Psychopharmacology (2021) 238:1923–1936 https://doi.org/10.1007/s00213-021-05820-z ORIGINAL INVESTIGATION Daidzein modulates cocaine-reinforcing effects and cue-induced cocaine reinstatement in CD-1 male mice Miquel Martin 1,2 & Miriam Gutiérrez-Martos 2 & Roberto Cabrera 2 & Klaus Langohr 1,3 & Rafael Maldonado 2,4 & Magi Farre 1,5,6 & Rafael de la Torre 1,4,7 Received: 2 October 2020 / Accepted: 8 March 2021 / Published online: 11 April 2021 # The Author(s) 2021 ABSTRACT Rationale Cocaine addiction is a chronic relapsing disorder that lacks of an effective treatment. Isoflavones are a family of compounds present in different plants and vegetables like soybeans that share a common chemical structure. Previous studies have described that synthetic derivatives from the natural isoflavone daidzin can modulate cocaine addiction, by a mechanism suggested to involve aldehyde-dehydrogenase (ALDH) activities. Objectives Based on these previous studies, we investigated the effects of three natural isoflavones, daidzin, daidzein, and genistein, on the modulation of the cocaine reinforcing effects and on cue-induced reinstatement in an operant mouse model of cocaine self-administration. Results Chronic treatment with daidzein or genistein decreased operant responding to obtain cocaine intravenous infusions. On the other hand, daidzein and daidzin, but not genistein, were effective in decreasing cue-induced cocaine reinstatement. Complementary studies revealed that daidzein effects on cocaine reinforcement were mediated through a mechanism that involved dopamine type-2/3 receptors (DA-D2/3) activities. Conclusions Our results suggest that these natural compounds alone or in combination can be a potential therapeutic approach for cocaine addiction. Further clinical studies are required in order to ascertain their potential therapeutic use. Keywords cocaine . daidzein . daidzin . dopamine receptors . genistein . isoflavones . reinstatement . reinforcing . self-administration Miquel Martin and Miriam Gutiérrez-Martos equally contributed to this study Rafael Maldonado, Magi Farre, and Rafael de la Torre equally supervised this study. * Rafael de la Torre 3 Department of Statistics and Operations Research, Universitat RTorre@imim.es Politècnica de Cataluña (UPC)/BarcelonaTech, Jordi Girona 1-3, 08034 Barcelona, Spain 1 Integrative Pharmacology and Systems Neuroscience Research 4 Universitat Pompeu Fabra (CEXS-UPF), C/Dr. Aiguader 88, Group, Hospital del Mar Medical Research Institute (IMIM), Parc de 08003 Barcelona, Spain Recerca Biomedica de Barcelona (PRBB), C/Dr. Aiguader 88, 5 08003 Barcelona, Spain Universitat Autònoma de Barcelona (UDIMAS-UAB), C/Dr. Aiguader 88, 08003 Barcelona, Spain 6 2 Laboratory of Neuropharmacology, Parc de Recerca Biomedica de Clinical Pharmacology Unit, Hospital Universitari Germans Trias i Barcelona (PRBB), Universitat Pompeu Fabra, C/Dr. Aiguader 88, Pujol (IGTP), Carretera de Canyet s/n, 08916 Badalona, Spain 08003 Barcelona, Spain 7 CIBER Fisiopatologia de la Obesidad y la Nutrición (CIBERobn), Choupana s, /n 15706 Santiago de Compostela, Spain 1924 Psychopharmacology (2021) 238:1923–1936 INTRODUCTION effects on dopamine neurotransmission (Lammers et al. 1999) and on cocaine addiction (Bagley et al. 2019). Cocaine addiction is a chronic relapsing disorder character- These earlier investigations suggest that, by a direct or in- ized by compulsive drug seeking and use. Cocaine inhibits the direct mechanism, isoflavones can regulate DA neurotrans- monoamine transporters producing an increase in the concen- mission in the brain reward system and subsequently could tration of dopamine (DA) and other monoamines in the syn- be of potential interest for the treatment of cocaine addiction. aptic cleft in different brain areas, including the The aim of this study was to evaluate the effects of daidzin, mesocorticolimbic pathway, a brain circuit crucially involved daidzein and genistein in cocaine reinforcing effects and cue- in drugs reinforcing effects (Camí and Farré 2003). These induced seeking behavior using an operant mouse model of pharmacological actions are considered to mediate the rein- cocaine self-administration. We have also performed comple- forcing effects of cocaine and also to be involved in the de- mentary studies to elucidate the possible mechanisms of ac- velopment of cocaine addiction (Dackis and O'Brien 2001). tion of these isoflavones on the modulation of cocaine rein- At present time, no effective treatment for this disorder is forcement. Results obtained were compared with those pro- available. duced by a reference ALDH inhibitor, disulfiram. Isoflavones are biologically active polyphenols present in soybeans and other plants such as Kudzu (Pueraria lobata). Daidzin, daidzein, and genistein, among others, are members METHODS of this family of compounds. Studies performed in the lasts decades have described important effects of these isoflavones Animals regulating a variety of neurophysiological responses, includ- ing the activity of the mesocorticolimbic dopaminergic sys- Male CD-1 mice from 20 to 22 g (around 28-day old) at the tem. In this regard, Kudzu root extracts have been used in beginning of the experiments were used. Mice were main- traditional Chinese medicine for centuries to treat alcoholism tained in a temperature (21 ± 1°C) and humidity (65 ± 10%) (Keung and Vallee 1994). Further research in rodents demon- controlled room with reversed light-dark cycle (lights on from strated an important role of daidzin in these responses, reduc- 20:00 to 08:00 h) except for the locomotor activity studies ing alcohol consumption by selectively inhibiting the activity where animals were exposed to a normal light dark cycle of the aldehyde dehydrogenases (ALDH), a family of en- (lights on from 8:00 to 20:00 h). Animals trained in the oper- zymes involved in dopamine (DA) and ethanol metabolism ant self-administrations boxes were single-housed after intra- (Keung et al. 1997). More recent investigations with a syn- venous catheter surgery. Mice exposed to the locomotor ac- thetic derivative of daidzin called CVT-10216 or GS 455534 tivity studies were housed 4 per cage. Different sets of mice also described important inhibitory effects of this compound were used on each study of locomotion and self-administra- on alcohol and cocaine consumption, as well as in palatable tion. Behavioral tests and animal care were conducted in ac- food binge eating behavior. Changes in ALDH activities and cordance with the standard ethical guidelines (European in DA neurotransmission in the mesocorticolimbic system Commission Directive 86/609 EC) and approved by the local were considered the main neurobiological mechanism in- ethical committee (Comité Etico de Experimentación Animal, volved in the responses observed after CVT-10216 adminis- PRBB-UPF). All behavioral studies were performed in blind tration (Arolfo et al. 2009; Lange and Diamond 2009; Yao conditions. All mice were handled and habituated to the con- et al. 2010; Bocarsly et al. 2014). ditions of the animal facility for at least 1 week before the Daidzein, another isoflavone present in Kudzu roots, experiments started. also produces potent antidipsotropic effects for ethanol in rodents (Keung and Vallee 1993) although, in contrast Drugs with daidzin, the mechanism of action does not involve the inhibition of ALDH activities (Keung et al. 1997) and Cocaine hydrochloride was obtained from the Spanish remains to be clarified. Further investigations, however, Agency of Medicines and Medical Devices (AEMPS, have described modulatory effects of this isoflavone on Madrid, Spain), dissolved in sterile saline, and administered DA synthesis and release (Bare et al. 1995) that could at 0.5 mg/kg (intravenously, i.v.). All natural isoflavones account for the inhibitory effects of daidzein on alcohol (daidzin, daidzein, and genistein) were purchased from LC consumption. On the other hand, genistein also regulates Laboratories (Woburn, MA) or PhytoLab DA synthesis and release, as was described in mouse (Vestenbergsgreuth, Germany), dissolved in 50 μl of Tween striatal slices (Goldstein et al. 2012). Moreover, both, ge- 80/0.5% carboxymethylcellulose (CMC) in sterile saline and nistein and daidzein, show potent agonistic effects on es- administered at different doses by intraperitoneal (i.p.) route. trogen receptors (ER) activities (Kuiper et al. 1998). The Disulfiram was purchased from TOCRIS (Bio-Techne, estrogen receptors are known to exert important regulatory Minneapolis, MN), dissolved in 50 μl of Tween 80/0.5% Psychopharmacology (2021) 238:1923–1936 1925 CMC in sterile saline and administered at different doses by tubing (0.96 mm o.d., Portex Fine Bore Polythene Tubing, i.p. route. Phenylephrine (1 mg/kg, subcutaneous (s.c.), not Portex Limited, Kent, England) to a single channel liquid permeable to cross the Blood Brain Barrier), atipamezole swivel (375/25, Instech Laboratories, Plymouth Meeting, (0.4 mg/kg, s.c.), isoproterenol (0.25 mg/kg, i.p.), PA, USA), and to the mouse i.v. catheters. The swivel was SKF38393 (0.1 mg/kg, s.c.), quinpirole (0.01 mg/kg, i.p.) mounted on a counter-balanced arm above the operant and tamoxifen (1 mg/kg, i.p.) were purchased from TOCRIS chamber. and dissolved in 50 μl of Tween 80/0.5% CMC in sterile saline. Surgery Locomotor activity Mice were anesthetized with a ketamine/xylazine mixture (0.1 ml/10 g body weight, i.p.) and then implanted with indwelling Locomotion was evaluated in locomotor activity boxes (9 × i.v. silastic catheters in the right jugular vein, as previously 20 × 11 cm; Imetronic, France) containing a line of photocells described (Trigo et al. 2006). After surgery, animals were 2 cm above the floor to measure horizontal movements, and individually housed and allowed 4 days for recovery before another line located 6 cm above the floor to measure vertical starting the operant training. The patency of the catheters was activity (rearing). Mice were individually placed in the boxes evaluated at the end of the operant cocaine self-administration and the total activity was recorded during 60 min in a low experimental sequence, and whenever drug self- luminosity environment (20–25 lux). Total locomotor activity administration behavior appeared to deviate more than 50% was evaluated as the sum of the horizontal and vertical move- from the one previously observed, by infusing 0.1 ml of thio- ments. All the locomotor activity tests were performed be- pental (5 mg/ml) through the catheter. If prominent signs of tween 09:30 and 16:30 h. Isoflavones and disulfiram were anesthesia were not apparent within 3 s of the infusion, the administered 60 min before locomotor activity measurements. animal was removed from the experiment. Phenylephrine, atipamezole, isoproterenol, SKF38393, quinpirole, and tamoxifen were administered 30 min before Procedure locomotor activity was evaluated. On each locomotion exper- iment, a control group of mice (vehicle) was included. Mice Animals were trained to nose-poke under an FR1 schedule of from all experimental conditions were evaluated in a reinforcement to receive cocaine (0.5 mg/kg/infusion). Self- counterbalanced manner. All locomotor activity studies were administration session started with a priming infusion of the performed with no previous habituation to the activity boxes. drug, lasted for 60 min and was conducted 7 days a week. After each session, mice were returned to their home-cages. Cocaine self-administration procedure The number of reinforcers was limited to 50 infusions per session. Each infusion was followed by a 15-s time-out period Apparatus during which an active nose-poke had no consequence. Stable acquisition of self-administration behavior was achieved Self-administration training and testing occurred in operant when the three following conditions were met: (i) <20% de- chambers (Model ENV-307A-CT, Med-Associates, Georgia, viation from the mean of the total number of reinforcers VT, USA) equipped with two holes, one selected as the active earned in three consecutive sessions (80% stability), (ii) at hole for delivering the reinforcer and the other as the inactive least 75% responding on the active hole, and (iii) a minimum hole. Acquisition of drug self-administration was performed of four reinforcers earned per session. When stability criteria using a fixed ratio 1 (FR1) schedule of reinforcement such that were achieved, the effects of daidzin (75 mg/kg, i.p.), daidzein one nose-poke in the active hole resulted in one cocaine infu- (100 mg/kg, i.p.), genistein (100 mg/kg, i.p.), disulfiram (75 sion, while nose-poking in the inactive hole had no pro- mg/kg, i.p.), and vehicle were evaluated on cocaine self- grammed consequences. A stimulus light, located above the administration for 5 consecutive days. These compounds were active hole, was paired contingently with the delivery of the administered 60 min before starting the self-administration reinforcer. Pump noise and a stimulus-light located above ac- session on each day. In a different set of mice, quinpirole or tive hole were paired with the delivery of the infusion. When tamoxifen were injected 30 min after vehicle or daidzein (100 mice responded on the reinforced hole, the stimulus light went mg/kg, i.p.) administration during 5 consecutive days to eval- on, and a cocaine infusion was delivered. Each response on uate the involvement of dopaminergic or estrogen receptors the active manipulandum in this phase led to the presentation on the modulatory effects of this isoflavone on cocaine self- of the cue light for 2 s. Infusions were delivered in a volume of administration. 23.5 μl over 2 s. Cocaine was infused via a syringe that was Data from acquisition of cocaine self-administration (Fig 2) mounted on a microinfusion pump (PHM-100A, Med- were expressed as number of infusions and as area under the Associates, Georgia, VT, USA) and connected, via Tygon curve (AUC). AUC was calculated by using a standard 1926 Psychopharmacology (2021) 238:1923–1936 trapezoidal method. The following equation was used: AUC = the estrogen, dopaminergic, and adrenergic systems on [0.5 *(B1 + B2)* h] + [0.5 *(B2 + B3)* h] +….+ [0.5 *(Bn + daidzein-induced hypolocomotor effects were studied Bn + 1)* h], where Bn were the infusions received for each employing two-way ANOVA models including condition mouse and h was the time (days) passed between the consec- (vehicle vs. daidzein) and treatment (vehicle, phenylephrine, utive measurements (Gibaldi and Perrier 1975). atipamezole, isoproterenol, SKF38393, tamoxifen, A different set of mice were trained to self-administer co- quinpirole) as well the interaction of both factors. All post caine. After reaching the acquisition criteria, mice underwent hoc pairwise comparisons of treatment versus vehicle under to an extinction process. During this phase, nose-poking into both conditions were carried out in the framework of that the reinforced poke caused no consequences, meaning that the model. The interaction permitted to compare each treatment pump was turn off and the mice did not receive the infusion of versus the vehicle separately for both conditions by means of the drug nor the conditioned stimulus (light). Mice were ex- post-hoc comparisons. posed to 1 h daily extinction sessions 6 days a week until We analyzed the effects of the isoflavones or disulfiram on extinction criterion was reached. The extinction criterion active responding during cocaine self-administration by was achieved when mice made a mean number of active re- means of four two-way repeated measures ANOVA models sponses in three consecutive extinction sessions of less than including day (of treatment) and treatment (daidzein, daidzin, 30% of the responses performed during the last day of the genistein, or disulfram versus vehicle) as well as their interac- cocaine-training period. A small percentage of animals never tion as factors. In case the day-treatment interaction was sta- reached the extinction criteria after the exposure to the extinc- tistically significant, treatment comparisons were carried out tion protocol for a month and a half (less than around 10%) separately for each day, otherwise no post-hoc comparisons and were excluded from the experiment. were performed. The area under the curve (AUC) of the active After achieving the extinction criterion, mice were exposed responding was also calculated for each compound in the ac- to cue (stimulus light)-induced reinstatement of cocaine seek- quisition of cocaine self-administration and analyzed with ing behavior. Cue-induced reinstatement was conducted un- one-way repeated measures ANOVA model. der the same conditions used in the acquisition phase except Tamoxifen and quinpirole modulatory effects on active co- that cocaine was not delivered. Each response on the active caine self-administration were evaluated separately for control manipulandum in this phase led to the presentation of the cue vehicle- and daidzein-treated mice by using 2-way repeated light for 2 s and the activation of the pump (but with no measures ANOVA models that included day (of treatment) delivery of the drug). Animals were reexposed to several ses- and treatment (vehicle, tamoxifen, quinpirole) as well as the sions of cue-induced reinstatement using a Latin-square de- interaction of all both factors. Data shown on Figs. 2 and 3 and sign. Each reinstatement session was preceded by an extinc- the corresponding statistical analysis was performed only with tion period and not performed until mice reached the extinc- the number of active nose-poke responses when cocaine was tion criterion again. This methodological approach allowed available. Active nose-pokes during the time out period were evaluating in the same animal the effects of vehicle and one not included. isoflavone or disulfiram on cue-induced relapse. The doses The model assumptions of all models, that is, homoscedas- used and period of administration of these compounds were ticity and normality, were checked by means of different re- the same as when tested during cocaine self-administration sidual plots. Whenever the model assumption did not seem to (75 or 100 mg/kg, i.p., 60 min before testing), but the animals hold, the data were log-transformed prior to the analyses. All received only an acute injection. We also followed a Latin- post-hoc pairwise comparisons were carried out in the frame- square design to investigate the involvement of the dopami- work of the corresponding model and the computation of the nergic (quinpirole) or estrogen (tamoxifen) receptors modu- simultaneous confidence intervals and adjusted p values in lating daidzein or daidzin effects on cue-induced cocaine re- order to guarantee a family-wise error rate of 0.05 was based lapse. These compounds were injected 30 min after vehicle or on the multivariate t distribution of the vector of test statistics isoflavone administration. (Hothorn et al. 2008). All mice used on cocaine self-administration studies and on Isoflavone or disulfiram effects on the active nose-poke cue-induced relapse studies were distributed in the different responses after cue-induced cocaine reinstatement were ana- experimental groups homogeneously based on their responses lyzed by applying a paired Student's t-test analysis. during the cocaine self-administration period. Finally, the effects of quinpirole and tamoxifen modulating the active nose-poke responses on cue-induced cocaine rein- Statistical analysis statement were analyzed separately for vehicle- or daidzein- or daidzin-treated animals by applying a one-way ANOVA, Locomotor activity results were analyzed for each compound followed by Dunnett's post hoc analysis tests when required. evaluated by using one-way ANOVA followed by Dunnett's All data were analyzed using the statistical software R, multiple comparison tests when required. The involvement of version 3.4.3. (Vienna, Austria; http://www.rproject.org) or Psychopharmacology (2021) 238:1923–1936 1927 STATISTICA software. Results were considered statistically agonist, 1 mg/kg, s.c), atipamezole (an α-2 adrenergic recep- significant whenever p < 0.05. All the results are expressed as tor antagonist, 0.4 mg/kg, s.c.), isoproterenol (a β-adrenergic mean + SEM. agonist, 0.25 mg/kg, i.p.), SKF38393 (a DA type-I, D1, re- ceptor agonist, 0.1 mg/kg, s.c.), or quinpirole (a dopamine type-II/III, D2/3, receptor agonist, 0.01 mg/kg, i.p.). Thirty RESULTS minutes after the second administration locomotion was eval- uated for a period of 60 min (Fig. 1e). Daidzin, daidzein, genistein, and disulfiram effects on The statistical analysis revealed that the doses chose of locomotor activity in CD-1 male mice each estrogen, adrenergic, or dopaminergic compounds were subeffective, as no significant differences were observed in This preliminary experiment was carried out in order to select between the different groups of mice treated with vehicle the appropriate dose of each isoflavone that produce no loco- (Fig. 1e). Daidzein (200 mg/kg, i.p.) produced significant motor effects to be used later on in the cocaine self- hypolocomotor effects when compared with control vehicle administration studies. The range of doses of each isoflavone treated mice (p < 0.05). and disulfiram tested in this experiment was selected based on Two-way ANOVA also demonstrated a significant effect previous studies (Keung and Vallee 1993; Schroeder et al. of the treatment with daidzein (F(1,129) = 66.130, p < 0.001), 2010). Daidzein and genistein were administered at the doses with an effect of the posterior treatment with the different of 100, 200, and 400 mg/kg, i.p.; whereas daidzin and disul- agonists or antagonists (F(6,129) = 4.195, p < 0.001) with no firam at the doses of 75, 150, and 300 mg/kg, i.p.. Mice were interaction between both factors (F(6,129) = 1.324, n.s.). exposed to the locomotor activity boxes 60 min after the ad- Further statistical analysis also revealed that the treatment ministration of the corresponding compound. with a subeffective dose of the D2/3 dopaminergic receptor Results show that only daidzin and genistein produced agonist quinpirole (0.01 mg/kg) (Fig. 1e) modified daidzein- some hypolocomotor effects when administered at the highest induced hypolocomotion (p < 0.05 post hoc pairwise compar- doses (F(3,30) = 6.7093, p < 0.001; p < 0.01 vehicle vs. daidzin isons: daidzein-vehicle vs. daidzein-quinpirole). Tamoxifen 150 and 300 mg/kg) (Fig. 1b); (F(3,25) = 4.809, p < 0.01; p < treatment (1 mg/kg, i.p.) also modified daidzein 0.01 vehicle vs. genistein 200 mg/kg and p < 0.05 400 mg/kg) hypolocomotion, although it did not reach statistical signifi- (Fig. 1c). Daidzein and disulfiram did not produce substantial cance (p = 0.084). locomotor changes at any of the doses tested, although the Daidzein, like other isoflavones, can produce blood vessel highest ones tended to reduce locomotor activity (F(3,30) = relaxation that may alter blood pressure (Nevala et al. 2001) 3.8821, n.s and F(3,28) = 2,369, n.s, respectively) (Fig. 1a,d). and indirectly affect locomotion. In order to discard the in- Based on these results, daidzein and genistein at the dose of volvement of hypotensive changes on daidzein 100 mg/kg, i.p., 60 min, and daidzin and disulfiram at the dose hypolocomotor effects, the combination with phenylephrine, of 75 mg/kg, i.p., were chosen to be evaluated in the operant a peripheral α-1 adrenergic receptor agonist with well-known cocaine self-administration paradigm. hypertensive properties, was also evaluated. No statistically significant differences in locomotion were observed after Daidzein produces its hypolocomotor effects through phenylephrine administration (Fig. 1e), suggesting that daid- a mechanism that requires dopaminergic receptor zein hypolocomotor responses were not the consequence of activities in CD-1 male mice associated alterations in blood pressure. Moreover, any of the others agonists or antagonists tested Another preliminary locomotor study was conducted to delve in this locomotor study (atipamezole, isoproterenol or into the mechanism regulating isoflavone pharmacological SKF38393, Fig. 1e) did not produce statistically significant actions. We investigated the involvement of the estrogen, nor- effects when evaluated. adrenergic and dopaminergic systems in these responses. Because preliminary studies in the laboratory suggested that Chronic treatment with daidzein decreases cocaine daidzein effects modulating cocaine reinforcing were more reinforcement in CD-1 male mice relevant than those produced by daidzin or genistein, this study was conducted in animals treated with daidzein. In order to evaluate the effects of daidzein, daidzin, genistein, Animals were exposed to vehicle or to daidzein (200 and disulfiram on cocaine reinforcement, a new set of mice mg/kg, i.p., a dose known to produce hypolocomotor effects). underwent surgery and got implanted with i.v. catheters and Thirty minutes later animals received a second administration trained in operant boxes to actively nose-poke to obtain infu- of vehicle or a subeffective dose of one of the following com- sions of cocaine (0.5 mg/kg/infusion). After reaching the sta- pounds: tamoxifen (an estrogen receptor antagonist, 1 mg/kg, bility criteria, animals were divided into the different experi- i.p.), phenylephrine (a peripheral α-1 adrenergic receptor mental groups and received vehicle, daidzein (100 mg/kg, 1928 Psychopharmacology (2021) 238:1923–1936 Fig. 1: Locomotor effects induced after acute isoflavone or disulfiram administration. Involvement of dopamine D2/3 and estrogen-receptor systems in the locomotor effects induced by daidzein. Total locomotor activity 60 min after acute administration (i.p.) of different doses of (a) daidzein (n = 8-10), (b) daidzin (n = 8-10), (c) genistein (n = 8-10), or (d) disulfiram (n = 8-10). Effects of acute administration (30 min before locomotion was evaluated) of different agonist or antagonist for the adrenergic, do- paminergic or estrogen receptor systems (n = 9-15 per group) in the locomotor activity induced by (e) acute vehicle (60 min before locomotion) or daidzein (200 mg/kg, 60 min before locomo- tion) administration. A total of 9- 15 mice were included per exper- imental group. Results are expressed as average + SEM, ⋆ ⋆ p < 0.01, ⋆ p < 0.05 versus vehicle or versus daidzein-vehicle treated mice, post hoc pairwise comparisons i.p.), daidzin (75 mg/kg, i.p.), genistein (100 mg/kg, i.p.), or Chronic daidzin (75 mg/kg, i.p.) effects on active nose- disulfiram (75 mg/kg, i.p.) for 5 consecutive days 60 min poke responding were also analyzed and compared with ve- before being exposed to the self-administration task. hicle-treatment. Two-way ANOVA of repeated measures When the effects of chronic daidzein (100 mg/kg, demonstrated no effect of the days (F(4,80) = 0.410, n.s.), an i.p.) versus chronic vehicle treatment were compared effect of the treatment (F(1 20) = 4.538, p < 0.05) and an in the active nose-poke responding, two-way repeated interaction between both factors (F(4,80) = 3.687, p < 0.01). measures ANOVA revealed no day-treatment interaction Further analysis demonstrated a significant difference in the (F(4,84) = 0.862, n.s.). For this reason, the interaction active responding in between mice treated with vehicle and was removed from the model and the updated model daidzin on day 1 (Fig. 2b; day 1 (p < 0.01)) but not after the revealed a treatment effect (F (1,21) = 17.744, p < chronic exposure to this isoflavone. 0.001), but no effect of the day (F(4,88) = 0.991, n.s.). When the effects of chronic genistein (100 mg/kg, i.p.) The estimated treatment difference (on the log-scale) of versus chronic vehicle treatment were compared in the active daidzein versus chronic vehicle was −1.169 (95% CI: nose-poke responding, two-way ANOVA of repeated mea- [−1.712, −0.625]; Fig. 2a). sures revealed no day-treatment interaction (F(4,80) = 0.513, Psychopharmacology (2021) 238:1923–1936 1929 Fig. 2: Chronic treatment with isoflavones decreases operant responding for cocaine. (a–d) Effects of the chronic treatment with vehicle (n = 12) and daidzein (100 mg/kg/day, i.p., n = 11) (a), daidzin (75 mg/kg/day, i.p., n = 10) (b), genistein (100 mg/kg/day, i.p., n = 10) (c) and disulfiram (75 mg/kg/day, i.p., n = 9) (d) on co- caine self-administration. (e–h) Area under the curve of the 5 days of chronic treatment with vehicle (n = 12) and daidzein (100 mg/kg/ day, i.p., n = 11) (e), daidzin (75 mg/kg/day, i.p., n = 10) (f), ge- nistein (100 mg/kg/day, i.p., n = 10) (g) and disulfiram (75 mg/kg/ day, i.p., n = 9) (h) on cocaine self-administration. Animals re- ceived the injection of the vehicle, isoflavone or disulfiram for 5 consecutive days 1 h before the beginning of the operant training session. Data are represented as the mean number of nose-pokes in the active hole to obtain co- caine (0.5 mg/kg/infusion) during the preacquisition and acquisi- tion-training, and following the chronic treatment for 5 days with vehicle, isoflavone or disulfiram. Data are expressed as mean + SEM. ⋆ ⋆ p < 0.01, ⋆ p < 0.05 versus vehicle treated mice post hoc pairwise comparisons n.s.). For this reason, the interaction was removed from the vehicle. Update analysis (after removing the interaction) re- model and the updated model revealed a treatment effect vealed a treatment effect (F(1,19) = 6.648, p < 0.05), but no (F(1,20) = 14.969, p < 0.001), but no effect of the day (F(4,84) effect of the day (F(4,80) = 2.009, n.s.). The estimated treatment = 1.557, n.s.). The estimated treatment difference (on the log- difference (on the log-scale) of disulfiram versus chronic ve- scale) of genistein versus chronic vehicle was −0.786 (95% hicle was −0.760 (95% CI: [−1.338, −0.182]; Fig. 2d). CI: [−1.184, −0.3878]; Fig. 2c). The AUC of active cocaine self-administration responses Statistical analysis (two-way ANOVA repeated measures) was also calculated for each compound and compared with the revealed no significant day-treatment interaction (F(4,76) = control vehicle treated animals. Results obtained demonstrat- 1.051, n.s.) in the active responding of mice treated with di- ed that daidzein significantly decreased cocaine self- sulfiram when compared with the animals treated with administration responses (p < 0.05 versus vehicle treated 1930 Psychopharmacology (2021) 238:1923–1936 animals; Fig. 2e). Interestingly, the statistical analysis also mice exposed to daidzein (p < 0.05, daidzein-quinpirole vs. revealed a significant effect of genistein modifying cocaine daidzein-vehicle) (Fig. 3b). self-administration responses (p < 0.05 vs. vehicle; Fig. 2g). On the other hand, responses in the inactive nose-poke Modifications in the AUC after daidzin and disulfiram treat- were not modified by any treatment, displaying an average ment were also calculated. No significant effects in the one- response ranging from 0.7 to 1.8 in between the different way ANOVA were revealed after daidzin or disulfiram ad- experimental groups (see Suppl. Fig. 2). ministration (n.s. in both cases; Fig. 2f,h). Our results suggest that daidzein modifies cocaine self- On the other hand, responses in the inactive nose-poke administration behavior by a mechanism that requires DA were not modified by any treatment, displaying an average D2/3 receptor activities. response ranging from 1.7 to 2.8 in between the different experimental groups (see Suppl. Fig. 1). Daidzein, daidzin and disulfiram, but not genistein, These results suggest that isoflavone effects on drug rein- decreased cue-induced cocaine seeking behavior and forcement are heterogeneous and point to different mecha- relapse in CD-1 male mice nisms of action involved in the regulatory actions produced by each isoflavone. A new set of animals was exposed to cocaine self-administra- tion, followed by an extinction protocol. After reaching ex- Involvement of the D2/3 dopamine receptor on tinction criteria, the effects of an acute injection of daidzein daidzein-induced modulatory effects in cocaine self- (100 mg/kg, i.p.), daidzin (75 mg/kg, i.p.), genistein (100 administration in CD-1 male mice mg/kg, i.p.), or disulfiram (75 mg/kg, i.p.) were evaluated on cue-induced cocaine seeking behavior by applying a Latin Based on our previous results obtained in the locomotor ac- square experimental design. All compounds were adminis- tivity studies (Fig.1), we investigated the involvement of the tered 60 min before starting the relapse session. estrogen receptors (tamoxifen produced clear modulatory Paired Student t-test analysis revealed that responding in actions on daidzin locomotor effects that almost reached the active hole was not modified in between sessions in mice statistical significance, see Fig. 1e) and DA D2/3 receptors treated with vehicle (Fig. 4a). in the modulatory effects induced by daidzein (isoflavone)s Statistical analysis revealed that daidzein (100 mg/kg, i.p.) on cocaine reinforcement. decreased cue-induced relapse when compared with vehicle Daidzein was selected as a reference isoflavone for this administration (p < 0.05) (Fig. 4b). Moreover, daidzin (75 study because preliminary experiments (included in the mg/kg, i.p.) preexposure was also effective to decrease co- results shown in Figs. 2 and 4) demonstrated more relevant caine relapse (p < 0.05). Similar results were also observed effects modulating both, active cocaine self-administration in mice pretreated with disulfiram (75 mg/kg, i.p.), showing a and cue-induced seeking behavior than those produced by significant decreased in the active nose-pokes responses when daidzin or genistein. compared with vehicle pretreatment (p < 0.05) (Fig. 4e). Animals were trained to self-administer cocaine. After However, genistein (100 mg/kg, i.p.) did not modify the reaching stability criteria a chronic treatment for 5 consecutive behavioral responses in this paradigm. Mice treated with this days began. Tamoxifen (1 mg/kg, i.p.), quinpirole (0.01 isoflavone showed the same number of active responses as mg/kg, i.p.) or vehicle were administered 30 min after daid- when received vehicle injections (paired Student's t test, n.s.) zein (100 mg/kg, i.p.) or vehicle injections during these 5 (Fig. 4d). consecutive days. Thirty minutes later, animals were exposed to the self-administration task. Chronic exposure to quinpirole Involvement of D2/3 dopamine receptors on daidzein, (0.01 mg/kg, i.p.) or tamoxifen (1 mg/kg, i.p.) produced no but not daidzin, modulatory effects in cue-induced significant effects in control animals preexposed to vehicle cocaine seeking behavior in CD-1 male mice (Fig. 3a). Two-way ANOVA of repeated measures revealed a signif- The involvement of estrogen and DA D2/3 receptors on iso- icant effect of the day (F(4,72) = 3.319, p < 0.05), with no effect flavone effects modulating cue-induced cocaine seeking be- of the treatment (F(2,18) = 2.313, n.s.) nor interaction between havior were also investigated. The effects of tamoxifen and factors (F(8,72) = 1.746, n.s.) in the active nose-poke responses quinpirole were evaluated in daidzein, and also daidzin treated in mice exposed to daidzein (100 mg/kg, i.p.) for 5 days. mice, because of the similar chemical structure shared by both Further analysis show that chronic tamoxifen administration isoflavones. produced no effect in the operant responding in mice pre- After reaching extinction criteria, animals received vehicle, treated with this isoflavone (Fig. 3b). However, post-hoc daidzein or daidzin and 30 min later tamoxifen, quinpirole or pairwise comparisons revealed a significant effect on the 5th vehicle. Thirty minutes after the last injection, animals were day of the treatment with quinpirole in the active responses in exposed to cue-induced relapse. Both tamoxifen and Psychopharmacology (2021) 238:1923–1936 1931 Fig. 3: Involvement of the dopamine D2/3 receptor on daidzein-induced preacquisition and acquisition-training, and following the chronic treat- modulatory actions in cocaine self-administration paradigm. Effects of ment for 5 days with vehicle or daidzein (60 min before the beginning of the exposure to vehicle, quinpirole (0.01 mg/kg, i.p.) or tamoxifen (1 the training session) and subsequent administration of vehicle, quinpirole, mg/kg, i.p.) in the operant responding to obtain cocaine in mice previous- or tamoxifen (30 min before the beginning of the training session). ly treated with (a) vehicle (i.p., n = 7-10) or (b) daidzein (100 mg/kg/day, Results are expressed as average + SEM. ⋆ p < 0.05 versus daidzein- i.p., n = 8-9). Data are represented as the mean number of nose-pokes in vehicle treated animals on the same day, post hoc pairwise comparisons the active hole to obtain cocaine (0.5 mg/kg/infusion) during the quinpirole did not modify cocaine-seeking behavior in control DISCUSSION mice previously treated with vehicle (one-way ANOVA F(2,30) = 0.0639, n.s.) (Fig. 5a). Daidzein (100 mg/kg, i.p.) In the present study, we have demonstrated that the natural decreased the number of active responses after cue-induced isoflavones daidzein, daidzin and genistein can regulate the cocaine relapse when compared with vehicle-treated animals reinforcing properties of cocaine and also cocaine seeking (Fig. 5b). One-way ANOVA revealed a significant effect of behavior in an operant self-administration paradigm in mice. treatment with quinpirole or tamoxifen (F(2,26) = 3.9221, p < We have compared the effects of these compounds with 0.05). Subsequent post hoc analysis demonstrated that DA those produced by disulfiram, a drug known to yield different D2/3 receptor agonist quinpirole (Fisher's F test post hoc anal- pharmacological effects including the inhibition of ALDH ysis, p < 0.05), but not estrogen receptor agonist tamoxifen activities and to reduce alcohol intake (Hald and Jacobsen (Fisher's F test post hoc analysis, n.s.), significantly modified 1948; Johansson 1992). This compound has recently been daidzein effects in the operant responses when compared with evaluated for the treatment of cocaine addiction in humans vehicle treatment (Fig. 5b). (Pani et al. 2010) (however, see also Carroll et al. 2016). In addition, acute administration of daidzin also decreased Our study reveals that daidzein is a promising isoflavone reg- cue-induced cocaine seeking behavior when compared to ve- ulating cocaine reinforcing effects (Figs. 2b and 3b) and co- hicle treated mice. In contrast with daidzein, administration of caine relapse (Figs. 4b and 5b). We have also described that tamoxifen or quinpirole did not modify the effects of daidzin daidzein modifies these behavioral responses by a mechanism on operant responding (F(2,25) = 1.0989, n.s.) (Fig. 5c). that requires of DA D2/3 receptor, but not estrogen receptor, These data suggest that daidzein effects on cocaine seeking activities. This hypothesis was first supported from results behavior and relapse are mediated by a mechanism that in- obtained in the preliminary locomotor activity study. We ob- volves DA D2/3 receptor activities. Moreover, our data sug- served that exposure to a sub-effective dose of quinpirole po- gest that isoflavones, daidzein and daidzin, act through differ- tentiated daidzein hypolocomotor effects without affecting the ent mechanisms of action regulating cocaine seeking activity of the control vehicle treated mice (Fig. 1e). responses. Furthermore, dopamine D2/3 receptors also modulated 1932 Psychopharmacology (2021) 238:1923–1936 Fig. 4: Acute administration of daidzein, daidzin, and disulfiram, but not to cue-induced cocaine seeking behavior paradigm. All compounds were genistein, decreases cue-induced cocaine seeking and reinstatement be- administered acutely 1 h before the beginning of each cue-induced rein- havior. Comparison of the effects of the administration of vehicle versus statement session. Data represents the active nose-poke responding the (a) vehicle (n = 9), (b) daidzein (100 mg/kg, i.p., n = 9), (c) daidzin (75 day of exposure to cue-induced reinstatement and also during the extinc- mg/kg, i.p., n = 9), (d) genistein (100 mg/kg, i.p., n = 9) or (e) disulfiram tion period 3 days before and after. Results are expressed as average + (75 mg/kg, i.p., n = 8) in the active nose-poke responding after exposure SEM. ⋆ p < 0.05 versus vehicle-induced reinstatement, Student's t test daidzein pharmacological actions on both, cocaine self- system may play an important role. These modifications seem administration and cocaine induced seeking behavior and re- to be characteristic and exclusive of each phase of the addic- lapse. Interestingly, chronic exposure to quinpirole was re- tive process (Samuvel et al. 2008; Wydra et al. 2015; Siciliano quired to revert the effects of daidzein on cocaine reinforce- et al. 2016) and can also differentially influence the pharma- ment (Fig. 3a) whereas an acute administration of quinpirole cological effects of the administration of daidzein alone or in was sufficient to modify the actions of this isoflavone on cue- combination with quinpirole. However, indirect actions induced cocaine seeking behavior and relapse (Fig. 5b). The through other neuromodulators/neurotransmitter systems presence of cocaine in the organism during the active cocaine known to modify DA neurotransmission that can also be mod- self-administration and its absence when cue-induced relapse ulated by daidzein administrations, such as CRF or CCK was evaluated could account for the different effectivity of (Crawley 1991; Wanat et al. 2013; Ahmed et al. 2017), may quinpirole (single vs repeated administration) in modulating also encompass these effects. Our observations suggest daidzein actions on both behavioral paradigms. Other mecha- that daidzein is a natural isoflavone with a therapeutic nisms, however, may also be involved in these responses. In potential to treat or palliate cocaine reinforcing effects. this regard, neurobiological alterations, such as the changes Its pharmacological mechanism seems to involve alter- that develop during active cocaine self-administration or after ations in the dopaminergic neurotransmission; however, extinction on cue-induced seeking behavior in the dopaminer- future investigations are required in order to further gic and in other neurotransmitters systems in the brain reward characterize it. Psychopharmacology (2021) 238:1923–1936 1933 Fig. 5: Involvement of the estrogen and dopamine type-II- receptor systems on daidzein-, but not daidzin-, effects in cue- induced cocaine seeking and re- instatement behavior. Comparison of the effects of an acute administration of vehicle or a subeffective dose of quinpirole (0.01 mg/kg, i.p.) or tamoxifen (1 mg/kg, i.p.) on cue-induced co- caine seeking behavior and rein- statement in mice acutely treated with (a) vehicle, (b) daidzein (100 mg/kg, i.p.) or (c) daidzin (75 mg/kg, i.p.). Animals were injected with vehicle, daidzein, or daidzin and 30 min later with ta- moxifen, quinpirole, or vehicle. Thirty minutes after the last in- jection, animals were exposed to cue-induced cocaine seeking be- havior paradigm. Results are expressed as average + SEM. ⋆ p < 0.05 versus daidzein-vehicle induced reinstatemnet, Dunnett's multiple comparison test In sharp contrast with daidzein, disulfiram exposure pro- relapse by a different mechanism that involves inhibitory ef- duced small effects on cocaine self-administration (Fig. 2d,h). fects on dopamine β-hydroxylase (DBH) activities, the en- These results are similar to previous preclinical studies in rats zyme responsible for the conversion of DA to noradrenaline describing no effect of an acute disulfiram administration in (NE) in the final step of NE biosynthesis (Weinshenker and the operant cocaine self-administration paradigm (Schroeder Schroeder 2007; Gaval-Cruz and Weinshenker 2009). By de- et al. 2010). When administered in vivo, disulfiram is metab- creasing NE release, these authors suggest that disulfiram also olized into diethylthiomethylcarbamate and other compounds reduces mesolimbic dopaminergic tone and cocaine seeking with different pharmacological profiles. The lack of effects of behavior (Gaval-Cruz and Weinshenker 2009). Others, how- disulfiram could be a reflection of the wide spectrum of phar- ever, propose the opposite that disulfiram ability to reduce macological actions produced by this compound and its me- cocaine relapse is due to an increase in the dopaminergic tone tabolites, some of which may show antagonistic effects in the after DHB inhibition (Bourdélat-Parks et al. 2005; Sofuoglu modulation of cocaine reinforcing effects. On the other hand, and Kosten 2006; Devoto et al. 2012). Further research is our results demonstrate that responses in the cue-induced re- required in order to ascertain the correct mechanism of action lapse paradigm were decreased after disulfiram treatment (Fig. involved in these responses. 4e). These observations are also in agreement with previous Acute administration of daidzin decreased operant cocaine preclinical observations describing the inhibitory properties of self-administration (Fig. 2b). However, tolerance to its phar- this compound on cocaine craving and relapse (Suh et al. macological effects developed when mice were exposed to 2006; Schroeder et al. 2010). Different mechanisms of action after a more prolonged treatment (Fig. 2b). Furthermore, the have been suggested to explain these effects. Thus, previous acute administration of daidzin also decreased cocaine seeking investigations have proposed that disulfiram can decrease cue- behavior in the cue-induced relapse paradigm (Fig. 4c). induced cocaine seeking behavior by altering DA release after Previous studies performed with this isoflavone and with the inhibition of ALDH activities. However, other authors some of its synthetic analogues, such as CVT-10216, suggest suggest that disulfiram can regulate cue-induced cocaine that daidzin can decrease cocaine effects by a mechanism that 1934 Psychopharmacology (2021) 238:1923–1936 involves modulatory actions in ALDH-2 and DA activities consideration if administered at higher doses like in this study. (Keung et al. 1997; Arolfo et al. 2009; Yao et al. 2010). Our Each isoflavone produces its effects on cocaine addiction results, however, suggest that DA D2/3 receptors may play a modulating different neurotransmitter, receptor systems and marginal role in modulating daidzin pharmacological effect. intracellular pathways so further research is required in order This statement is based on our observations in the cue-induced to unravel each specific mechanism. Moreover, under a clin- relapse paradigm, where no differences were observed after ical therapeutic perspective, it will be important to take into exposure to quinpirole in daidzin treated mice (Fig. 5b). We consideration whether treatment to patients should be per- can suggest that other dopamine receptor, like type-I recep- formed with soybean extracts or with specific isoflavones. tors, may play a more prominent role in daidzin pharmacolog- In summary, there is a need for further studies on the mech- ical effects. Moreover, the regulation of ALDH-2 by this iso- anisms of plant extracts and their active compounds action, flavone can also lead to alterations in 5-HT metabolism like isoflavones, that might be a valuable alternative way for (Keung and Vallee 1998) that can influence cocaine addictive the prevention and treatment of various drug dependences. properties. In this sense, previous studies have shown that 5- HT modifies cocaine reinforcement, seeking behavior and re- lapse by a mechanism that requires interactions with DA and/ Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00213-021-05820-z. or glutamate neurotransmission (Howell and Cunningham 2015). ACKNOWLEDGMENTS We acknowledge the financial support of the Genistein was also effective decreasing cocaine reinforcing Spanish “Instituto de Salud Carlos III” (#RD16/0017/0020 to R.M, effects (Fig. 2c,g), an effect that developed after the second #RD16/0017/003 to M.F., and #CIBEROBN to R. dl T.), Spanish “Ministerio de Sanidad y Política Social-Investigación Clínica day of exposure to this isoflavone. However, in sharp contrast Independiente” (#EC10-166 to M.F.), the Catalan Government (#2017 with daidzein and daidzin, genistein did not modify the active SGR 669 and #TECCIT2014 and 2015-NeuroPhar to R.M., #2017-SRG- nose-poke response in the cue-induced cocaine relapse para- 316 to M.F., and #2017-SGR-138 and #TECCIT2014 and 2015-BAPP to digm (Fig. 4d). Different neurobiological mechanism may be R dl T.). M.M. was a “Beatriu de Pinós” fellow (#2010-BP-B00221). Partial support of FEDER funds is also acknowledged. The authors de- involved in the regulatory effects of genistein modulating co- clare no conflict of interest (financial and non-financial). caine reinforcing effects. In this regard, genistein is a potent agonist of the β-estrogen receptors (Wang et al. 1996), and AUTHOR CONTRIBUTIONS RT, MF, and RF elaborated the study design. previous studies have shown the involvement of these recep- MM and MG-M collected the experimental data. RG and MG-M per- tors modulating cocaine-rewarding effects by a mechanism formed the surgical procedures. MG-M and KL analyzed the data. MM, MG-M, KL, RT, and RM drafted the article. RT, MF, RF, MM, and MG- not fully understood. Thus, blocking the brain estrogen recep- M contributed to data interpretation. All authors critically reviewed the tors activity completely inhibits the development and expres- content and approved the final version for publication. sion of cocaine-induced locomotor sensitization and cocaine reinforcing properties in rodents (Segarra et al. 2014; Satta Declarations et al. 2018). On the other hand, genistein is also a potent inhibitor of tyrosine kinase (TK) activities and previous stud- CONFLICT OF INTEREST On behalf of all authors, the corresponding ies have shown that genistein can increase DA release from author states that there is no conflict of interest. mouse striatal slices by modifying the activity of this enzyme (Bare et al. 1995). Moreover, genistein can also modulate Open Access This article is licensed under a Creative Commons different intracellular signaling pathways, including the nucle- Attribution 4.0 International License, which permits use, sharing, adap- ar factor-kappa β (NF-kβ) (Kazi et al. 2003; Nagaraju et al. tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- 2013) which is known to be increased in the nucleus accum- vide a link to the Creative Commons licence, and indicate if changes were bens after cocaine administration. This increase in neuronal made. The images or other third party material in this article are included NF-kβ signaling pathways has been implicated in the long- in the article's Creative Commons licence, unless indicated otherwise in a term effects of cocaine that lead to the development of cocaine credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by addiction (Ang et al. 2001). Thus, different mechanisms might statutory regulation or exceeds the permitted use, you will need to obtain play a role in the effects produced by genistein on cocaine permission directly from the copyright holder. To view a copy of this reinforcing effects, however, further research is required in licence, visit http://creativecommons.org/licenses/by/4.0/. order to ascertain and deeply understand the complexity of these responses. A clinical study with these compounds, es- pecially daidzein, is required to ascertain their promising ther- apeutic use for the treatment of cocaine addiction in humans. REFERENCES Moreover, the use of natural compounds may be safer and promising for human purposes (Munro et al. 2003; Choi and Ahmed T, Javed S, Tariq A, Budzyńska B, D'Onofrio G, Daglia M, Nabavi SF, Nabavi SM (2017) Daidzein and its effects on brain. 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