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IN ST IT U T O D E C IÊ N C IA S B IO M ÉD IC A S A B EL S A LA Z A R

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THE ROLE PLAYED BY OXIDATIVE

STRESS AND HYPERTHERMIA

IN THE CARDIOTOXICITY OF

MDMA COMBINED WITH BZP

Sara Cristina Oliveira da Silva

M

2018

M

.ICB

AS

MESTRADO MEDICINA LEGAL

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SARA CRISTINA OLIVEIRA DA SILVA

THE ROLE PLAYED BY OXIDATIVE STRESS AND HYPERTHERMIA

IN THE CARDIOTOXICITY OF MDMA COMBINED WITH BZP

Candidature dissertation of Master in Legal Medicine

submitted to Institute of Biomedical Sciences Abel Salazar of

University of Porto

Supervisor: Dr Diana Dias da Silva

Category: Investigator & Auxiliary Professor

Affiliations: UCIBIO–Applied Molecular Biosciences Unit,

REQUIMTE Laboratory of Toxicology, Biological Sciences

Department, Faculty of Pharmacy, University of Porto &

IINFACTS, Department of Sciences, Institute of Research

and

Advanced

Training

in

Health

Sciences

and

Technologies, University Institute of Health Sciences

(IUCS-CESPU)

Co-Supervisor: Dr

Helena Maria Ferreira da Costa Ferreira

Carmo

Category: Auxiliary Professor

Affiliation: UCIBIO–Applied Molecular Biosciences Unit,

REQUIMTE Laboratory of Toxicology, Biological Sciences

Department, Faculty of Pharmacy, University of Porto

Co-Supervisor: Dr

Maria da Graça Lobo

Category: Auxiliary Professor

Affiliation: Laboratory of Pharmacology, Immuno-Physiology

and Pharmacology Department, Institute of Biomedical

Sciences Abel Salazar, University of Porto

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This work was funded by the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência

e a Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement

PT2020 UID/MULTI/04378/2013. The study is a result of the project NORTE-01-0145-FEDER-000024 supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBIOtecHealth—New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF).

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PUBLICATIONS

In accordance to the no 2, paragraph a, article 31 from Decree-Law no 115/2013, the

following submitted manuscript and conference communications were prepared under the scope of this thesis.

Poster in international conference proceedings

I. S. O. Silva, F. Carvalho, M. D. L. Bastos, H. Carmo, D. D. D. Silva (2018)

Benzylpiperazine (BZP) additively increases methylenedioxymethamphetamine

(MDMA) cardiotoxicity in H9c2 cells, an effect potentiated by hyperthermia. 54th

Congress of the European Societies of Toxicology (EUROTOX 2018). 2-5 September. Brussels, Belgium.

Oral communications in national conference proceedings

I. Silva, S.; Enea, M.;, Coelho, S.; Carmo, H.; Carvalho, F.; Bastos, ML; Dias da Silva,

D. (2018) Exposure of cardiomyocytes to ecstasy, in combination with benzylpiperazine, reveals potential cardiotoxic risks, especially under increased temperature conditions. XLVIII Annual Meeting of Portuguese Society for Pharmacology, XXXVI Annual Meeting of Clinical Pharmacology and XVII Annual Meeting of Toxicology. 5-7 February. Lisbon, Portugal.

II. Silva, S.; Enea, M.;, Coelho, S.; Carmo, H.; Carvalho, F.; Bastos, ML; Dias da Silva,

D. (2018) In vitro cardiotoxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) combined with benzylpiperazine (BZP) in hyperthermia. IJUP 2018, 11th Meeting of Young Researchers University of Porto. 7–9 February. Porto, Portugal.

III. Silva, S.; Enea, M.;, Coelho, S.; Carmo, H.; Carvalho, F.; Bastos, ML; Dias da Silva,

D. (2018) Hyperthermia exacerbates the cardiotoxicity of Benzylpiperazine (BZP) and Methylenedioxymethamphetamine (MDMA) combinations in H9c2 cells. III Annual Meeting of Portuguese Society for Forensic Sciences (APCF, Associação Portuguesa de Ciências Forenses). 24–25 May. Porto, Portugal.

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Author’s declaration

The author declares to have actively contributed to the conception and technical execution of the work, acquisition of data, analysis and interpretation of the results, as well as to the preparation of the published or submitted work included in this thesis, in close collaboration with all other authors.

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ACKNOWLEDGEMENTS

First and foremost, my deepest thanks to my supervisor Professor Dr Diana Dias da Silva for accepting me, for all the knowledge, guidance, motivation and availability. Without her constant belief, support and encouragement I would never had the opportunity to work with such impressive subjects and finish this hard journey. Thanks for all the strength when I needed the most. My genuine gratitude.

Many thanks to Professor Dr Maria José Pinto da Costa, coordinator of the Master in Legal Medicine, to Professor Dr Maria de Graça Lobo, from Laboratory of Pharmacology, and to Professor Dr Maria de Lourdes Bastos, from Laboratory of Toxicology, for all their effort to made this master possible.

I would like to express my gratitude to Professor Dr Helena Carmo for authorizing the project to be conducted in their laboratory research group. Thanks for accepting me, for all the help, trust and valuable advices as an excellent professional.

To all the people working in the laboratory for giving me such attention, time and materials, my many thanks, specially to Cátia and Margarida.

Maria Enea, I do not have enough words for thanking you. For your huge patience with me and foremost for your great friendship. Thanks for all the hours you spent with me and for all the advices and tips. Thanks for the chocolates and coffees, too. It was a privilege to know you and share this journey.

My thanks and appreciations to all my friends and laboratory colleagues for the constant (mental) support. Thanks to Patrícia Moreira. Thanks to Ana Filipa Mendes. Thanks to Ana Margarida Araújo. Thanks to Daniela Rodrigues. Thanks, Jorge Soares.

Finally, I must express my very profound gratitude to my parents, to my sister and to my husband for providing me with unfailing support and continuous encouragement throughout this very, very difficult year. This accomplishment would not have been possible without them.

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RESUMO

As substâncias sintéticas psicoativas, incluindo a 3,4-metilenodioximetanfetamina (MDMA, ecstasy) e a N-benzilpiperazina (BZP) são frequentemente consumidas em associação. Considerando que cada uma dessas drogas per se é capaz de provocar cardiotoxicidade severa e que a hipertermia é uma condição amplamente conhecida como potenciadora da toxicidade das anfetaminas, colocámos a hipótese de que a exposição de cardiomioblastos a esta combinação revelaria efeitos tóxicos potencialmente diferentes daqueles provocados individualmente por cada uma das drogas, sobretudo em condições de temperatura aumentada. Deste modo, os efeitos cardiotóxicos in vitro das drogas isoladas e da sua combinação foram avaliados em cardiomioblastos H9c2. Depois da exposição das células à BZP e à MDMA durante 24 horas, a 37 ºC e a 40.5 ºC, foi determinada a viabilidade celular e os efeitos estimados para a mistura calculadas de acordo com os modelos de Ação Independente (IA) e de Adição da Concentração (CA). Alterações no estado antioxidante celular, energético e mitocondrial também foram avaliadas. Os nossos

resultados mostram que a MDMA (EC50 1.74 mM a 37 ºC e 1.17 mM a 40.5 ºC) é

significativamente mais tóxica do que a BZP (EC50 2.72 mM a 37 ºC e 1.93 mM a 40.5 ºC).

As toxicidades previstas pelos modelos IA e CA foram coincidentes e ambos estimaram com precisão os efeitos de mistura obtidos experimentalmente. A hipertermia agravou significativamente o efeito cardiotóxico in vitro destas drogas, individualmente e em mistura, afetando o estado de redução celular, com aumento da produção de espécies reativas e de glutationa oxidada (GSSG), depleção de glutationa reduzida (GSH) e de ATP, com comprometimento mitocondrial e lisossomal. A cascata de sinalização apoptótica foi despoletada, sobretudo em condições de normotermia. Em suma, de uma perspetiva clínica e médico-legal, a cardiotoxicidade aditiva observada suscita preocupação relativamente a uma potencial deterioração da saúde dos consumidores, sobretudo nas condições de consumo dos ambientes recreativos, que favorecem a hipertermia.

Palavras-chave: N-Benzilpiperazina (BZP); 3,4-Metilenodioximetanfetamina (MDMA,

ecstasy); Efeitos de mistura; Cardiomioblastos H9c2; Cardiotoxicidade; Hipertermia; Stress

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ABSTRACT

Synthetic psychotropic substances, including 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and benzylpiperazine (BZP) are frequently co-abused. Considering that each of these drugs on its own can lead to severe cardiotoxicity, and that hyperthermia is a well-known condition that potentiates the toxicity of amphetamines, we hypothesised that exposure of cardiac cells to this mixture would reveal effects that potentially differ from single drug toxicities, especially under increased temperature settings. Therefore, the in vitro cardiotoxic effects of the single drugs and their combination were assessed in H9c2 cardiomyoblasts. After cell exposure to BZP and MDMA for 24h, at 37ºC and at 40.5ºC, viability was recorded and mixture expectations were calculated using independent action (IA) and concentration addition (CA) models. Changes in antioxidant, energetic and

mitochondrial function were also evaluated. Our data showed that MDMA (EC50 1.74 mM

at 37 ºC and 1.17 mM at 40.5 ºC) was significantly more toxic than BZP (EC50 2.72 mM at

37 ºC and 1.93 mM at 40.5 ºC). Toxicities predicted by both IA and CA models were coincident and accurately estimated the experimental mixture effects. Hyperthermia significantly aggravated the in vitro cardiotoxic effect of both drugs, individually and in mixture, affecting the cellular redox status, with increased generation of reactive species and oxidized glutathione (GSSG), reduced glutathione (GSH) and ATP depletion, and mitochondrial and lysosomal impairment. Apoptosis cascade signalling was also triggered, particularly under normothermic conditions. Overall, from a clinical and forensic perspective, the observed additive cardiotoxicity raises concern about a potential deterioration of the health of abusers, specially under recreational conditions that favour hyperthermia.

Keywords: N-Benzylpiperazine (BZP); 3,4-Methylenedioxymethamphetamine (MDMA,

ecstasy); Mixture effects; H9c2 cardiomyoblasts; Cardiotoxicity; Hyperthermia;

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TABLE OF CONTENTS

PUBLICATIONS ... 3 ACKNOWLEDGEMENTS ... 5 RESUMO ... 6 ABSTRACT ... 7 TABLE OF CONTENTS ... 8 FIGURE INDEX ... 11 TABLE INDEX ... 14 LIST OF ABBREVIATIONS ... 15 CHAPTER 1 ... 17 GENERAL INTRODUCTION ... 17

1.1. 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) ... 18

1.1.1. Hyperthermia ... 20

1.1.2. Cardiotoxicity ... 21

1.2. N-Benzylpiperazine (BZP) ... 22

1.2.1. Hyperthermia ... 23

1.2.2. Cardiotoxicity ... 24

1.3. Association of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and N-benzylpiperazine (BZP) ... 25

CHAPTER 2 ... 27

OBJECTIVES OF THE THESIS ... 27

CHAPTER 3 ... 28

MATERIALS AND METHODS ... 28

3.1. Chemicals ... 28

3.2. H9c2 cells ... 28

3.3. H9c2 cell culture ... 29

3.4. Mixture testing ... 29

3.5. Drug exposures ... 30

3.6. Cytotoxicity by the MTT reduction assay ... 30

3.7. Prediction of mixture effects ... 31

3.8. Lysosomal damage through the neutral red (NR) incorporation assay ... 31

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3.10. Determination of intracellular reduced glutathione (GSH) and oxidized glutathione

(GSSG) ... 32

3.11. Evaluation of mitochondrial integrity ... 33

3.12. Measurement of intracellular adenosine triphosphate (ATP) ... 33

3.13. Determination of caspase-8, -9, and -3 activities ... 34

3.14. Statistical Analysis ... 34

CHAPTER 4 ... 36

RESULTS ... 36

4.1. Hyperthermia exacerbated 3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) toxicities in H9c2 cells ... 36

4.2. 3,4-Methylenedioxymethamphetamine (MDMA) interacts addictively with N-benzylpiperazine (BZP), both at normothermic and hyperthermic conditions ... 37

4.3. The combination of non-cytotoxic concentrations of 3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) results in significant toxicity, an effect further exacerbated under hyperthermia ... 39

4.4. 3,4-Methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP), individually and combined, disrupt lysosomal integrity, an effect further exacerbated under hyperthermia ... 40

4.5. Oxidative stress underlines toxicity of the combination of 3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) ... 41

4.6. Combination of 3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) impairs mitochondrial functioning ... 44

4.7. 3,4-Methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP), individually and combined, increase apoptosis by activating intrinsic and extrinsic cell death mechanisms ... 45

CHAPTER 5 ... 47

DISCUSSION ... 47

CHAPTER 6 ... 54

CONCLUSION AND FUTURE PERSPECTIVES ... 54

CHAPTER 7 ... 56

REFERENCES ... 56

CHAPTER 8 ... 62

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APPENDIX A ... 67

Routine Cell Culture ... 67

Aseptic work area ... 67

Culture media ... 67

Thawing frozen H9c2 cells ... 68

H9c2 cell routine ... 68

H9c2 cell seeding ... 69

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FIGURE INDEX

Figure 1. Cytotoxicity elicited by 3,4-methylenedioxymethamphetamine (MDMA) and

N-benzylpiperazine (BZP) in H9c2 cells. Data obtained in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, after 24 h-incubation at 37 °C (black lines, labelled ‘Normothermia’) or at 40.5 °C (red lines, labelled ‘Hyperthermia’), are from a minimum of four independent experiments (performed in triplicate) and are presented as percentage of cell death, relative to the negative controls. Curves were fitted to the Logit model. The dashed lines are the upper and lower limits of the 95% confidence belts of the best estimate of mean responses. The dotted red lines represent 50% and 100% effect……….37

Figure 2. Predicted and observed cytotoxicity of three distinct mixtures of

3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP) in H9c2 cells, at 37 °C (Normothermia) or at 40.5 °C (Hyperthermia). Mix ß: BZP and MDMA were combined

in proportion to their EC50 values. Mix 𝛿: BZP and MDMA were combined in proportion to

their EC01 values. Mix α: BZP and MDMA were combined at a MDMA:BZP 4:1 ratio (based

on concentrations found in blood of intoxicated patients). The experimental effects were attained by the MTT reduction assay after 24 h-incubations. On the basis of the single drug concentration–response relationships (Fig. 1), additive combination effects were predicted using the models of concentration addition (CA, solid red line) and independent action (IA, solid purple line). Normalised data are presented as percentage of cell death, relative to negative controls, and are from a minimum of four independent experiments, performed in triplicate. Curves were fitted to the dosimetric Logit model (solid black line) and the dashed black lines are the upper and lower limits of the 95% confidence belts of the best estimate

of mean responses. The dotted lines represent 50% and 100%

effect………..39

Figure 3. Cytotoxicity elicited by 3,4-methylenedioxymethamphetamine (MDMA) and

N-benzylpiperazine (BZP) when tested individually at their EC01 or in combination in H9c2

cells, after 24 h at 37 °C (A) or at 40.5 °C (B). CA: prediction of the mixture effect by concentration addition. IA: prediction of the mixture effect by independent action. MIX:

experimentally observed effect when BZP and MDMA were present at their individual EC01

concentrations (Table 2). The individual concentrations of the binary mixture components tested at 37 °C (A) were 247 μM MDMA and 296 μM BZP; and at 40.5 °C (B) were 87 μM MDMA and 98 μM BZP. The dashed line corresponds to the sum of the individual effects of

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the mixture components (2%). Data (mean results) were from four independent experiments, run in six replicates. Error bars represent the standard error of the mean (SEM). Statistical comparisons were made using the Kruskal–Wallis test followed by the Dunn’s multiple comparison post hoc test. ****Show statistically significant differences (p<0.0001) between the mixture and all other treatments, i.e., individual drugs and control………...41

Figure 4. Lisossomal integrity, indirectly assessed by the neutral red incorporation in the

organelle, in H9c2 cells after 24 h-incubations with 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1 at 37 °C (blue light bars) or at 40.5 °C (blue dark bars). Results from at least four independent experiments, run in six replicates are expressed as percentage control ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA test followed by the Tukey’s multiple comparison post hoc test. **p<0.01; ****p<0.0001, vs. control. All drugs at 40.5 ºC significantly impaired lysosome integrity (p<0.0001), compared with treatment at 37 ºC………42

Figure 5. Intracellular reactive species, as assessed by the oxidation of the DCFH dye, in

H9c2 cells, after 24 h-incubations at 37 °C (blue light bars) or at 40.5 °C (blue dark bars) with 100 μM of 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1. Results from at least four independent experiments, run in six replicates, are expressed as percentage control ± standard error of the mean (SEM). Statistical comparisons were made using the Kruskal–Wallis test followed by the Dunn’s

multiple comparison post hoc test. ****p<0.0001, vs. control. #p<0.05, vs. mixture at 40.5

°C……….43

Figure 6. Intracellular contents of total (tGSH), reduced (GSH, blue bars) and oxidized

glutathione (GSSG, white bars), in H9c2 cells, after 24 h-incubations at 37 °C or at 40.5 °C with 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1. Results from four independent experiments are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA test followed by the Tukey’s multiple comparison post hoc test. *p<0.05;

***p<0.001; ****p<0.0001, vs. GSH control at the same temperature. ##p<0.01; ###p<0.001,

vs. GSSG control at the same temperature. &p<0.05; &&&p<0.001, &&&&p<0.0001, vs. 37

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Figure 7. Mitochondrial membrane potential (Δψm) indirectly assessed by the TMRE

incorporation in mitochondria of H9c2 cells, after 24 h-incubations at 37 °C (blue light bars) or at 40.5 °C (blue dark bars) with 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1. Results from at least four independent experiments, run in triplicates are expressed as percentage control ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA test followed by the Tukey’s multiple comparison post hoc test. *p<0.05; **p<0.01; ***p<0.001;

****p<0.0001, vs. control. &p<0.05; &&p<0.01; &&&p<0.001, &&&&p<0.0001, vs. 37

oC……….45

Figure 8. Intracellular ATP levels, in H9c2 cells, after 24 h-incubations at 37 °C (blue light

bars) or at 40.5 °C (blue dark bars) with 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1. Results from four independent experiments are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made using the Kruskal–Wallis test followed by the Dunn’s multiple comparison post hoc test. **p<0.01; ***p<0.001; ****p<0.0001, vs. control at the same

temperature. ###p<0.001, vs. mixture at the same concentration and temperature conditions.

&p<0.05, vs. 37 oC………..46

Figure 9. Caspase-8, -9, and -3 activation, in H9c2 cells, after 24 h-incubations at 37 °C

(blue light bars) or at 40.5 °C (blue dark bars) with 3,4-methylenedioxymethamphetamine (MDMA), N-benzylpiperazine (BZP) and the mixture MDMA 4: BZP 1. Results from three independent experiments are expressed as percentage control ± standard error of the mean (SEM). Statistical comparisons were made using the Kruskal–Wallis test followed by the Dunn’s multiple comparison post hoc test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, vs.

control. #p<0.05; . ##p<0.01, vs. mixture. &p<0.05; &&p<0.01; &&&&p<0.0001, vs. 40.5

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TABLE INDEX

Table 1. Individual concentrations (mM) of 3,4-methylenedioxymethamphetamine (MDMA)

and N-benzylpiperazine (BZP) in each concentration of the mixture MDMA 8: BZP tested in

H9c2, after 24h-incubations, at 37 oC or 40.5 oC……….31

Table 2. Parameters derived from the nonlinear fits of

3,4-methylenedioxymethamphetamine (MDMA) and N-benzylpiperazine (BZP)………...37

Table 3. Parameters derived from the nonlinear fits of the mixtures of

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LIST OF ABBREVIATIONS

Δѱm Mitochondrial Membrane Potential Δp Electrochemical Proton Motive Force

ΔpHm Mitochondrial pH Gradient

Ac-DEVD-pNA Acetyl-Asp-Glu-Val-Asp-p-Nitroaniline Ac-IETD-pNA Acetyl-Ile-Glu-Thr-Asp-p-Nitroaniline Ac-LEDH-pNA Acetyl-Leu-Glu-His-Asp-p-Nitroaniline 5-HT Serotonin

ATP Adenosine 5’-Triphosphate

BSA Bovine Serum Albumine BZP N-benzylpiperazine CA Concentration Addition

CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate Hydrate CNS Central Nervous System

COMT Catechol O-Methyl Transferase CYP Cytochome P450

CYP1A1 Cytochrome P450 isoform 1A1 CYP1A2 Cytochrome P450 isoform 1A2 CYP1B1 Cytochrome P450 isoform 1B1 CYP2D6 Cytochrome P450 isoform 2D6 CYP3A4 Cytochrome P450 isoform 3A4 DA Dopamine

DBZP Dibenzylpiperazine

DCFH-DA 2’,7’-dichlorodihydrofluorescein Diacetate

DMEM Dulbecco’s Modified Eagle’s high glucose Medium

DMSO Dimethyl Sulfoxide

DTNB 5,5-dithio-bis(2-nitrobenzoic Acid) DTT Dithiothreitol

EC50 Half-maximum-effect concentration

EC01 Concentration that produces 1% of the maximal effect in the assay

EDTA Ethylenediaminetetraacetic Acid

EMCDDA European Monitoring Centre for Drug and Drug Addiction FBS Foetal Bovine Serum

GSH Reduced Glutathione GSSG Oxidized Glutathione

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GST Glutathione S-transferase

HBSS Hank’s Balanced Salt Solution

HClO4 Perchloric Acid

HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic Acid IA Independent Action

MAO Monoamine Oxidase

mCPP meta-chlorophenylpiperazine

MDMA 3,4-methylenedioxymethamphetamine MS Mass Spectrometry

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide NA Noradrenaline

NEAA Nonessential Amino Acids

NADPH Nihydronicotinamide-Adenine Dinucleotide Phosphate NPS New Psychoactive Substances

NR Neutral Red

RNS Reactive Nitrogen Species ROS Reactive Oxygen Species SEM Standard Error of the Mean SERT Serotonin Transporter SH Sulfhydryl groups

SINTES/INPS Système d’Identification National des Toxiques Et Substances/ Institut

National de la Police Scientifique

TFMPP 1-(3-trifluoromethylphenyl)-piperazine tGSH Total Glutathione

TMRE Tetramethylrhodamine Ethyl Ester Perchlorate TNB 5-thio-2-nitrobenzoic Acid

TRIS 2-amino-2-hydroxymethyl-propane-1,3-diol

TRITON X-100 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene Glycol UCPs Uncoupling Proteins

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CHAPTER 1

GENERAL INTRODUCTION

Drug abuse is a growing menace fuelled by multifactorial reasons that vary greatly for every individual. Either to try to fit in with their peers as a means of feeling “a part of”, or as a way to relax or to self-medicate, many people are at risk for substance abuse, and their vulnerability for developing addiction disorders might originate, among others, from the genetic endowment, family background, psychological factors, and social patterns (Sussman et al., 2004). Importantly, drug abuse has an enormous health, social and economic impact. According to the World Health Organization, 275 million people were estimated to have used illicit drugs in 2016 (WHO, 2018).

In this context, a recent phenomenon of important toxicological significance, is the abuse of new psychoactive substances (NPS). These NPS, which became very popular among young people, mainly in recreational environments such as “rave” parties and at clubs and other nightlife venues, have been known in the market by terms such as ‘‘legal highs’’, ‘‘designer drugs’’, ‘‘bath salts’’, and “research chemicals” (Gross et al., 2002; Hopfer et al., 2006; Schifano, 2004; TF and Engels, 2005), These substances are predominantly novel synthetic derivatives and analogues of existing controlled drugs (such as cocaine and amphetamines) that have emerged in the drug black market mainly to evade drug control legislation. Nevertheless, as their abuse and health complications spread at unprecedented rates, many of them also became illegal in many countries. Despite the strong efforts by the authorities to restrict the production and availability of NPS, there have been frequent reports of tabletting and manufacture of these substances within European borders (EMCDDA, 2018).

Although is well acknowledged that NPS pose a significant risk to public health, often little is known about the adverse health effects and social harms of NPS, posing a considerable challenge for drug policy in what concerns prevention and treatment.

3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is among the most commonly used illicit stimulants in Europe, but in the last decade the abuse of some lesser-known NPS, including benzylpiperazine (BZP), have dramatically proliferated as an alternative to the classic drug. The abuse of these stimulants generally occurs in particular nightlife settings, such as rave dance and electronic music parties, and is linked to health problems. In general, complications range from seizures, agitation, aggression, and acute psychosis, to severe hyperthermia, hyponatremia, rhabdomyolysis, and cardiac arrest.

Although much information is already available for MDMA by virtue of decades of research and clinical observation, in what concerns NPS, the users have frequently been hospitalized

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with severe intoxications, but data on toxicity and life-threatening potential of the majority of the congeners are not available or are very limited, and information on long-term adverse effects or risks are also still largely unknown due to the very recent emergence of this drug abuse trend. Furthermore, since purity and composition of products containing NPS are often undisclosed, users are placed at high risk, as evidenced by the number of emergency room admissions and deaths, repeatedly associated with poly-substance use (Nugteren-van Lonkhuyzen et al., 2015).

1.1. 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy)

Amphetamines became very popular in the early 1990’s due to the ease and low expense on synthesizing these drugs in home-made or amateur laboratories (Courtney and Ray, 2014). Among them, MDMA is a synthetic substance chemically related to d-amphetamine, but divergent to some extent in its effects. Although originally synthesized by Merck in 1912 for pharmaceutical purposes, MDMA had a popularity boom as a drug of abuse in the mid-1990s (Carvalho et al., 2012). It is a white crystalline powder in its pure form; although being consumed as such by snorting, it is mainly ingested compressed in tablets or diluted in drinks (EMCDDA, 2018). New MDMA tablet designs with several colours, shapes and brand logos are continuously being introduced into the drug market. From 2013 on, after a period of low availability linked to a lack of chemical precursors needed for its manufacture, which coincided with the beginning of the NPS phenomenon, the MDMA market has seen a revival, leading to an increase of the intoxication reports. Signs of this recent drug market recovering is the dismantling of several large MDMA production centres in Belgium and the Netherlands (EMCDDA, 2018). In fact, Europe is a major producer of some synthetic stimulant drugs like MDMA, exporting products and expertise to other parts of the world (EMCDDA, 2018).

Positive and negative psychological and physical effects experienced by MDMA users vary greatly among different individuals (Schifano, 2004). The acute subjective effects more frequently described for the drug are euphoria, increased alertness, energy and sexual drive, anxiety, depression, closeness to others, fear, calmness, and facilitation of social interaction. Overall, MDMA stimulant effects lead to the sense of well-being, feelings of trust and empathy, increased intimacy and self-awareness, decreased inhibitions and moderate hallucinogenic effects, all responsible for the drug popularity (Green et al., 2003). Somatic unpleasant effects are also amply reported, including anorexia, hyperthermia, cardiac arrhythmia, hypertension, nausea and vomiting, bruxism, muscle aches, headache,

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sweating, fatigue, dizziness, and dry mouth, all compatible with monoamine overstimulation (Baylen and Rosenberg, 2006).

In what concerns pharmacological action, MDMA enhances the release of dopamine (DA), serotonin (5-HT) and noradrenaline (NA) at the central nervous system (CNS). The drug produces an acute, rapid enhancement in the release of both 5-HT and DA from nerve endings in the brain, acting mainly as a potent indirect monoaminergic agonist. This also leads to a long-term depletion of these neurotransmitters through the inhibition of the reuptake of HT and in lower extension of DA (Green et al., 2003). Since inhibitors of 5-HT transport block the effect of amphetamine derivatives on acute 5-5-HT release and the destruction of serotonergic terminals, the 5-HT transporter (SERT) has been implicated in this process (Rudnick and Wall, 1992). Coherent with MDMA effects, 5-HT participates in the regulation of several behavioural functions, such as mood, anxiety, and sleep. In turn, the disturbing of DA circuits related to the reward pathway also contribute to the MDMA response, as this is the principal neurotransmitter involved in motivational processes, such as reward and reinforcement.

According to data from The French Monitoring Centre for Drugs (SINTES/INPS) (Di Trapani et al., 2018), the content of MDMA in pills of ecstasy seized increased from the average dose of 50–60 mg per tablet in the 2000s to 100–150 mg in 2012, with some pill contents reaching 200 mg. As a consequence, also a dramatic increase in the number of MDMA intoxication cases with serious complications has been verified.

Of note, MDMA blood concentrations greatly differ among abusers who suffered non-fatal symptomatic and fatal intoxications and, consequently, lethal concentrations are currently unknown for this drug (De Letter et al., 2004; Schifano, 2004). This lack of linearity between the severity of the intoxication and the blood concentrations might be attributed, among others, to polymorphisms of enzymes, transporters and receptors involved in the pharmacodynamic response or metabolism of the drug, to other interindividual variability (age, gender, ethnicity, etc.), and to the presence of other drugs that were deliberately co-ingested and/or inadvertently present as contaminants in the pills. As an example, eighty-two MDMA-related deaths were reported in Australia between 2000 and 2005 (Kaye et al., 2009); although more than 80% of those were due to the direct drug toxicity, in almost 60% of the cases, MDMA was found in association with other drugs.

In what concerns the metabolism of MDMA, cytochrome P450 isoform (CYP) 2D6 (i.e. CYP2D6) and catechol-O-methyltransferase (COMT) are the main responsible for the metabolic fate of MDMA in the body (Carvalho et al., 2012), and both exhibit genetic polymorphism. Thus, as referred, these enzymes influence the individual responses to the

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severe intoxications (Di Trapani et al., 2018), due to the higher cytotoxicity of MDMA metabolites from oxidative reactions. Moreover, since MDMA pharmacokinetics is nonlinear, partially by virtue of the drug-induced CYP2D6 inhibition, the severity of the damage caused to patients is not necessarily related to the amount of ingested dose (Cherner et al., 2010). Naturally, this apparent lack of relationship between the alleged ingested dose and the severity of the acute toxic reaction is one of the main dangers of the abuse of MDMA (Carvalho et al., 2012).

Most acute severe intoxications exhibit common signs and symptoms that include those of 5-HT syndrome and exertional hyperpyrexia (Dickinson, 1989), leading to rhabdomyolysis and multi-organ failure, hyponatremia and cerebral oedema, isolated acute liver failure, cerebrovascular accidents, acute anxiety, panic disorder, and sudden death (Carvalho et al., 2012; Di Trapani et al., 2018).

1.1.1. Hyperthermia

As referred, complications following MDMA consumption include a wide range of symptoms, whose extent and severity may substantially vary between abusers. Nevertheless, from the acute physiological consequences of the drug, cardiovascular toxicity and severe hyperthermia are among the most life-threatening (Carvalho et al., 2012).

Unfortunately, the dysregulation of body temperature to values above 40 ºC is a common adverse effect associated to severe acute MDMA intoxication. This body overheating is probably related to the drug-induced disruption of serotonergic circuits at the thermoregulatory centre, located in the hypothalamus (Carvalho et al., 2012), as some animal studies have shown that this temperature increase is mediated by actions at the 5-HT receptors within the CNS (Schmidt et al., 1990). Additionally, the hyperthermia induced by the drug may be due to its action on peripheral changes in blood flow. Several studies revealed that by activating anterior hypothalamus neurons, MDMA induces NA release,

impairing heat dissipation through vasoconstriction secondary to the activation of α1

-adrenoreceptors, and slightly increases T4 serum levels (Carvalho et al., 2012). Also, NA together with thyroid hormones modulates brown fat thermogenesis by regulating the expression of uncoupling proteins (UCPs), which incorporate in mitochondria, dissociating the mitochondrial proton gradient from adenosine 5’-triphosphate (ATP) synthesis (mitochondrial uncoupling) and releasing the free energy as heat, in skeletal muscle and brown fat (Carvalho et al., 2012).

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Additionally, the environmental conditions of the places where MDMA intake frequently occurs, for instance, the overcrowded and overheated recreational venues, also contribute to potentiate drug-induced hyperthermia (Bellis et al., 2002). Furthermore, the excessive heating produced by the muscular hyperactivity associated with the extravagant dancing performed by MDMA abusers is also an adjuvant factor to the exacerbation of body temperature increase. All together, these conditions trigger a cascade of other

life-threatening complications, such as disseminated intravascular coagulation,

rhabdomyolysis, myoglobinuria, and acute renal failure (Carvalho et al., 2012).

1.1.2. Cardiotoxicity

Ecstasy has been linked to major cardiovascular events and other co-morbidities (Esse et al., 2011). Due to sympathetic stimulation and increased myocardial oxygen demand, MDMA may lead to tachycardia, vasoconstriction, changes in blood pressure, and arrhythmias. In severe intoxications, vasospasm leads to acute myocardial infarction and also to irreversible dilated cardiomyopathy (Ghuran and Nolan, 2000).

Evidence also indicates that cardiotoxicity of MDMA is related to the metabolism of catecholamines (Carvalho et al., 2004c). Accordingly, catecholaminergic stimulation is a potential source of drug-induced oxidative stress in the heart, leading to macrophage infiltration and myocardial necrosis through several mechanisms, such as ischaemia

secondary to coronary vasoconstriction, Ca2+ overload, and the production of oxygen and

nitrogen free radicals, either by auto-oxidation of catecholamines or their degradation by monoamine oxidase (MAO) (Jiang and Downing, 1990; Shenouda et al., 2008). Furthermore, reactive species may also be produced by mitochondrial dysfunction, leukocyte recruitment and activation (Badon et al., 2002), and by ischaemia induced by coronary vasospasm (Jiang and Downing, 1990). In addition, the redox active metabolites of MDMA have been implicated in the cardiotoxic effects of this drug (Carvalho et al., 2004c; Shenouda et al., 2008), as the drug itself is metabolized in the liver and at lower extent also in the heart, into ortho-quinones, that are also highly redox active molecules, and can undergo redox cycling, generating massive amounts of reactive oxygen and nitrogen species (Bolton et al., 2000). In addition, it has been shown that MDMA also significantly increases the nitrotyrosine content in the heart, along with the nitration of contractile proteins, such as troponin-T, tropomyosin-α1 chain, myosin light polypeptide, and myosin regulatory light chain (Varga et al., 2015). All these deleterious effects of MDMA in the heart lead to both structural and functional alterations in the myocardium. Furthermore, animal

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studies shown that the repeated binge administration of MDMA lead to myocarditis with inflammatory infiltrates, as well as areas of necrosis and disrupted cytoarchitecture (Shenouda et al., 2008).

1.2. N-Benzylpiperazine (BZP)

The most popular piperazine derivative is BZP. This drug, also known as “A2”, “Frenzy” and “Nemesis”, presents effects comparable to those of S(+)-amphetamine, and is available either as the hydrochloride salt or the free base (Monteiro et al., 2013). It was originally synthesized as an antihelmintic agent for use in farm animals and was then tested in the early 1970s as an antidepressant drug (Wikstrom et al., 2004). The popularity of BZP as a recreational drug is relatively recent. In the last decade, the drug emerged on the European drug market as a licit alternative to MDMA; BZP is cheaper and greatly succeeds in attempts to reproduce the MDMA subjective experience. Additionally, some drug users have also sought BZP in an effort to find a safer alternative to the classic drug (Baumann et al., 2005) as at the time it was introduced as a drug of abuse, BZP was claimed by some retailers as a “natural” product or as a “herbal high”, leading to a false perception of a weaker and safer product, when in fact the drug is entirely synthetic (Butler and Sheridan, 2007; Gee and Fountain, 2007). Then, the drug became illegal in many countries and it more often appeared as a contaminant in ecstasy pills (Arbo et al., 2012).

It is believed that BZP shares common features of its biological action with MDMA. Similar to the classic drug, the stimulant, euphoric, and socializing effects of BZP promoted its widespread use as a recreational drug among young consumers. These drug actions derive from the increase in releasing of DA, 5-HT and NA and from the inhibition of the reuptake of these neurotransmitters (Berney-Meyer et al., 2012). In general, BZP action on serotonergic pathways is similar to that of MDMA but the drug has a lower effect on NA and DA reuptake (Baumann et al., 2004). BZP is also characterized by its high affinity

antagonism at the α2-adrenergic receptor, leading to an increase in NA release by inhibition

of the negative feedback conveyed by the receptor (Wai Yeap et al., 2018). On the other

hand, BZP binding to 5-HT2A receptors explains the mild hallucinogenic effect elicited at

high doses of the drug, and the partial agonism at the 5-HT2B receptors is responsible for

BZP peripheral side effects (e.g., stomach pain felt by abusers), as this receptor is expressed in the gut. In turn, the frequent occurrence of headaches following intake of BZP

likely results from 5-HT3 receptor activation, since the activity of this receptor is linked to the

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Studies undertaken on animals indicate that BZP can substitute for methamphetamine in addicted rats, although it is ten times less potent and correspondingly produces weaker addictive effects (Brennan et al., 2007). Also, Campbell et al (1973) performed a blind-study that indicated that former adult addicts in amphetamines could not distinguish between BZP and dexamphetamine in what concerns the subjective, behavioural and autonomic effects elicited by the drugs.

Classical side effects of BZP are diverse and may vary in severity, ranging from mild and/or benign to lethal. Usually, the ingestion of low doses of BZP (50–200 mg) causes moderate toxicity (Berney-Meyer et al., 2012) with common side effects including anxiety, agitation, tremor, gastrointestinal upset (nausea and stomach pain), headache, autonomic instability (diaphoresis, vomiting and diarrhoea), musculoskeletal complications (including rhabdomyolysis), and fluid and electrolytes disorders (such as hyponatraemia and hyperkalaemia) (EMCDDA, 2018). Other clinical findings frequently described for the drug are compatible with cardiotoxicity, including palpitations, tachycardia, chest pain, increased pulse rate and hypertension, acute coronary syndrome, and dysrhythmias (Berney-Meyer et al., 2012). BZP can also lead to seizures, psychosis, renal toxicity, tremor, hallucinations, fever, and jaw clenching (EMCDDA, 2018). Even at typically consumed doses, BZP can induce serious toxicity (Berney-Meyer et al., 2012) and the increasing of the drug dose can extend the reported side effects in time or even aggravate them with fatal consequences (Berney-Meyer et al., 2012).

There are several case reports describing the ingestion of BZP (Balmelli et al., 2001; Berney-Meyer et al., 2012; Chatterton et al., 2012; Elliott and Smith, 2008; Gee et al., 2010; Gee et al., 2005; Karch, 2011; Lecompte et al., 2008; Wikstrom et al., 2004; Wood et al., 2008) but none of these publications is conclusive to establish BZP as the direct cause of the described deaths, mainly because most of the situations concern polydrug abuse. Nevertheless, it seems that BZP plays an important role in these fatal outcomes, and in some cases it was hypothesised that pharmacological and toxicokinetic synergistic interactions with amphetamine derivatives were at play (Lecompte et al., 2008).

In what concerns metabolic interactions, CYP2D6 and COMT are the main enzymes involved in the biotransformation of BZP, but CYP1A2 and CYP3A4 are also relevant (Lin et al., 2011).

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As explained above, hyperthermia is thought to derive from the disturbance of the central thermoregulatory system, being potentiated by the physical activity/dancing performed by BZP abusers at the recreational venues, and by the drug-derived neuromuscular hyperactivity (seizures, tremor, myoclonus, muscle rigidity hyperreflexia, and fasciculation) that occurs secondarily to sympathomimetic and 5-HT toxicity. Body temperature increase that follows these events may then lead to metabolic acidosis, rhabdomyolysis, cardiovascular collapse, renal failure, intracranial haemorrhage and, finally, to death (Balmelli et al., 2001; Berney-Meyer et al., 2012).

There is only one case of life-threatening toxicity associated with BZP abuse reporting the impairment of body temperature promoted by the drug (Gee et al., 2010). This report describes the intoxication of an adult female who developed status epilepticus, hyperthermia, disseminated intravascular coagulation, rhabdomyolysis, and renal failure after BZP ingestion (Gee et al., 2010). Despite this case suggesting that BZP alone has the potential to cause serious hyperthermia-related toxicity, there are no other reported occurrences supporting temperature deregulation following single drug intake. Accordingly, the other case-reports available in the literature relating BZP with hyperthermia, describe intoxications in which the drug was not taken alone (Berney-Meyer et al., 2012; Gee et al., 2010).

A study conducted in our laboratory indicated the worsening of the in vitro hepatotoxic effects elicited by several piperazine derivatives, when the drugs were tested under hyperthermia (unpublished data).

1.2.2. Cardiotoxicity

Undoubtedly, cardiotoxicity secondary to sympathomimetic and serotonergic disturbance is the most fearsome complication of BZP. At recreational doses, BZP and other piperazine derivatives have been shown to cause tachycardia and mild elevation of blood pressure (Dargan and Wood, 2013). Both palpitations and increased blood pressure result from dopaminergic and serotonergic stimulation at the peripheral adrenergic system (Gee et al., 2010; Schep et al., 2011). Accordingly, an increase in systolic and diastolic blood pressure and heart rate was observed in a randomized, double-blind, placebo-controlled study performed by Lin et al. (2009) after a single administration of 200 mg BZP or BZP combined with 1-(3-trifluoromethylphenyl)-piperazine (TFMPP) to volunteers (Lin et al., 2011; Thompson et al., 2010). Despite there have been no recorded incidents of significant

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arrhythmias at such doses, either extreme hypertension or hypotension may occur, resulting from the drug-induced autonomic instability (Dargan and Wood, 2013).

Sympathomimetic effects, such as dilated pupils and tachycardia, were also observed in three young adults after the consumption of four tablets containing BZP in association with (TFMPP) (Wood et al., 2008) and in a non-fatal case related to an overdose with another piperazine derivative (Kovaleva et al., 2008). Moreover, in a recent in vitro study, Arbo and collaborators (2014) observed that piperazine drugs, including BZP, were capable of

producing concentration-dependent cytotoxic effects in rat cardiomyoblasts (EC50 343.9 μM

for BZP), proving for the first time the cardiotoxic effects of piperazine designer drugs at the cellular level. Although no changes in intracellular oxidative stress markers were observed,

BZP caused significant increases in intracellular Ca2+ levels, accompanied by decreases in

ATP and in mitochondrial membrane potential (Δѱm) that seemed to involve the

mitochondrial permeability transition pore (Arbo et al., 2014). The authors also observed early apoptosis and a high number of cells undergoing secondary necrosis (Arbo et al., 2014).

1.3. Association of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)

and N-benzylpiperazine (BZP)

One of the most complex scenarios concerning drug abuse is the co-consumption of drugs, either due to the will of the user to engage in such pattern or to the lack of purity of the drugs sold in the illicit market that render abusers prone to unintended exposure to multiple substances (WHO, 2018). Generally, the majority of MDMA users are polydrug abusers (Scholey et al., 2004; Winstock et al., 2001; Wu et al., 2006), and many of them intentionally blend MDMA with piperazines, such as BZP (Wikstrom et al., 2004).

Not surprisingly, the content of MDMA among pills is widely variable, and usually contain a range of other psychoactive substances, in addition to, or in place of MDMA, including other methamphetamines (Parrott, 2004), ephedrine, caffeine, ketamine (Makino et al., 2003) and even piperazines (Wikstrom et al., 2004). As a result, consumption effects are unpredictable (Carvalho et al., 2012; Mohamed et al., 2011) and often culminate in lethality (Kaye et al., 2009).

Besides the presence of piperazine-derived drugs as cutting agents in ecstasy pills, these NPS may also occur as the main active substance of the formulation, and drugs sold as BZP are frequently contaminated with other chemicals, too. In Europe, BZP is largely commercialized in the drug black market combined with other piperazines, such as

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meta-chlorophenylpiperazine (mCPP), TFMPP, and dibenzylpiperazine (DBZP), and less commonly mixed with MDMA, amphetamine, ketamine or caffeine (EMCDDA, 2018). On the other hand, pills containing BZP and TFMPP have been reported to produce a ‘high’ similar to that produced by MDMA (Lin et al., 2011).

Although less frequent, the combination of MDMA with BZP is described as highly toxic due to the excessive sympathomimetic and serotonergic stimulation, and a number of studies and reports suggest that co-ingestion of BZP with MDMA or other amphetamine derivatives can be even more toxic than the single drug intake (Berney-Meyer et al., 2012). In this line, piperazine-derivatives are liable for drug–drug interactions with inhibitors or other substrates of CYP2D6 such as MDMA, which might increase the risk of toxicity (Staack, 2007), especially since they seem to have a narrow safety margin when used recreationally (Gee et al., 2005), possibly due to intrinsic pharmacodynamic properties, self-dosing variability, or genetic polymorphism. Several sudden deaths associated with the recreational use of BZP combined with MDMA have been reported worldwide, including in Sweden (Wikstrom et al., 2004), Switzerland (Balmelli et al., 2001), USA, Canada, and New Zealand (Gee et al., 2010). Forensic data from these fatal occurrences revealed signs of brain oedema (Balmelli et al., 2001), cardiac failure and hyperthermia-derived complications (rhabdomyolysis, disseminated intravascular coagulation, acute renal injury and multiple organ failure) (Gee et al., 2010). Together, these cases raise concerns about the likely occurrence of toxic synergisms or additivity from the combination of these two stimulants (Gee et al., 2010; Staack, 2007).

Since BZP has serotonergic effects, the drug may exacerbate the hyperthermia induced by other recreational serotonergic drugs, such as MDMA (Gee et al., 2010), or even by therapeutic drugs, such as fluoxetine (Berney-Meyer et al., 2012) leading to an increased risk of complications. In New Zealand, a young man required prolonged hospital care after developing toxicity that resulted from the combined use of BZP and MDMA (Gee et al., 2010). It is probable that the disruption of thermoregulation might have been promoted by both co-administered substances, which in association were able to overpass the stimulant threshold for hyperthermia. In fact, a core temperature above 38.5 ºC is already sufficient to trigger the onset of toxic events, which may then progress to severe hyperthermia and multi-organ failure (Berney-Meyer et al., 2012; Dargan and Wood, 2013; Gee et al., 2010).

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CHAPTER 2

OBJECTIVES OF THE THESIS

N-Benzylpiperazine (BZP) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) are two popular drugs of abuse that are consumed all over the world, frequently in combination with one another. So far, the effects elicited by such combination have not been elucidated and, worryingly, remain of unpredictable consequence. Both drugs present cardiotoxic risk, and potential toxicokinetic and toxicodynamic interactions. Hyperthermia is a (non-)physiological outcome secondary to the abuse of these drugs, and the conditions at which the recreational consumption of these drugs takes place, further raise the body temperature of the abusers. As a result, we hypothesised that cardiotoxicity of each single drug can be further aggravated by the drug combination and also by the increase in body temperature that follows administration.

Considering these facts, the aim of this thesis was to clarify the nature of the interaction of MDMA and BZP, through the evaluation of the in vitro cardiotoxic effects of the single drugs and their combination, both in normothermic and hyperthermic conditions. Specifically this study intended to:

a. Analyse the potential in vitro cardiotoxic of MDMA and BZP, using H9c2 rat cardiomyoblasts, individually and in combination

b. Elucidate the effect of hyperthermia (40.5ºC) in the observed cardiotoxicity, as a result of the thermogenic action of the drugs under the conventional settings of recreational consumption;

c. Determine the accuracy of two different mathematical models, Independent Action model (IA) and Concentration Addition model (CA), in predicting interaction (i.e. synergism, additivity, or antagonism);

d. In case of relevant toxicity, to delineate the mechanism(s) underlying the elicited effects.

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CHAPTER 3

MATERIALS AND METHODS

3.1. Chemicals

All chemicals used in this study were of analytical grade. N-Benzylpiperazine (BZP, 99.3 % purity) was purchased from Chemos GmbH (Regenstauf, Germany) and MDMA hydrochloride was extracted and purified from high purity ecstasy tablets that were provided by the Portuguese Criminal Police Department. The obtained salts were purified and fully characterized by nuclear magnetic resonance (NMR) and mass spectrometry (MS) methodologies. Unless stated otherwise, reagents used in cell culture were purchased from Gibco (Alfagene, Lisbon, Portugal) and all other chemicals from Sigma-Aldrich (Lisbon, Portugal).

3.2. H9c2 cells

H9c2 is an established and commercially available, clonal cardiomyoblast cell line derived from embryonic BDIX inbred rat heart ventricles (Kimes and Brandt, 1976). Considering the large experimental and clinical body of evidence documenting that MDMA and BZP lead to cardiovascular dysfunction and toxicity (Shenouda et al., 2010), H9c2 cells were selected as the in vitro model for this study. H9c2 is a representative model widely used in cardiotoxicity in vitro assays (Arbo et al., 2014; Begieneman et al., 2016), because these cells exhibit similar morphological features to immature embryonic cardiomyoblasts and preserve manifold biochemical characteristics of the cardiac adult cells (Hescheler et al., 1991). These cells are myoblasts with characteristics of skeletal muscle due to the expression of nicotinic receptors and the ability to express of a muscle-specific creatine phosphokinase isoenzyme when the mononucleated myoblasts fuse (Kimes and Brandt, 1976). According to Kimes and Brandt (1976), these cells form confluent multinuclear myotubes and present electrical and signalling mechanisms characteristic of the cardiomyocyte. Furthermore, H9c2 are cardio-specific for the L-type voltage-dependent

Ca2+ channels and for the pattern of signal-transducing G proteins. These cells are

well-endowed with rough endoplasmic reticulum, their surface is frequently enlarged by microvilli, and the sugar residues coating these cells are similar to those found in isolated rat cardiomyocytes (Hescheler et al., 1991). Moreover, genes encoding for cardiac

sarcomeric proteins, as for example troponin T, Ca2+ transporters and associated

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expressed in H9c2 cells at similar levels with those expressed in the heart. As an example, CYP1A1 and CYP1B1 are constitutively expressed in both H9c2 cells and the heart. For all of those reasons, this model adequately mimics the metabolic capacity of the rat heart (Zordoky and El-Kadi, 2007). Notwithstanding, there are some limitations concerning H9c2 cells. These cells lack morphological properties of freshly prepared cardiomyocytes. For instance, H9c2 cells do not express gap junctions or myofibrils with organized sarcomeres. Also, increased passage number could cause dedifferentiation, preventing the formation of confluent, multinuclear cells (Hescheler et al., 1991). H9c2 cells also could fuse to form myotubes if they were allowed to reach confluence.

3.3. H9c2 cell culture

H9c2 cells (ATCC, Manassas, VA, USA) were used between passages 12–22 and cultured as described elsewhere (Martins et al., 2018). Briefly, H9c2 cells were cultured with Dulbecco’s Modified Eagle’s high glucose medium (DMEM) (Sigma Aldrich) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 1% antibiotic solution (10,000 U/mL

penicillin; 10,000 μg/mL streptomycin) and 1% nonessential amino acids (NEAA) in 75 cm2

Corning® flasks (VWR, Lisbon, Portugal). H9c2 cells were incubated at 37 °C in a

humidified incubator with 5% CO2 atmosphere. The cell culture medium was removed when

cells reached 70–80% confluence, and then the cells were washed with pre-warmed Hank’s

balanced salt solution (HBSS, without Ca2+ and without Mg2+). After that, cultures were

passaged by trypsinization (0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) solution) and sub-cultured over a maximum of 10 passages.

3.4. Mixture testing

The drugs were combined at a MDMA:BZP 4:1 realistic ratio, as this was the proportion found in plasma samples from an authentic intoxication case (Peters et al., 2003). This mixture (Mix α) was tested in all experiments performed in the current study. For the sake of assessment of the type of mixture effect that emerges from combination of both drugs (synergism, antagonism or additivity), two other mixtures were also designed. In the first

mixture (Mix ß), the drugs were combined at their respective EC50 (representing the

half-maximum-effect concentrations from the mortality curves, such that the drugs were present at concentrations that elicited the same effect, i.e. equipotent concentrations). For this purpose, a master stock solution was prepared containing the individual components at

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section). The other mixture (Mix 𝛿) was prepared in a similar way by mixing BZP and MDMA

in proportion to the respective EC01 (see Table 2 in the Results section) to test possible joint

effects when individual components are present at statistically ineffective (i.e. non toxic) concentrations.

3.5. Drug exposures

H9c2 cells were seeded at a density of 2x104 cells/cm2 in 96-well plates and incubated

overnight at 37 °C with 5% CO2 for cell adhesion. In the next day, cells were exposed for

24 h to each drug and their mixture(s), under normothermic (37 °C) and hyperthermic (40.5 °C) conditions. All stock solutions of the test drugs and their mixture(s) were made in HBSS,

with Ca2+ and Mg2+, stored at -20 °C, and freshly diluted on the day of the experiment. By

employing the fixed mixture ratio design (da Silva et al., 2014b; Dias da Silva et al., 2013c) a range of concentrations of the mixture was subsequently prepared for testing, maintaining constant the ratio between each constituent (MDMA 8: BZP 2; Table 1). The percentage of

solvent (HBSS, with Ca2+ and Mg2+) in the medium was always lower than 5 %, and in all

cases solvent controls were performed in parallel to negative controls.

Table 1. Individual concentrations (mM) of 3,4-methylenedioxymethamphetamine (MDMA) and

N-benzylpiperazine (BZP) in each concentration of the mixture MDMA 8: BZP tested in H9c2, after 24h-incubations, at 37 oC or 40.5 oC. Mixture MDMA BZP 0.1 0.82 0.18 0.2 1.64 0.3 6 0.5 4.11 0.89 0.8 6.57 1.43 1.0 8.21 1.79 2.0 16.42 3.58 3.0 24.63 5.37 4.0 32.84 7.16

3.6. Cytotoxicity by the MTT reduction assay

To evaluate the general damage elicited by drug exposures, H9c2 cells seeded onto 96-well plates were exposed for 24 h to sixty-four concentrations of each drug and their mixtures (from 1.2 µM to 20 mM for MDMA; from 3.7 µM to 25 mM for BZP; from 1.7 µM to 10 mM for the mixtures), previous to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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2017). Briefly, at the selected time point, the culture medium was aspirated and the attached cells were rinsed with 100 µL HBSS, followed by the addition of fresh culture medium containing 0.5 mg/L MTT and the incubation for 30 min at 37 °C in a humidified 5 % CO2 atmosphere. The formed intracellular formazan crystals were then dissolved in 100 µL 100 % dimethyl dulfoxide (DMSO) and the absorbance was measured at 570 nm, using a

multi-well plate reader (BioTek SynergyTM HT, BioTek Instruments, Inc). To reduce

inter-experimental variability, each concentration was tested in triplicate at least in four independent experiments. The results were normalised with negative (only cell culture medium) and positive 1% 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) controls and results were graphically presented as percentage of cell death versus concentration (mM).

3.7. Prediction of mixture effects

The expected mixture effects were calculated using two pharmacological models of additivity, concentration addition (CA) and independent action (IA), as previously described (Dias da Silva et al., 2013c).

3.8. Lysosomal damage through the neutral red (NR) incorporation assay

Lysosomal integrity was evaluated through the neutral red (NR) uptake assay in 96-well plates, as previously described (Arbo et al., 2016). Briefly, after drug exposure, the medium was replaced by fresh medium containing 50 mg/mL NR. Then, the cells were incubated at

37 ºC in a humidified 5%, CO2–95% air atmosphere for 3 h, allowing the lysosomes of viable

cells to take up the dye. Subsequently, H9c2 cells were carefully washed with 200 µL of HBSS to eliminate extracellular dye and lysed with a ethanol: glacial acetic acid: water 5:1:4 solution. Triton X-100 at 1% was used as positive control. The absorbance was measured

at 540 nm in a multi-well plate reader (BioTek SynergyTM HT, BioTek Instruments, Inc).

Each one of the eight concentrations (100 μM – 4 mM) was tested in six replicates at least in four independent experiments. Data were expressed as percentage of negative control.

3.9. Measurement of reactive oxygen and nitrogen species (ROS and RNS)

Measurement of intracellular oxygen and nitrogen reactive species (ROS and RNS) was made by the 2’,7’-dichlorofluorescin diacetate (DCFH-DA) assay in 96-well plates, as previously described (da Silva et al., 2014a). Briefly, H9c2 cells were rinsed twice with

(33)

HBSS and incubated with 10 mM DCFH-DA (Sigma Aldrich) for 30 min, at 37 ºC, previous to all drug exposures. DCFH-DA was stored in an opaque airtight container at - 20 ºC. Since DCFH-DA is a non water-soluble powder, a 4 mM stock solution was initially prepared in DMSO (Merck) and immediately before each experiment the final concentration was made up by adding fresh culture media (ensuring that the final concentration of DMSO did not exceed 0.05%). After a 24 h-drug treatment period, fluorescence was recorded at 37 ºC on

a fluorescence microplate reader (BioTek SynergyTM HT, BioTek Instruments, Inc) set to

485 nm excitation and 530 nm emission. For each drug, a concentration of 100 μM was tested in six replicates at least in four independent experiments. A blank (without cells) and a negative control (no test drugs) were also included in the 96-well plate. Data were expressed as percentage of negative control.

3.10. Determination of intracellular reduced glutathione (GSH) and oxidized

glutathione (GSSG)

Determination of the reduced glutathione (GSH) and oxidized glutathione (GSSG) was performed according to the protocol described elsewhere (Dias da Silva et al., 2017), after seeding H9c2 cells onto 6-well plates and exposure to three concentrations of each drug (500 μM – 4 mM). Briefly, on the day of the assay, the cells were rinsed twice with HBSS

and scraped/precipitated with 5% perchloric acid (HClO4, w/v). After centrifugation for 5 min

at 16,000 g (at 4 ºC), the supernatants were collected and kept frozen at -80 ºC until further quantification, and the pellet was used for protein quantification. The thawed acidic supernatant was neutralized with an equal volume of 0.76 M potassium bicarbonate

(KHCO3) and centrifuged for 5 min at 16,000 g (at 4 ºC). Total glutathione (tGSH) was

determined by transferring, in triplicate, 100 µL of the neutralized supernatants, standards

or blank (5% HClO4, w/v) to a 96-well plate, followed by the addition of 65 µL of freshly

prepared reagent containing 0.24 mM nihydronicotinamide-adenine dinucleotide phosphate (NADPH) and 1.3 mM 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB) in phosphate buffer (71.5

mM Na2HPO4, 71.5 mM NaH2PO4 and 0.63 mM EDTA, pH 7.5). Then, the plates were

incubated for 15 min, at 30 ºC, in a microplate reader (Power Wave XTM, BioTek

Instruments, Inc.), prior to the addition of 40 µL per well of a freshly prepared 10 U/mL glutathione reductase solution (Sigma Aldrich) in phosphate buffer. The stoichiometric formation of 5-thio-2-nitrobenzoic acid (TNB) was followed every 10 sec for 3 min at 415 nm (at 30 ºC), and compared with a standard curve performed for all readings. For the determination of GSSG, 10 µL of 2-vinylpyridine were added to 200 µL aliquots of the acidic supernatants and mixed continuously for 1 h (at 0 ºC) for derivatization of the sulfhydryl

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