Methadone Requires the Co-Activation of µ-Opioid and Toll-Like-4 Receptors to Produce Extracellular DNA Traps in Bone-Marrow-Derived Mast Cells.

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates µ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express µ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of µ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between µ-opioid and TLR4 receptors.

Effects of fentanyl and the adulterant levamisole on the rewarding and locomotor effects of methamphetamine in rats.

BACKGROUND: People who use psychostimulant substances can be exposed to unknown adulterants, such as the synthetic opioid fentanyl (FEN) and the anthelmintic cholinergic agent levamisole (LEV). This work explores the rewarding and locomotor effects of methamphetamine (METH) in combination with FEN or LEV. METHODS: We used adult male Wistar rats in the conditioned-place preference (CPP) paradigm (conditioning, extinction, and reinstatement phases) and in the open field test to study effective doses of METH, FEN, or LEV, or ineffective doses of METH+FEN or METH+LEV in combination. RESULTS: METH and LEV, at 1mg/kg METH each, and 30µg/kg FEN produced CPP. Extinction to METH- or LEV-induced CPP occurred after eight saline injections, but it took 8-26 sessions to extinguish FEN-induced CPP. A challenge dose of 0.5mg/kg METH reinstated CPP. The same occurred with 15µg/kg FEN but not with 0.5 or 1mg/kg LEV. Training animals with ineffective doses of METH (0.01mg/kg) combined with either FEN (0.3µg/kg) or LEV (0.01mg/kg) produced CPP. Sub-effective doses of METH or FEN alone did not induce reinstatement after extinction. However, animals challenged with LEV, METH+FEN, or METH+LEV mixtures did it. Combining FEN (3µg/kg) with 0.1mg/kg METH increased locomotor activity. CONCLUSION: Ineffective FEN and LEV doses mixed with METH produce effects larger than would be expected based on the effects of either drug alone. This outcome suggests a supra-additive interaction, which could increase the risk of developing a METH use disorder.

First drug-checking study at an electronic festival and fentanyl detection in the central region of Mexico.

BACKGROUND: Perception of drug adulteration has increased in Mexico, but there is little research on adulterants and toxicity. The aim of this study was to identify drug composition in an electronic music outdoor festival nearby Mexico City. METHODS: The participants completed a questionnaire with demographic data, harm reduction strategies, drug-use patterns, history, and the drug they expected to find. We took a small sample of each substance and prepared it for drug checking. A two-section drug testing station was placed within the grounds of the festival. Interaction with participants occurred at the front part. Drug checking was conducted at the rear part. The service was free of charge, voluntary and confidential. Forty persons aged 22 to 48 years participated (mode = 28), of which 92.5% were male, most (82.5%) were single. Through the Substance Analysis Program of "ReverdeSer Collective," we conducted the testing with the attendants that provided 51 drug samples, following ethical and biosafety protocols. We used colorimetry, Fourier Transform Infrared Spectroscopy, and fentanyl immunoassay strips for sample analysis. RESULTS: Substances of choice among attendants were psychostimulants (MDMA and other amphetamine-like drugs) and hallucinogens. Most samples contained what the users expected plus adulterants. Main adulterants were methylene-dioxy-ethyl-amphetamine, methylene-dioxy-propyl-amphetamine, hydroxyamphetamine, and the selective serotonin reuptake inhibitor venlafaxine. Fentanyl was present in 2 out of 4 cocaine samples and in 14 of the 22 confirmed MDMA samples. CONCLUSIONS: Some of the adulterants found pose serious health risks, especially fentanyl, amphetamine-like substances, and venlafaxine. Therefore, it is urgent to monitor these adulterants at electronic music festivals and to implement prevention, treatment, and harm reduction public policies. Naloxone distribution and drug-assisted therapies should be part of government programs in Mexico.

Xylazine potentiates the lethal but not the rewarding effects of fentanyl in mice.

BACKGROUND: Fentanyl is commonly laced with xylazine. People who use this combination report heightened effects, but it also increases death risk. Although no medication has been approved to counteract overdoses produced by fentanyl and xylazine, naloxone is frequently used. This paper studies the preclinical rewarding and lethal effects of fentanyl combined with xylazine and the efficacy of yohimbine or naloxone to prevent death. METHODS: Male Swiss Webster mice were treated with (in mg/kg, i.p.) xylazine (0.3, 1, 3, or 5.6), fentanyl (0.01, 0.3, or 0.1), or 1 xylazine plus 0.01 (non-effective) or 0.1 (effective) fentanyl doses during the conditioned-place preference (CPP) test. In addition, independent groups received (in mg/kg, i.p.): xylazine (31.6, 60, 74.2, or 100), fentanyl (3.1 or 10), or both substances at two doses: 31.6 xylazine + 3.1 fentanyl, or 60 xylazine + 10 fentanyl to analyze lethal effects. We determined whether yohimbine or naloxone (each medication tested at 10 or 30mg/kg) could prevent the lethality produced by fentanyl/xylazine combinations. Female mice were also tested in key experiments. RESULTS: Xylazine neither induced CPP nor altered fentanyl's rewarding effects. In contrast, lethality was potentiated when fentanyl was combined with xylazine. Naloxone, but not yohimbine, effectively prevented the lethality of the fentanyl/xylazine combinations. CONCLUSIONS: At the doses tested, xylazine does not increase the rewarding effect of fentanyl on the CPP in male mice but potentiates the risk of fatal overdose in male and female mice. A high naloxone dose prevents death induced by coadministration of fentanyl and xylazine in both sexes.

The Resurgence of Hallucinogen Drugs in Clinical Research.

Since the dawn of civilization, ancient cultures have utilized hallucinogens from plants and fungi in the context of religious and healing practices. Recently, their use has expanded to other cultures. Hallucinogens are natural or synthetic substances that alter the perception of reality at nontoxic doses, producing intense psychological and physiological effects. The initial research on hallucinogens began in the 1950s. However, their non-medical use, studies without proper controls, and negative social opinion resulted in legal restrictions that limited their use for clinical and preclinical research for more than two decades. A renewed interest in studying hallucinogens as potential therapeutic agents for treating different psychiatric conditions has recently re-emerged. This review summarizes the effects of main hallucinogen drugs and their therapeutic potential. Classic hallucinogens such as LSD, dimethyltryptamine, psilocin, and mescaline have chemical structures similar to serotonin and directly activate 5-hydroxy-tryptamine (5-HT2A) receptors. Ketamine is a dissociative anesthetic with antagonist effects at the glutamatergic N-methyl-D-aspartate receptor, indirectly activating 5-HT2A receptors. Ketamine has rapid antidepressant effects and reduces suicidal ideation, but its effects are short-lasting. Other hallucinogens are under study. It is necessary to continue this research with a more rigorous methodology and include studying the long-term effects of psychedelics use.

Amphetamine-type Stimulants: Novel Insights into their Actions and use Patterns.

This review focuses on the effects and mechanisms of action of amphetamine-type stimulants (ATS) and their adverse effects on the cardiovascular, nervous, and immune systems. ATS include amphetamine (AMPH), methamphetamine (METH, "crystalmeth," or "ice"), methylenedioxymethamphetamine (MDMA, "ecstasy," or "Molly"), MDMA derivatives (e.g., methylenedioxyamphetamine [MDA] and methylenedioxy-N-ethylamphetamine [MDEA]), khat, and synthetic cathinones. The first section of this paper presents an overview of the historical aspects of ATS use, their initial clinical use, and regulations. The second part reviews the acute and chronic impact and the most salient clinical effects of ATS on the central nervous and cardiovascular systems, skin, and mouth. The chemical structure, pharmacokinetics, and classic and non-canonical pharmacological actions are covered in the third section, briefly explaining the mechanisms involved. In addition, the interactions of ATS with the central and peripheral immune systems are reviewed. The last section presents data about the syndemic of ATS and opioid use in the North American region, focusing on the increasing adulteration of METH with fentanyl.

Fentanyl and other New Psychoactive Synthetic Opioids. Challenges to Prevention and Treatment.

Synthetic opioids have played a significant role in the current opioid crisis in the United States (U.S.) and Canada and are a matter of concern worldwide. New psychoactive opioids (NPOs) are classified in the internationally recognized new psychoactive substances (NPSs) category. This group comprises compounds that may have been synthesized decades ago but appeared only recently in the illicit drug market. Such is the case of fentanyl, fentanyl analogs, and non-fentanyl opioids. Most NPOs have effects similar to morphine, including euphoria and analgesia, and can produce fatal respiratory depression. Here, we present an overview of the systemic and molecular effects of main NPOs, their classification, and their pharmacological properties. We first review the fentanyl group of NPOs, including the four compounds of clinical use (fentanyl, alfentanil, sufentanil, and remifentanil) and the veterinary drug carfentanil. We also provide essential information on non-medical fentanyl analogs and other synthetic opioids such as brorphine, etonitazene, and MT-45, used as adulterants in commonly misused drugs. This paper also summarizes the scarce literature on the use of NPOs in Mexico. It concludes with a brief review of the challenges to prevention and treatment posed by NPOs and some recommendations to face them.

Fentanyl and other New Psychoactive Synthetic Opioids. Challenges to Prevention and Treatment

ABSTRACT Synthetic opioids have played a significant role in the current opioid crisis in the United States (U.S.) and Canada and are a matter of concern worldwide. New psychoactive opioids (NPOs) are classified in the internationally recognized new psychoactive substances (NPSs) category. This group comprises compounds that may have been synthesized decades ago but appeared only recently in the illicit drug market. Such is the case of fentanyl, fentanyl analogs, and non-fentanyl opioids. Most NPOs have effects similar to morphine, including euphoria and analgesia, and can produce fatal respiratory depression. Here, we present an overview of the systemic and molecular effects of main NPOs, their classification, and their pharmacological properties. We first review the fentanyl group of NPOs, including the four compounds of clinical use (fentanyl, alfentanil, sufentanil, and remifentanil) and the veterinary drug carfentanil. We also provide essential information on non-medical fentanyl analogs and other synthetic opioids such as brorphine, etonitazene, and MT-45, used as adulterants in commonly misused drugs. This paper also summarizes the scarce literature on the use of NPOs in Mexico. It concludes with a brief review of the challenges to prevention and treatment posed by NPOs and some recommendations to face them.

Amphetamine-type Stimulants: Novel Insights into their Actions and use Patterns

ABSTRACT This review focuses on the effects and mechanisms of action of amphetamine-type stimulants (ATS) and their adverse effects on the cardiovascular, nervous, and immune systems. ATS include amphetamine (AMPH), methamphetamine (METH, "crystalmeth," or "ice"), methylenedioxymethamphetamine (MDMA, "ecstasy," or "Molly"), MDMA derivatives (e.g., methylenedioxyamphetamine [MDA] and methylenedioxy-N-ethylamphetamine [MDEA]), khat, and synthetic cathinones. The first section of this paper presents an overview of the historical aspects of ATS use, their initial clinical use, and regulations. The second part reviews the acute and chronic impact and the most salient clinical effects of ATS on the central nervous and cardiovascular systems, skin, and mouth. The chemical structure, pharmacokinetics, and classic and non-canonical pharmacological actions are covered in the third section, briefly explaining the mechanisms involved. In addition, the interactions of ATS with the central and peripheral immune systems are reviewed. The last section presents data about the syndemic of ATS and opioid use in the North American region, focusing on the increasing adulteration of METH with fentanyl.

The Resurgence of Hallucinogen Drugs in Clinical Research

ABSTRACT Since the dawn of civilization, ancient cultures have utilized hallucinogens from plants and fungi in the context of religious and healing practices. Recently, their use has expanded to other cultures. Hallucinogens are natural or synthetic substances that alter the perception of reality at nontoxic doses, producing intense psychological and physiological effects. The initial research on hallucinogens began in the 1950s. However, their non-medical use, studies without proper controls, and negative social opinion resulted in legal restrictions that limited their use for clinical and preclinical research for more than two decades. A renewed interest in studying hallucinogens as potential therapeutic agents for treating different psychiatric conditions has recently re-emerged. This review summarizes the effects of main hallucinogen drugs and their therapeutic potential. Classic hallucinogens such as LSD, dimethyltryptamine, psilocin, and mescaline have chemical structures similar to serotonin and directly activate 5-hydroxy-tryptamine (5-HT2A) receptors. Ketamine is a dissociative anesthetic with antagonist effects at the glutamatergic N-methyl-D-aspartate receptor, indirectly activating 5-HT2A receptors. Ketamine has rapid antidepressant effects and reduces suicidal ideation, but its effects are short-lasting. Other hallucinogens are under study. It is necessary to continue this research with a more rigorous methodology and include studying the long-term effects of psychedelics use.

Endocannabinoid modulation of allergic responses: Focus on the control of FcεRI-mediated mast cell activation.

Allergic reactions are highly prevalent pathologies initiated by the production of IgE antibodies against harmless antigens (allergens) and the activation of the high-affinity IgE receptor (FcεRI) expressed in the surface of basophils and mast cells (MCs). Research on the mechanisms of negative control of those exacerbated inflammatory reactions has been intense in recent years. Endocannabinoids (eCBs) show important regulatory effects on MC-mediated immune responses, mainly inhibiting the production of pro-inflammatory mediators. However, the description of the molecular mechanisms involved in eCB control of MC activation is far from complete. In this review, we aim to summarize the available information regarding the role of eCBs in the modulation of FcεRI-dependent activation of that cell type, emphasizing the description of the eCB system and the existence of some of its elements in MCs. Unique characteristics of the eCB system and cannabinoid receptors (CBRs) localization and signaling in MCs are mentioned. The described and putative points of cross-talk between CBRs and FcεRI signaling cascades are also presented. Finally, we discuss some important considerations in the study of the effects of eCBs in MCs and the perspectives in the field.

Sexual satiety modifies methamphetamine-induced locomotor and rewarding effects and dopamine-related protein levels in the striatum of male rats.

RATIONALE: Drug and natural rewarding stimuli activate the mesolimbic dopaminergic system. Both methamphetamine (Meth) and copulation to satiety importantly increase dopamine (DA) release in the nucleus accumbens (NAc), but with differences in magnitude. This paper analyzes the interaction between Meth administration and the intense sexual activity associated with sexual satiety. OBJECTIVES: To evaluate possible changes in Meth-induced behavioral effects and striatal DA-related protein expression due to sexual satiety. METHODS: Meth-induced locomotor activity and conditioned place preference (CPP) were tested in sexually experienced male rats that copulated to satiety (S-S) or ejaculated once (1E) the day before or displayed no sexual activity (control group; C). DA receptors and DA transporter expression were determined by western blot in the striatum of animals of all sexual conditions treated with specific Meth doses. RESULTS: Meth's locomotor and rewarding effects were exacerbated in S-S animals, while in 1E rats, only locomotor effects were enhanced. Sexual activity, by itself, modified DA-related protein expression in the NAc core and in the caudate-putamen (CPu), while Meth treatment alone changed their expression only in the NAc shell. Meth-induced changes in the NAc shell turned in the opposite direction when animals had sexual activity, and additional changes appeared in the NAc core and CPu of S-S rats. CONCLUSION: Sexual satiety sensitizes rats to Meth's behavioral effects and the Meth-induced striatal DA-related protein adaptations are modified by sexual activity, evidencing cross-sensitization between both stimuli.

Co-administration of morphine and levamisole increases death risk, produces neutropenia and modifies antinociception in mice.

Levamisole is a veterinary anthelmintic drug and a common adulterant of misused drugs. This study analyses the lethal, antinociceptive and haematological effects produced by acute or repeated levamisole administration by itself or combined with morphine. Independent groups of male Swiss Webster mice were i.p. injected with 100 mg/kg morphine, 31.6 mg/kg levamisole (lethal doses at 10%, LD10 ) or the same doses combined. Naloxone pretreatment (10 mg/kg, i.p.) prevented morphine-induced death, as did 2.5 mg/kg, i.p. mecamylamine with levamisole. Co-administration of levamisole and morphine (Lvm + Mor) increased lethality from 10% to 80%. This augmented effect was prevented by 30 mg/kg, i.p. naloxone and reduced with 10 mg/kg naloxone plus 2.5 mg/kg, i.p. mecamylamine. In independent groups of mice, 17.7 mg/kg, i.p. levamisole antagonized the acute morphine's antinociceptive effect evaluated in the tail-flick test. Repeated 17.7 mg/kg levamisole administration (2×/day/3 weeks) did not affect tolerance development to morphine (10 mg/kg, 3×/day/1 week). Blood samples obtained from mice repeatedly treated with levamisole showed leukopenia and neutropenia. Morphine also produced neutropenia, increased erythrocyte count and other related parameters (e.g. haemoglobin). Lvm + Mor had similar effects on leukocyte and neutrophil counts to those seen with levamisole only, but no erythrocyte-related alterations were evident. Blood chemistry analysis did not indicate liver damage but suggested some degree of electrolyte balance impairment. In conclusion, Lvm + Mor increased death risk, altered morphine-induced antinociceptive effects and produced haematologic abnormalities. The importance of studying combinations of drugs of abuse lies in the fact that drug users frequently combine drugs, which are commonly adulterated.

Morphine and Fentanyl Repeated Administration Induces Different Levels of NLRP3-Dependent Pyroptosis in the Dorsal Raphe Nucleus of Male Rats via Cell-Specific Activation of TLR4 and Opioid Receptors.

Morphine promotes neuroinflammation after NOD-like receptor protein 3 (NLRP3) oligomerization in glial cells, but the capacity of other opioids to induce neuroinflammation and its relationship to the development of analgesic tolerance is unknown. We studied the effects of morphine and fentanyl on NLRP3 inflammasome activation in glial and neuronal cells in the dorsal raphe nucleus (DRN), a region involved in pain regulation. Male Wistar rats received i.p. injections of morphine (10 mg/kg) or fentanyl (0.1 mg/kg) 3 × daily for 7 days and were tested for nociception. Two hours after the last (19th) administration, we analyzed NLRP3 oligomerization, caspase-1 activation and gasdermin D-N (GSDMD-N) expression in microglia (CD11b positive cells), astrocytes (GFAP-positive cells) and neurons (NeuN-positive cells). Tolerance developed to both opioids, but only fentanyl produced hyperalgesia. Morphine and fentanyl activated NLRP3 inflammasome in astrocytes and serotonergic (TPH-2-positive) neurons, but fentanyl effects were more pronounced. Both opioids increased GFAP and CD11b immunoreactivity, caspase-1 and GSDMD activation, indicating pyroptotic cell death. The opioid receptor antagonist (-)-naloxone, but not the TLR4 receptor antagonist (+)-naloxone, prevented microglia activation and NLRP3 oligomerization. Only (+)-naloxone prevented astrocytes' activation. The anti-inflammatory agent minocycline and the NLRP3 inhibitor MCC950 delayed tolerance to morphine and fentanyl antinociception and prevented fentanyl-induced hyperalgesia. MCC950 also prevented opioid-induced NLRP3 oligomerization. In conclusion, morphine and fentanyl differentially induce cell-specific activation of NLRP3 inflammasome and pyroptosis in the DRN through TLR4 receptors in astrocytes and through opioid receptors in neurons, indicating that neuroinflammation is involved in opioid-induced analgesia and fentanyl-induced hyperalgesia after repeated administrations.

Sodium chloride injection to treat opioid overdose; Does it work? A preclinical study.

Opioid overdoses (ODs) are increasing in Mexico's northern border. Because naloxone is usually not available, witnesses inject common salt (NaCl) into a vein of OD victims in an attempt to help them regain consciousness. Despite this widespread practice, no preclinical studies have addressed the efficacy of NaCl as an opioid antidote. Here we tested saline solutions at different concentrations. Because the highest (31.6 %) caused tail necrosis, we selected 17.7 % as a hypertonic saline solution (HSS) to determine if it could prevent the lethal effect of morphine (Mor), fentanyl (Fen), or Mor + Fen in adult Wistar male rats. We also evaluated if NaCl could modify the opioid antagonist effect of naloxone. Our results show that HSS: a) sensitizes animals to thermal but not mechanical stimuli; b) does not prevent mortality caused by high morphine or fentanyl doses; c) decreases the latency to recovery from the sedative effects caused by low doses of morphine or fentanyl; and d) increases naloxone's efficacy to prevent the lethality produced by Mor or Fen, but not by Mor + Fen. These results suggest that HSS is marginally effective in shortening the recovery time from nonfatal opioid ODs and increases naloxone's efficacy to counteract opioid-induced ODs.

The last two decades on preclinical and clinical research on inhalant effects.

This paper reviews the scientific evidence generated in the last two decades on the effects and mechanisms of action of most commonly misused inhalants. In the first section, we define what inhalants are, how they are used, and their prevalence worldwide. The second section presents specific characteristics that define the main groups of inhalants: (a) organic solvents; (b) aerosols, gases, and volatile anesthetics; and (c) alkyl nitrites. We include a table with the molecular formula, structure, synonyms, uses, physicochemical properties and exposure limits of representative compounds within each group. The third and fourth sections review the direct acute and chronic effects of common inhalants on health and behavior with a summary of mechanisms of action, respectively. In the fifth section, we address inhalant intoxication signs and available treatment. The sixth section examines the health effects, intoxication, and treatment of nitrites. The seventh section reviews current intervention strategies. Finally, we propose a research agenda to promote the study of (a) solvents other than toluene; (b) inhalant mixtures; (c) effects in combination with other drugs of abuse; (d) age and (e) sex differences in inhalant effects; (f) the long-lasting behavioral effects of animals exposed in utero to inhalants; (g) abstinence signs and neurochemical changes after interrupting inhalant exposure; (h) brain networks involved in inhalant effects; and finally (i) strategies to promote recovery of inhalant users.

Minocycline prevents neuronal hyperexcitability and neuroinflammation in medial prefrontal cortex, as well as memory impairment caused by repeated toluene inhalation in adolescent rats.

Toluene can be intentionally misused by adolescents to experience psychoactive effects. Toluene has a complex mechanism of action and broad behavioral effects, among which memory impairment is reported consistently. We have previously reported that repeated toluene inhalation (8000 ppm) increases layer 5 prelimbic pyramidal cells' excitability in the medial prefrontal cortex (mPFC) of adolescent rats. Toluene also produces reactive oxygen species (ROS), which activate glial cells. Here, we tested the hypothesis that the anti-inflammatory agent minocycline would decrease toluene's effects because it inhibits NF-κB (nuclear factor enhancer of the kappa light chains of activated B cells) and reduces pro-inflammatory cytokine and ROS production. Our results show that minocycline (50 mg/kg, ip, for 10 days) prevents the hyperexcitability of mPFC neurons observed after repeated 8000 ppm toluene exposure (30 min/day, 2×/day for 10 days). Minocycline prevents toluene-induced hyperexcitability by a mechanism that averts the loss of the slow calcium-dependent potassium current, and normalizes mPFC neurons' firing frequency. These effects are accompanied by significant decreased expression of astrocytes and activated microglia in the mPFC, reduced NLRP3 inflammasome activation and mRNA expression levels of the pro-inflammatory cytokine interleukin 1ß (IL-1ß), as well as increased mRNA expression of the anti-inflammatory cytokine transforming growth factor ß (TGF-ß). Minocycline also prevents toluene-induced memory impairment in adolescent rats in the passive avoidance task and the temporal order memory test in which the mPFC plays a central role. These results show that neuroinflammation produces several effects of repeated toluene administration at high concentrations, and minocycline can significantly prevent them.

Fentanyl is used in Mexico's northern border: current challenges for drug health policies.

BACKGROUND AND AIMS: Results from a recent study among 750 heroin users in three Mexico's northern border cities revealed an increase in white powder availability (also known as China white) and preference for this product among heroin users, as well as a general perception of increased overdose cases among this population. Here, we questioned whether those findings reflect an increased presence of heroin laced with fentanyl, which is associated with greater risks of overdose but that, until now, has not been described in Mexico. DESIGN: We tested fentanyl using highly sensitive test strips in syringe plungers, metal cookers and drug wrappings associated with heroin use. SETTING: Three injection sites in Tijuana, Baja California, México. PARTICIPANTS: Eighty-nine heroin users who interchanged paraphernalia for new syringes. MEASUREMENTS: We tested 59 residues of 'pure' white powder. The rest were white powder with black tar (n = 5) or white powder with crystal meth (n = 9), black tar with crystal meth (n = 1), black tar only (n = 13) and crystal meth only (n = 2). FINDINGS: Users believed that they consumed either white powder heroin, white powder heroin with crystal meth, white powder with black tar heroin or black tar heroin only. Analyses revealed that 93% (n = 55) of the 'pure' white powder samples had fentanyl. All (n = 9) the white powder samples mixed with crystal meth and 40% (n = 2) of the white powder with black tar were also laced with fentanyl. CONCLUSIONS: In a sample of 89 heroin users in Mexico, most white powder heroin users were unknowingly exposed to fentanyl, with fentanyl detected in 93% of white powder samples.