>1E-05) NA (>1E-05) NA (>1E-05)

tamoxifen 1.6E-08* 4.0E-09* 4.0E-09*NA (>1E-06) NA (>1E-06) NA (>1E-06)NA (>1E-06) NA (>1E-06) NA (>1E-06)

bisphenol A 2.0E-08 1.6E-08 3.2E-06 8.3E-07 7.8E-07 1.2E-05 NA (>1E-04) NA (>1E-04) NA (>1E-04)

genistein 3.4E-08 3.7E-08 1.4E-07 1.0E-04 1.0E-04 1.0E-04 NA (>1E-04) NA (>1E-04) NA (>1E-04)

4-tert-octylphenol4.3E-08 1.0E-06 NA (>1E-06) 8.6E-07 5.5E-07 NA (>1E-05) NA (>1E-05) NA (>1E-05) NA (>1E-05)

19-nortestosterone6.1E-08 8.5E-09 1.4E-08 NA (>4E-05) NA (>2E-05) NA (>3E-05) 4.0E-05 2.0E-05 3.2E-05

kepone 8.4E-08 6.3E-08 6.3E-08 2.0E-06 3.6E-06 3.2E-06 4.0E-06 4.0E-06 5.0E-06

4-cumylphenol1.8E-076.3E-07 6.9E-06 1.4E-06 1.6E-06 1.0E-05 NA (>3E-05) NA (>3E-05) NA (>3E-05)

butylbenzyl phthalate 5.0E-07 2.5E-05 4.0E-05 4.2E-06 NA (>1E-04) NA (>1E-04)NA (>1E-04) NA (>1E-04) NA (>1E-04)

p,p'-methoxychlor 6.3E-07 2.0E-08 2.5E-07 2.1E-06 2.0E-07 1.6E-06 1.0E-04 1.0E-04 1.0E-04

testosterone6.3E-072.5E-07 4.0E-07 1.5E-09* 5.7E-09* 1.1E-08* 3.2E-05 1.0E-04 3.2E-05

kaempferol 7.6E-07 1.0E-06 6.4E-06 4.0E-06 3.3E-06 7.9E-06 NA (>1E-04) NA (>1E-04) NA (>1E-04)

ethyl paraben 5.5E-06 3.2E-05 3.8E-05 2.4E-05 6.3E-05 1.0E-04 NA (>1E-04) NA (>1E-04) NA (>1E-04)

corticosteroneNA (>1E-04)NA (>1E-04) NA (>1E-04)4.0E-07 4.0E-07 4.0E-07 7.9E-05 1.0E-04 7.9E-05

linuron NA (>1E-04) 3.2E-06 1.0E-05 1.1E-06 2.0E-06 2.0E-06 NA (>1E-04) NA (>1E-04) NA (>1E-04)

spironolactoneNA (>1E-04) 1.0E-05 NA (>1E-04) 3.2E-08 7.9E-07 2.4E-08 2.5E-05 NA (>1E-04) 5.0E-05

ketoconazole NA (>1E-05) 4.0E-07 1.3E-06 3.2E-06 3.2E-06 2.7E-06 1.6E-05 1.6E-05 1.0E-05

reserpine NA (>1E-05) NA (>1E-05) NA (>1E-05) 5.0E-06 3.2E-06 4.0E-06 1.3E-05 1.0E-05 1.6E-05

flutamide NA (>1E-05) 3.2E-06 NA (>1E-05) 4.1E-07 3.0E-08 2.3E-08 NA (>1E-05) NA (>1E-05) NA (>1E-05)

atrazine NA (>3E-05) 1.3E-05 2.2E-05 NA (>3E-05) NA (>3E-05) NA (>3E-05)NA (>3E-05) NA (>3E-05) NA (>3E-05)

vinclozolinNA (>3E-05) 3.2E-06 8.5E-06 7.6E-08 8.4E-08 6.3E-08 NA (>3E-05) NA (>3E-05) NA (>3E-05)

Fig. 3E. Five compounds showed less estrogenic activity upon Phase Phase II enzymes inactivated one compound. No compounds were

I metabolism. activated, i.e. converted into more toxic metabolites, by the S9

Phase II enzymes mainly resulted in decreased activity of the enzymes.

compounds: 17 compounds showed less estrogenic activity with Table 3 focuses on the nine chemicals proposed as reference

Phase I + II enzymes than when tested without S9 or with Phase I compounds in [17]. The list contains ER-agonists and −antagonists,

enzymes only. Kaempferol (Fig. 3A) and bisphenol A (Fig. 3B) are and AR agonists and −antagonists. Five of these compounds have

examples of compounds inactivated by Phase II enzymes. been described to become less active upon metabolism, while

Twenty-three compounds were active on the AR CALUX assay; four are known to become more active. Table 3 shows that the

22 of those were active as antagonists; testosterone was active CALUX-results qualitatively match the expected results in all cases.

as an AR agonist. For this assay, metabolism seemed to have less The only discrepancy was that vinclozolin is known to be con-

pronounced effects on the activity than for the ER␣ CALUX: six verted into a more potent anti-androgenic metabolite, while no

compounds were inactivated and two compounds were activated difference in AR-antagonism was observed in the AR CALUX. This

by Phase I enzymes, and ﬁve compounds were inactivated by Phase could be explained by the fact that a second metabolite is also

II enzymes. For example, butylbenzyl phthalate was inactivated by formed, and this second metabolite is a less potent anti-androgen

Phase I enzymes (Fig. 3C), while ﬂutamide was converted into a [32]. The effects of both metabolites combined could cancel each

10-fold more potent anti-androgen (Fig. 3F). other out. Both metabolites are estrogenic, and indeed an increase

Only 10 of the compounds were active on the Cytotox CALUX, in estrogenicity is seen upon Phase I metabolism in the ER␣

meaning that they caused >20% cytotoxicity at the highest con- CALUX, indicating that vinclozolin is indeed metabolised by Phase

centration used. Phase I enzymes inactivated two compounds, and I enzymes in the current experimental set-up.

46 B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48

Fig. 3. Dose-response curves of kaempferol, bisphenol A, p,p-methoxychlor and linuron in ER␣ CALUX, and butylbenzyl phthalate and ﬂutamide in anti-AR CALUX, assayed

in the absence of metabolic enzymes (black, open circles) and in presence of phase I metabolic enzymes (red, closed circles) or phase I and phase II metabolic enzymes

(blue, squares). The compounds in the top row are inactivated by the metabolic enzymes, while the compounds in the bottom row are activated. (For interpretation of the

references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

Table 3

expected versus observed effects of Phase I and Phase II metabolism on ER␣-CALUX- and AR-CALUX activity for nine compounds suggested as preliminary reference substances

for validation of metabolising systems to be incorporated into endocrine assays.

Compound Activity Metabolism (expected) Metabolism (observed)

17␤-estradiol ER agonist Phase I/II to less active metabolites Decreased ER␣ CALUX activity (Ph I/II)

4-tert-octylphenol ER agonist Phase I/II to inactive metabolites Decreased/no ER␣ CALUX activity (Ph I/II)

Bisphenol A ER agonist Phase II to inactive metabolite Decreased ER␣ CALUX activity (Ph II)

Genistein ER agonist Phase I to similarly active metabolite; Similar ER␣ CALUX activity (Ph I); Decreased ER␣ CALUX

Phase II to less active metabolites activity (Ph II)

p,p’-methoxychlor ER agonist Phase I to more active metabolite Increased ER␣ CALUX activity (Ph I)

Tamoxifen ER antagonist Phase I to more active metabolite Increased ER␣ CALUX activity (Ph I)

Testosterone AR agonist Phase I/II to less active metabolites Decreased AR CALUX activity (Ph I/II)

Vinclozolin AR antagonist; ER-agonist Phase I to more active metabolites No effect on AR CALUX antagonism; Increased ER␣ CALUX

agonism (Ph I)

Flutamide AR antagonist Phase I to more active metabolites Increased AR CALUX antagonism (Ph I)

4. Discussion Therefore, no interference caused by receptor cross-reactivity, or

up/down regulation of ER␣ expression, is expected to occur.

␣

Recently, the ER CALUX assay has been included in the OECD Since many endocrine active substances are known to be

guidelines TG455, meeting all performance standards for stably affected by hepatic metabolism, it would be an important step for-

transfected transactivation in vitro assays to detect estrogen ago- ward in risk assessment if this step could be incorporated into

nists and scoring 100% concordance of classiﬁcation [14,28]. Both in vitro test systems for endocrine active substances. Not only

the ER␣ and AR CALUX human reporter gene assays have proven would this improve the quality and predictive capacity of in vitro

to be very useful for the detection of chemicals acting on the toxicity testing; it would also reduce the need of performing addi-

hormone system; both assays have been applied successfully in tional animal tests. However, like most immortalized cell lines, the

studies involving assessment of EDC effects and reproductive tox- host cell line U2-OS used to generate CALUX assays does not express

icity [12,13,26,28,33–35]. The assays have the potential to play a signiﬁcant amounts of metabolic enzymes. Therefore, in vivo hep-

valuable role in in vitro toxicity screening for endocrine active sub- atic metabolism is not mimicked in most stable reporter gene stances. assays.

␣

The ER CALUX results on the 36 reference compounds com- To compensate for the lack of metabolic capacity of the CALUX

pare favourably to the results published by the US-EPA ToxCast cell lines, a metabolic fraction was added modularly to the reporter

program. It turned out that the ER␣ CALUX predictions were at least gene assays. This modular approach has several advantages over

as accurate, when only one assay was used instead of the panel of 18 systems expressing metabolic enzymes endogenously. By compar-

assays used for ToxCast. The motivation to use a battery of multiple ing the reporter gene assay results of an analysis in the absence

assays was to eliminate assay interference. However, as opposed and presence of metabolic enzymes, information is obtained on the

to several other ER␣ reporter gene cell lines, the ER␣ CALUX host extent of bio(in)activation, as opposed to measuring only the activ-

cell line U2-OS does not express nuclear receptors endogenously. ity of the mixture after metabolism, and thus remaining unaware

of the activity of the parent compound. Additionally, the modu-

B.M.A. van Vugt-Lussenburg et al. / Reproductive Toxicology 75 (2018) 40–48 47

lar approach enables to distinguish between Phase I (activating or 17␤-estradiol, 17␣-estradiol, diethylstilbestrol, tamoxifen, genis-

inactivating) and Phase II (only inactivating) metabolism. This way, tein, 4-cumylphenol, p,p’-methoxychlor and testosterone. Atrazine

information is obtained on the possible bioactivation of a com- and ﬂutamide were inactive in the database and in our study,

pound, as well as on its metabolic stability. Such information is although both compounds can be activated by Phase I metabolism,

important for risk assessment, because it facilitates prediction of and subsequently inactivated by Phase II metabolism. Bisphenol A

the fate of a compound in vivo. Another advantage is that the modu- was classiﬁed as ambiguous, with 37 positive results versus 6 nega-

lar approach with S9 fraction results in reproducible enzyme levels, tive results. Our results show that bisphenol A is a potent estrogen,

while endogenous expression often depends on cell status and/or but it is inactivated by Phase II metabolism. For 4-tert-octylphenol,

the presence of inducers. three positive and one negative result were found in the database;

For 23 of the 27 compounds tested in this study the endocrine Table 2 shows that also this compound is a potent estrogen, but

activity was affected by metabolism. Twelve compounds (44%) it is efﬁciently inactivated by both Phase I and II metabolism. For

were signiﬁcantly bioactivated by Phase I enzymes in ER␣ CALUX, butylbenzylphthalate, kaempferol and ethyl paraben, only negative

of which six more than one order of magnitude. Inactivation entries were found in the database. In our study, these compounds

occurred in 18 (67%) compounds by Phase II enzymes. These per- are clearly positive in the absence of S9 fraction, but they are con-

centages underline the importance of incorporating metabolism in verted into inactive metabolites in the presence of S9 fraction.

in vitro bioassays; for example the 30-fold increase in estrogenic Overall, the ER␣ CALUX data presented in this study is in good

potency of p,p’-methoxychlor, or the 160-fold decrease in potency agreement with the uterotrophic database [40]. The added value

of bisphenol A has serious consequences for risk assessment of such of the addition of S9 fraction to the reporter gene assay is shown

compounds. in the case of butylbenzylphthalate, kaempferol and ethyl paraben,

Many of the observed effects have been previously observed where the results only match when Phase I metabolism is taken

in in vivo studies described in literature. For example the con- into account. Additionally, like in the case of bisphenol A and 4-

version of tamoxifen by Phase I enzymes into the more potent tert-octylphenol, information on metabolism can help to explain

anti-estrogen 4-OH-tamoxifen [36], or the metabolic activation ambiguous results in the uterotrophic database.

of p,p’-methoxychlor into the more potent estrogen and anti- The procedure is simple, cost-effective and high-throughput-

androgen 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) compatible. Because of the low concentration of S9 fraction used

[25]. Also the conversion of testosterone into less androgenic (0.03% v/v), unwanted phenomena such as cytotoxicity, or potency

metabolites [37,38] and the Phase II glucuronidation of bisphenol A shifts due to test compounds binding to proteins in the S9 fraction,

to the inactive bisphenol A-glucuronide have been described before were not observed. The procedure could be extended to include

[39]. The metabolic fate of all nine (anti-)androgenic/estrogenic endpoints related to the thyroid hormone system, such as thy-

chemicals proposed as reference compounds in [17] is predicted roid receptor (ant)agonism [28,34] and Transthyretin (TTR) binding

correctly by the reporter gene assays when supplemented with rat [41,42]. In addition, options to look at inhibition of steroidogene-

liver S9 fraction. sis enzymes using reporter gene assays [43] can be supplemented

In a recent study where the ER␣ CALUX and anti-AR CALUX with S9 fraction in similar fashion.

were combined with S9 fraction, similar results were shown In conclusion, the modular addition of S9 fractions with cofac-

for the metabolic activation of ﬂutamide (10-fold more anti- tors for Phase I and Phase II metabolic enzymes is a straightforward

androgenic) and p,p’-methoxychlor (30-fold more estrogenic) [19]. procedure to improve the predictive capacity of in vitro cell-based

Also in an MCF-7 based ER reporter gene assay, activation of reporter gene bioassays. The technique can be applied to cell-based

p,p’-methoxychlor was observed upon addition of 0.1% rat liver reporter gene assays covering a variety of endpoints related to

S9 to the assay [24]. However, for 17␤-estradiol a 35-fold right- endocrine disruption, and may greatly facilitate regulatory toxicity

shift was reported, while we observed a shift that was merely testing whilst reducing animal testing.

two-fold, and therefore classiﬁed as ‘not signiﬁcant’. The authors

reported that the shift was consistent, but of variable magnitude. Funding

In another study, an ER reporter gene assay was preceded by a

preincubation with a high concentration (3%) of rat liver S9 for

This work was supported by the BE-Basic consortium; and by

p,p’-methoxychlor and 17␤-estradiol [25]. Also here, an increased

EU-ToxRisk (European Union’s Horizon 2020 research and innova-

potency was observed for methoxychlor, and a decrease in potency

tion programme) [grant number 681002].

for 17␤-estradiol, but in this study the potency of 17␤-estradiol

decreased only 3-fold, which is more in line with our ﬁndings.

Appendix A. Supplementary data

Apparently the ratio of the various metabolites formed from 17␤-

estradiol is variable and/or dependent on the exact conditions of

Supplementary data associated with this article can be found,

the incubation, and may have a profound effect on the estrogenic

in the online version, at https://doi.org/10.1016/j.reprotox.2017.

activity of the ﬁnal mixture.

11.005.

Taxvig et al. published a study where the T-Screen reporter

gene assay was preceded by preincubations with either human or

rat liver S9 fraction [23]. Their ﬁndings showed that, according to References

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