2-isopropyl-9H-thioxanthen-9-one

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The aim of the present study was to contribute to this, starting with the hazard identification of ITX. Initial toxicological studies were performed with in vitro models, aiming at reducing the use of laboratory animals. Based on the structural similarities between ITX and known Arylhydrocarbon receptor agonists (AhR) like dioxins and furanocoumarins, it was envisaged
that ITX is able to activate the Ah-receptor (AhR). This was first tested in H4IIE rat hepatoma cells, using the induction of CYP1A related EROD activity and the DR CALUX bioassay, and subsequently
DNA microarray studies. Furthermore, using yeast-based estrogen and androgen bioassays, it was determined whether ITX displayed (anti)estrogenic and (anti)androgenic activities.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: Endocrine effects

Species:

other: Rat H4IIE hepatoma cells (ATCC, Manassas, VA) and DR CALUX cells,

Details on test animals or test system and environmental conditions:

Rat H4IIE hepatoma cells (ATCC, Manassas, VA) and DR CALUX cells, which are H4IIE cells stably transfected with a luciferase reporter plasmid containing a Dioxin Responsive Element (DRE), were grown routinely at 37 °C, 5% CO2 and 100% relative humidity in Alpha Minimal Essential Medium (AMEM; BioWhittaker, Verviers, Belgium) supplemented with fetal calf serum (FCS; 10% v/v; Invitrogen, Breda, The Netherlands), penicillin (5000 U, Sigma), and streptomycin (5 mg/L, Sigma).

Details on results:

AhR-activation by ITX in the DR CALUX and EROD assays
On the basis of structural similarities between ITX and the AhRagonists TCDD and the furanocoumarin bergamottin, the DR CALUX reporter gene assay was used to determine whether ITX is a potential AhR-agonist. The DR CALUX assay is based on rat H4IIE hepatoma cells stably transfected with an AhR-controlled luciferase reporter gene construct and enables quantification of the level of AhR-mediated gene expression. Exposure of cells to ITX for 24 h resulted in a full dose–response curve with a maximum response as high as that for TCDD. The half-maximal
response (EC50) was observed at 7 lM for ITX, as compared to 11 pM for TCDD. The relative potency of ITX in this particular bioassay was thus 6.105-fold less than that of TCDD.
The EROD induction assay was used to study the effect of ITX on the activity of the AhR-responsive enzyme cytochrome P450 1A1 in the wild-type rat H4IIE cell line, i.e. the original cells used for the construction of the cells of the DR CALUX assay. Also in this assay, exposure to ITX resulted in a clear response after 24 h of exposure (Fig. 2B). Within the ITX concentration range applied (0.005–50 lM), the EROD activity reached a maximum at 5 lM, but contrary to the response in the DR CALUX assay, decreased at higher concentrations (15–50 lM). The maximum EROD activity by ITX was 5-fold lower as that observed for TCDD. The EC50 values for ITX and TCDD were 1 lM and 20 pM respectively, indicating that in the EROD assay ITX was 50.000-fold less potent than TCDD.
However, this apparent higher (relative) potency of ITX in the EROD assay as compared to the DR CALUX assay is due to the decrease in activity at higher concentrations in the EROD assay, thus
decreasing the EC50 value for ITX. After 6 h of exposure in the EROD assay, no response was obtained with ITX, whereas TCDD showed a small and much lower response than observed after 24 h.

(Anti)estrogenic and (anti)androgenic activities of ITX and TCDD in the yeast assays
The potential estrogenic and androgenic, as well as the antiestrogenic and anti-androgenic activities of ITX was tested with the in-house developed yeast hormonal bioassays (Bovee et al.,
2004; 2007) and compared to TCDD. Neither ITX, nor TCDD showed estrogenic or androgenic activity. However, when tested in the presence of respectively 17b-estradiol (E2) or testosterone (T), ITX displayed both anti-estrogenic and anti-androgenic activities. The anti-estrogenic activity was observed at ITX concentrations higher than 1000 nM in the presence of a 17bestradiol
level of 1 nM but not at 3 nM. Anti-androgenic activity was also observed at ITX-concentrations above 1000 nM but in this case both at an intermediate (70 nM) and high (1000 nM) level of the agonist testosterone. At the lower testosterone level, the androgenic activity was completely blocked. In contrast, TCDD did not show such activities. Similar to TCDD, the dioxin-like PCB 126 showed no agonistic or antagonistic activities. Previous studies with the wellknown anti-estrogenic compounds ICI 182,780 and RU 58,668 showed antagonistic effects under similar conditions (1.5 nM E2) at concentrations higher than 100 nM (Bovee et al., 2008). In both
cases no complete inhibition was obtained. The known anti-androgens flutamide and hydroxyflutamide showed antagonistic effects in combination with 70 nM testosterone at concentrations higher than 1000 nM (Bovee et al., 2010), i.e. similar as ITX in the present
study.

Effect of ITX on gene expression in H4IIE cells using DNA microarrays
Binding of TCDD to the Ah-receptor is known to result in the induction of a number of biotransformation enzymes. Regarding the similarities between the response of ITX and TCDD in the DR CALUX and EROD assays, it was examined whether exposure of wild-type H4IIE cells to ITX and TCDD would result in similar gene-expression profiles. Based on the maximum response in the EROD assay, cells were exposed for 24 h to 5 lM ITX, 150 pM TCDD, 0.5% DMSO (control exposure) or AMEM (incubation medium) only. The hybridization data from the 44 K rat oligomicroarrays were subjected to univariate analysis, and pathway analysis. Univariate analysis of the data showed that the number of genes significantly differentially expressed (fold change
criterion of P1.5 and a p-value 60.01) as a result of exposure to ITX and TCDD, was 15 and 55, respectively. Most of these genes were up-regulated and only one was down-regulated. Thirteen genes were up-regulated by both TCDD and ITX, among them Cyp1a1, Cyp1a2, Cyp1b1, Cyp3a62, Gsta2, LOC286989, and Nqo1 which are genes encoding enzymes involved in biotransformation processes.
Only two out of the 15 genes differentially expressed by ITX, were not affected by TCDD.
The set of 54 genes up-regulated by TCDD was also subjected to Gene Set Enrichment Analysis (GSEA) to further substantiate the similarity of effects of ITX and TCDD on gene expression. GSEA demonstrated that this set of 54 genes was significantly up-regulated by ITX as well (p < 0.001). According to the GSEA statistics 42 of 54 genes were ‘‘enriched” in the ITX treatment
group compared to the control. It is evident that many genes expressed after exposure to
TCDD were also expressed after exposure to ITX. However, only 13 out these genes fulfilled the criteria of a ratio P1.5 and a p-value 60.01.
Taken together, from the microarray study it can be concluded that cells exposed to ITX exhibit to a large extent a similar gene expression profile as cells exposed to TCDD.

Confirmation of microarray results with RT-PCR
Two genes were selected for confirmation by RT-PCR, i.e. Cyp1a1 and Cyp3a62. Both genes were found to be up-regulated by TCDD as well as by ITX using the microarrays. The up-regulation
of Cyp3a62 is of particular interest, as gene expression of the Cyp3a family is considered not to be under control of the Ah-receptor pathway.
The Cyp1a1 PCR data showed the characteristic high induction by TCDD. Exposure to ITX also resulted in an increased level of this mRNA, but to a lower extent than TCDD. This is in agreement
with the lower response observed in the DR CALUX and EROD assays, and the lower gene expression in the microarray study at an ITX-concentration of 5 lM as compared to 150 pM
TCDD. The mRNA levels of CYP3a62 were also increased by both TCDD and ITX, although to a lower extent than Cyp1a1. Again, this is in accordance with the microarray data.

Degradation of ITX by the DR CALUX cells
The metabolism of ITX by the engineered H4IIE cells was studied at two different concentrations and incubation times. At the lower concentration of 5 lM and an incubation period of 24 h, medium concentrations of ITX decreased by 89%, as compared to 27% after 6 h at this concentration. The decrease was relatively slow at the higher concentration of 50 lMITX, showing 15% and 59% lower ITX concentrations after 6 and 24 h exposures, respectively. Several unknown peaks were observed after incubation, one major peak at a retention time (RT) of 16 min and minor metabolites at RTs of 9.9, 16.5, 17.4 and 28.0 min. Based on peak area, these compounds could explain 69% of the disappeared ITX at 50 lM after 24 h. However, this is only indicative since the relative response may change due to changes in the molecular properties of metabolites. At this stage, no attempt was made to identify and quantify these metabolites or to determine their biological effects in comparison to ITX. However, the formation of these metabolites confirms the degradation of ITX by the DR CALUX cells.

Conclusions:

ITX showed a clear response in the DR CALUX bioassay and also induced EROD activity in H4IIE rat hepatoma cells indicating that ITX has the potency to activate the Ah-Receptor.
Moreover, using yeast-based estrogen and androgen bioassays, it was demonstrated that ITX has potential antiestrogenic and anti-androgenic properties.

Executive summary:

2-Isopropylthioxanthone (2-ITX) has been widely used as a photoinitiator in printing ink of packaging materials. A few years ago, this compound got special attention since it was detected in milk and fruit drinks. Since little is known about possible effects of this compound on human health, studies were initiated to investigate its properties, starting with in vitro studies. Structural similarities between ITX and the AhR-agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and furanocoumarins, prompted us to investigate whether ITX could have the potency to activate the AhR.

ITX showed a clear response in the DR CALUX bioassay and also induced EROD activity in H4IIE rat hepatoma cells. Microarray studies in these cells showed a gene expression profile similar to that of TCDD. Moreover, using yeast-based
estrogen and androgen bioassays, it was demonstrated that ITX, but not TCDD, has potential antiestrogenic and anti-androgenic properties. The observed in vitro effects warrant further in vivo studies for confirmation and hazard characterization.

Reference 2

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effects of 2-ITX on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively.

GLP compliance:

not specified

Type of method:

in vitro

Endpoint addressed:

other: endocrine activity

Species:

other: see below

Details on results:

In Vitro Yeast Androgen and Estrogen Bioassays
The potential effects of the five TX compounds on the androgen receptor and the estrogen receptor alpha were tested in yeast-based bioassays. The positive controls, T and E2, gave clear dose-response curves, but none of the TX derivatives showed androgenic or estrogenic
activity. However, when tested in the presence of T, four TX derivatives acted as antiandrogens. Concentrations of 5µM and higher of 2-ITX and completely inhibited the responses of 0.07µM T and 1µM T.
When the effects of the compounds in the estrogen yeast assay were tested in the presence of 1 or 3nM E2, antiestrogenic activity was observed for 2-ITX at 5µM and higher. No toxicity toward the yeast cells was observed with 2-ITX, as checked by the OD 630 nm measurements.

Effects on H295R Cells Cytotoxicity.
Noncytotoxic concentrations were chosen in order to properly study the effect of TX compounds on steroidogenesis in H295R cells. Therefore, their possible cytotoxicity was evaluated first using WST-1 and ATPlite assays.
Combined results of these two assays revealed cytotoxic effects of ITX at concentrations above 10µM, 4-ITX at concentrations above 100µM. Compound concentrations determined to be cytotoxic for the H295R cells in both the WST-1 and the ATPlite assay were excluded from further
experiments. After 48-h exposure of the H295R cells to noncytotoxic concentrations of the TX compounds, the medium was collected for GC-MS/MS analysis, and RNA was isolated from
the cells for real time RT-PCR analysis.

Steroid synthesis.
Levels of 5 and 10µM 2-ITX resulted in a significant decrease in levels of dehydroepiandrosterone
(DHEA), androstenedione, and T and a significant increase in estrone and E2 levels Incubation of H295R cells with 2-ITX also significantly increased levels of pregnenolone and progesterone
(both at 10µM 2-ITX) and decreased levels of 17a-OHprogesterone (5µM 2-ITX)


Real time RT-PCR analysis of enzymes involved in steroidogenesis.
To determine whether changes in the steroid profile were correlated with changes in the expression level of key steroidogenic genes, RNAs from exposed H295R cells were subjected to quantitative RT-PCR analysis. H295R cells exposed to the positive control forskolin (aromatase inducer) significantly induced gene expression of CYP11A1, CYP17A1, 3ß-HSD, and CYP19A1 (aromatase), resulting in a 2.7-, 4.2-, 76.4-, and 18-fold induction, respectively, compared with
the DMSO solvent control. Exposure to 10µM 2-ITX significantly reduced the gene expression level
of CYP17A1, whereas the expression of CYP19A1 was significantly induced by 10µM 2-ITX.


Aromatase Activity
To analyse whether the increase in CYP19A1 gene expression upon exposure of H295R cells to 2-ITX was also reflected at the enzymatic level, the catalytic activity of aromatase was assessed. Exposure to 10µM of the aromatase inhibitor formestane resulted in a significantly decreased aromatase activity, whereas 10µM of the aromatase inducer forskolin significantly increased aromatase activity. 2-ITX concentrations lower than 1µM did not significantly alter aromatase activity, but 5 and 10µM 2-ITX significantly increased the aromatase activity in the H295R cells.

Conclusions:

The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.

Executive summary:

Photoinitiators used in food packaging ink, such as 2-isopropylthioxanthone (2-ITX), have been shown to migrate into food and beverages. In this work, the effects of 2-ITX on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. 2-ITX didn’t show androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities.

In an adapted version of the H295R steroidogenesis assay, using gas chromatography tandem mass spectrometry analysis of H295R media, 2-ITX increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays. The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.

Description of key information

Recently, several studies indicated that 2-ITX might be an endocrine-disrupting chemical.

AhR-agonistic, anti-androgenic and anti-estrogenic potencies of 2-isopropylthioxanthone (ITX) as determined by in vitro bioassays and gene expression profiling (Peijnenburg 2010). ITX showed a clear response in the DR CALUX (Dioxin Receptor Chemical Activated Luciferase gene eXpression) bioassay and also induced EROD (ethoxyresorufin-O-deethylase) activity in H4IIE rat hepatoma cells indicating that ITX has the potency to activate the Ah-Receptor. Moreover, using yeast-based estrogen and androgen bioassays, it was demonstrated that ITX has potential antiestrogenic and anti-androgenic properties.

In the publication of Reitsma (2013) , the effects of 2-ITX on steroidogenesis and androgen and estrogen receptor-mediated transcription activation have been studied using human H295R adrenocarcinoma cells and yeast hormone bioassays, respectively. 2-ITX didn’t show androgenic or estrogenic activities, but clear antiandrogenic and antiestrogenic activities. In an adapted version of the H295R steroidogenesis assay, using gas chromatography tandem mass spectrometry analysis of H295R media, 2-ITX increased levels of 17ß-estradiol and estrone. H295R cells incubated with 2-ITX also showed significantly reduced androgen and increased pregnenolone and progesterone levels. Expression of particular steroidogenic genes, including the one encoding for aromatase (CYP19A1), was significantly upregulated after incubation of H295R cells with 2-ITX. In line with the increased CYP19A1 mRNA expression, 2-ITX increased catalytic activity of aromatase in H295R cells as measured by cognate aromatase assays.  

The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.