Mode of action (MoA) in toxicology: general concept

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2 Toxicogenomics toxicology at a molecular level (mrna, mirna, proteins, metabolites ) Mode of action (MoA) in toxicology: general concept Chemical substance Key event 1 (Molecular initiating event) Key event 2 Key event n Adverse effect Associated event

3 1. Description of all key events 2. Dose-response relationship 3. Temporal association 4. Strength, consistency and specificity of association of adverse effects with key events 5. Biological plausibility and coherence 6. Other MoA considered and dismissed

4 Chemical substance Key event 1 (Molecular initiating event) Key event 2 Key event n Adverse effect * Case study : Testicular toxicity *S. Ludwig et al. Toxicol. Lett. 213, 2012

5 1,3-Dinitrobenzene Detailed mechanism of action unknown

6 Oral Gavage 4 days Control + 0.1, 1, 4, 8 mg/kg/d 10 Adult male Wistar rats Body and organ weights Testicular histopathology Testicular transcriptome Plasma testosterone 8mg/kg/d Testicular wt 4mg/kg/d Multiple lesions

7 A B C D Caspase 3

8 Oral Gavage 4 days Control + 0.1, 1, 4, 8 mg/kg/d 10 Adult male Wistar rats Body and organ weights Testicular histopathology Testicular transcriptome Plasma testosterone 8mg/kg/d Testicular wt 4mg/kg/d Multiple lesions Clear transcriptomic changes at 4mg/kg/ d

9 When applying ANOVA p-value of : 4mg/kg/day: 3918 genes significantly deregulated 0.1 & 1mg/kg/day: No significant changes in gene expression Normalised Expression DNB (mg/kg/day)

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11 Mitotic Role of Polo-Like Kinase: Effects at 4mg/kg/day Centrosome Separation & Maturation Down-regulation Up-regulation Mitotic Entry Septum-Inducing Network & Cytokinesis Metaphase Anaphase Transition & Mitotic Exit

12 Oral Gavage 4 days Control + 0.1, 1, 4, 8 mg/kg/d 10 Adult male Wistar rats Body and organ weights Testicular histopathology Testicular transcriptome Plasma testosterone 8mg/kg/d Testicular wt 4mg/kg/d Multiple lesions Clear transcriptomic changes at 4mg/kg/ d No effects

13 6 Plasma Testosterone (ng/ml) ± SD Dose DNB (mg/kg/day)

14 Confirmation of previously published histopathological changes Clear differences between 1 and 4mg/kg/day both histopathologically and gene expression Correlation between testicular lesions and gene changes Multiple doses of DNB do not affect testosterone levels These results indicate a direct testicular toxicity (no apparent endocrine mediated effect)

15 Single Oral Gavage Control + 4 mg/kg/d Sacrifice: 8, 24, 48, 72h after dosing 10 Adult male Wistar rats Body and organ weights Testicular histopathology Testicular Gene expression Plasma testosterone No effects No effects Changed

16 n = 10 Testosterone (% control) ** hrs 24 hrs 48 hrs 72 hrs Time after single 4mg/kg oral dose **: p 0.01

17 Single Oral Gavage Control + 4 mg/kg/d Sacrifice: 8, 24, 48, 72h after dosing 10 Adult male Wistar rats Body and organ weights Testicular histopathology Testicular Gene expression Plasma testosterone No effects No effects Effects Changed

18 Star Cyp11a1 Relative quantification (treated/control) *** 8 hrs 24 hrs 48 hrs 72 hrs Time after a single 4mg/kg oral dose Relative quantification (treated/control) hrs 24 hrs 48 hrs 72 hrs Time after a single 4mg/kg oral dose Cyp17a1 Hsd3b1 Relative quantification (treated/control) hrs 24 hrs 48 hrs 72 hrs Relative quantification (treated/control) hrs 24 hrs 48 hrs 72 hrs Time after a single 4mg/kg oral dose Time after a single 4mg/kg oral dose ***: p 0.001

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20 n = 3 studies % control Hormone Progesterone Testosterone Estradiol * * *** DNB Concentration (µm) 1,3-DNB affects steroid hormone secretion in vitro in H295R cells *: p 0.5 ***: p Evidence for endocrine-mediated effects? Cytotoxicity from 100µM

21 Testosterone (ng/ml) ± SD ** ** *** *** *** *** *** FORSK DNB Concentration (µm) 1,3-DNB affects steroid hormone secretion in vitro in primary Leydig cells **: p ***: p Evidence for endocrine-mediated effects? n = 4 replicate plates; 3 wells/concentration/plate

22 Key event 1 (Molecular initiating event) Key event 2 Key event n Adverse effect Molecular initiating events Sertoli cells Molecular target(s) Leydig cells Molecular target(s)? Germ cell cycle affected (polo-like kinase) Steroidogenesis affected - Gene transcript - testosterone level Testicular toxicity

23 DNB is considered as direct acting testicular toxicant (following repeat dosing) - histopath. - cell cycle affected at a molecular level - but initiating molecular target not yet known DNB also interferes with steroidogenesis (acute in vivo/in vitro) - Testosterone and genes involved in steroidogenis affected transiently. Is this interference a key event contributing to the testicular toxicity or just an associated event?

24 Chemical substance Key event 1 (Molecular initiating event) Key event 2 Key event n Adverse effect H. Tinwell et al., Regul. Toxicol. Pharmacol., 66, 2013

25 Estrogen receptor binding (rat uterus) her transcriptional activation (human cell lin HeLa) Androgen receptor binding (rat prostate) Steroidogenesis (human cell line (H295R)) Aromatase (Human recombinant) Uterotrophic (immature rat) Hershberger (castrated rat) Pubertal female (rat) Pubertal male (rat) Amphibian metamorphosis (frog) Fish short term reproduction The targeted (MOA) assays have been validated using known EDs and negative (vehicle) controls, but the correlation between the targeted data and the apical data is often missing.

26 In vitro steroidogenesis Testosterone Estradiol Paracetamol Positive 200 Gingerol Positive 160 Caffeine Capsaicin Eugenol Positive Positive Positive 120 % Control 80 Cinnamaldehyde Positive 40 Resveratrol Curcumin Cuminaldehyde Positive Positive Positive PARACETAMOL (µm) Vitamin C Weak Positive Testosterone Estradiol Echinacoside Saccharose Ibuprofen Vitamins B9, 6, 3 Lipoic acid Gingkolide A Allyl sulfide Allyl disulfide Weak Positive Negative Negative Negative Negative Negative Negative Negative % Control CAFFEINE (µm) % Control Testosterone hcg stimulated primary rat Leydig cells µM 250µM 500µM 1000µM Caffeine concentration

27 Estrogen receptor binding (rat uterus) her transcriptional activation (human cell lin HeLa) Androgen receptor binding (rat prostate) Uterotrophic (immature rat) Hershberger (castrated rat) Pubertal male (rat) Pubertal female (rat) Steroidogenesis (human cell line (H295R)) Aromatase (Human recombinant). Amphibian metamorphosis (frog) Fish short term reproduction. The correlation between in vitro and in vivo data is often missing and the mechanistic link between these 2 sets of data is not always obvious.

28 Cortisol/corticosterone Testicular Leydig cell hyperplasia/ tumours Cholesterol Pregnenolone/progesterone Testosterone Changes in circulating steroid hormones Changes in pituitary hormones LH, FSH, ACTH Adverse effects on the reproductive tissues e.g. uterine/ovarian tumours Estradiol Adverse effects on the adrenals

29 PND 23 PND 30 PND 55 PND 55 Sprague-Dawley rats 12/group 0, 5, 20, 100 mg/kg/d caffeine Preputial separation Sacrifice Testes/SAT/adrenal/liver weights Plasma hormone measurements qpcr: Testes, adrenals, pituitary

30 Effect of Caffeine on Ventral Prostate Weight Effect of Caffeine on Seminal Vesicle Weight Ventral Prostate wt (mg) + SD ** Seminal Vesicles Wt (mg) +SD ** 0 Controls 5mg/kg/d 20mg/kg/d 100mg/kg/d 0 Controls 5mg/kg/d 20mg/kg/d 100mg/kg/d A clear treatment related effect on SAT weights No clear effects on preputial separation

31 Caffeine affects parameters measured in MALE Rat Pubertal assay(2) Progesterone (pg/ml) % 69% Caffeine (mg/kg/day) No apparent effect on testosterone Progesterone data are consistent with literature 37% 3 2 RQ RQ 1 0 Hsd3b1 Pregnenolone Progesterone Caffeine (mg/kg/d) Slight at 100 mg/kg/d Cyp17a1 Progesterone androstenedione Caffeine (mg/kg/d) at 100 mg/kg/d *

32 Conclusion from the MALE Rat Pubertal assay

33 PND 22 PND 51 PND 52 Sprague-Dawley rats 12/group 0, 5, 20, 100 mg/kg/d caffeine Vaginal opening/estrus cycle Sacrifice Ovary/uterus/adrenal/liver weights Plasma hormone measurements qpcr: Ovary, adrenals, pituitary

34 BWt (g) at Complete VO Control 5mg/kg/d Caffeine 20mg/kg/d caffeine 100mg/kg/d caffeine Age (PND) at Complete Vaginal Opening A clear treatment related delay in vaginal opening recorded at 100 mg/kg/day caffeine Prolonged estrus cycle also observed at 100 mg/kg/day Data consistent with literature

35 Fshb Prl 3 3 ** RQ 2 RQ 2 ** Caffeine (mg/kg/d) Caffeine (mg/kg/d)

36 Tshb ** Thyroid hormone effects reported Prolactin ** Pituitary (adenomas reported) ACTH ** Mammary gland tumors (mice) reported Fsh ** Lh no effect Ovaries 8d Delay in VO: ** Prolonged estrus cycle: ** Ovarian atrophy: * Gene transcript changes in pituitary support the phenotypic changes observed in pubertal female and the adverse effects following chronic dosing

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38 Key event 1 (Molecular initiating event) Key event 2 Key event n Adverse effect Steroidogenesis in vitro (H295R, Leydig cells) affected steroidogenesis in vivo affected progesterone level gene transcript changes (Hsd3bl, Cyp17a1) Prostate weight Seminal vesicle weight Spermatogenesis affected (1) steroidogenesis in vivo affected unclear? Delay in vaginal opening? FSH Transcript Pituitary Prolactin Transcript Pituitary adenomas in rat (2) Mammary gland tumours mice (3) (1) Ezzat & Gohary, Funct. Dev. Morphol., 4, 1994 (2) T. Yamagami et al., Surg. Neurol., 20, 1983 (3) C. Welsch et al., Cancer Res., 48, 1988

39 Caffeine conclusion The prediction of adverse effects from the results of in vitro and in vivo screening assays is very challenging even though some mechanistic information are contained in these assays. The use of a series of in vitro and in vivo methods in combination with omic and traditional tools are required to identify key events of a mode of action which eventually lead to an adverse effect.

40 For the time being investigation on the mode of action of toxicity from chemicals still requires the use of traditional and molecular (omics) tools in in vitro and in vivo assays. Such an approach will identify the key events and elucidate the mechanistic link between the different events which eventually lead to an adverse effect at a given dose level. It is anticipated that in addition to omic tools computational life sciences through the modeling of pathways will play a role to accelerate the identification of mode of action of toxicity.