A validated dilute-and-shoot LC-MS/MS urine screening for the analysis of 95 illicit drugs and medicines: insights from clinical and forensic Brazilian cases.

Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace, and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC-MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 minutes. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision CVs% were lower than 20%, and bias was within ± 20%. Only four of the 95 analytes presented significant ionization suppression or enhancement (> 25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%), and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential to understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.

Mitochondrial impairment related to the immunotoxicity of the herbicides clomazone, glyphosate and sulfentrazone in THP-1 cells.

Background: Pesticides are indispensable for the cultivation of crops, especially those of economic importance, such as soybeans. Data on the annual use of herbicides in crops show that they correspond to 50%, making it the most used in agriculture. Aim: Therefore, the aim of this study was to evaluate the toxicity of the three commercial herbicides (clomazone, glyphosate, and sulfentrazone) in THP-1 cells. Methods: Cells were incubated with 0-5,000 mg/L of the herbicides for 24 h at 37 °C for cytotoxicity evaluation. Additionally, a few toxicological pathways such as reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of pesticides, were also evaluated. Results: A potential immunotoxic effect of the herbicides on THP-1 cells was observed, especially glyphosate, as it is a powerful agent of cellular immunotoxicity. It was also possible to verify an increase in oxidative stress and IL-8 levels and mitochondrial dysfunction. Conclusion: All herbicides showed cytotoxic effects in THP-1 monocytes, which were related to mitochondrial impairment.

L-cysteine improves boar semen motility at 5 ºC but does not affect the oxidative status.

Hypothermic storage has been proposed as a method to reduce bacterial loads and promoting prudent use of antibiotics. Reducing temperature, however, can lead to cold shock damage and oxidative stress in boar semen. This study verified the effect of L-cysteine on the quality of semen stored at 5 °C for 120 h. Twenty-one normospermic ejaculates were diluted in Beltsville Thawing Solution into five treatments: Positive control (Pos_Cont, storage at 17 °C without L-cysteine) and groups with 0, 0.5, 1, and 2 mmol/L of L-cysteine supplementation stored at 5 °C. Variables were analyzed as repeated measures, considering treatment, storage time, and interaction as main factors. The effects of different L-cysteine concentrations were also evaluated using polynomial orthogonal contrasts. Sperm motility and pH were higher in the Pos_Cont compared to the groups stored at 5 °C (P < 0.05). In polynomial orthogonal contrast models, total motility was affected by the interaction between L-cysteine and storage time (P = 0.04), with a linear increase in motility when increasing the amount of L-cysteine at 72 and 120 h. Progressive motility increased quadratically as the L-cysteine reached 1 mmol/L (P < 0.01). In the thermoresistance test at 120 h, sperm motility increased quadratically up to an L-cysteine dose of 1 mmol/L (P < 0.05). Sulfhydryl content linearly increased with L-cysteine supplementation (P = 0.01), with no effect on intracellular ROS and sperm lipid peroxidation (P ≥ 0.06) in 5ºC-stored doses. In conclusion, L-cysteine supplementation has a positive effect on sperm motility up to 120 h of storage at 5 °C.

Immunomodulatory effect of imidacloprid on macrophage RAW 264.7 cells.

The neonicotinoid imidacloprid was promoted in the market because of widespread resistance to other insecticides, plus its low mammalian impact and higher specific toxicity towards insects. This study aimed to evaluate the immunomodulatory effect of imidacloprid on macrophages. RAW 264.7 cells were incubated to 0-4000 mg/L of imidacloprid for 24 and 96 h. Imidacloprid presented a concentration-dependent cytotoxicity after 24 h and 96 h incubation for MTT reduction (3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide) (EC50 519.6 and 324.6 mg/L, respectively) and Neutral Red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) assays (EC50 1139.0 and 324.2 mg/L, respectively). Moreover, imidacloprid decreased the cells' inflammatory response and promoted a mitochondrial depolarization. The complex II and succinate dehydrogenase (SDH) activities in RAW 264.7 cells incubated with imidacloprid increased more at 24 h. These results suggest that imidacloprid exerts an immunomodulatory effect and mitochondria can act as regulator of innate immune responses in the cytotoxicity mediated by the insecticide in RAW 264.7 cells.

Antiproliferative activity and toxicity evaluation of 1,2,3-triazole and 4-methyl coumarin hybrids in the MCF7 breast cancer cell line.

Four coumarin-triazole hybrids were selected from our in house library and screened for cytotoxic activity on A549 (lung cancer), HepG2 (liver cancer), J774A1 (mouse sarcoma macrophage), MCF7 (breast cancer), OVACAR (ovarian cancer), RAW (murine leukaemia macrophage), and SiHa (uterus carcinoma) and their in vitro toxicity was assessed on 3T3 (healthy fibroblasts) cell lines. SwissADME pharmacokinetic prediction was performed. Effects on ROS production, mitochondrial membrane potential, apoptosis/necrosis and DNA damage were evaluated. All of the hybrids have good pharmacokinetic predictions. Each of them showed cytotoxic activity against the MCF7 breast cancer cell line, with IC50 between 2.66 and 10.08 µM, lower than cisplatin (45.33 µM) for the same test. One can observe an order of reactivity from the most potent: LaSOM 186 > LaSOM 190 > LaSOM 185 > LaSOM 180, with a better selectivity index than the reference drug cisplatin and the precursor hymecromone, and caused cell death by apoptosis induction. Two compounds showed antioxidant activity in vitro and three disrupted the mitochondrial membrane potential. None of the hybrids caused genotoxic damage to healthy 3T3 cells. All hybrids showed potential for further optimization, mechanism elucidation, in vivo activity and toxicity tests.

Characterization and in vivo toxicological evaluation of multi-walled carbon nanotubes: a low-dose repeated intratracheal administration study.

This study characterized and investigated the toxicity of two multi-walled carbon nanotubes (MWCNT) NM-401 and NM-403 at 60 and 180 µg after four repeated intratracheal instillations; follow-up times were 3, 7, 30, and 90 days after the last instillation. NM-401 was needle-like, long, and thick, while NM-403 was entangled, short, and thin. Both MWCNT types induced transient pulmonary and systemic alterations in renal function and oxidative lipid damage markers in recent times. Animals showed general toxicity in the immediate times after exposures, in addition to increased pulmonary LDH release at day 3. In further times, decreased liver and kidney relative weights were noted at higher MWCNT doses. Lung histological damages included pulmonary fibrosis, for both MWCNT types, similarly to asbestos; single liver and kidney histological alterations were present. Repeated instillations led to persistent pulmonary damage at low doses, and possibly the extrapulmonary effects may be associated with the consecutive exposures.

A multi-analyte LC-MS/MS method for the determination of 57 pharmaceuticals and illicit drugs in plasma, and its application to poisoning cases.

The diagnostic methods in an emergency scenario must be simple, fast, and efficient to provide an effectiveness and efficient treatment, thus reducing the consequences of exposure. Considering the sample analysis, the protein precipitation combined with LC-MS/MS has been shown to be a good strategy for the simultaneous determination of compounds of toxicological interest, such as medicines and drugs of abuse. In this study, a rapid and simple multi-analyte method was developed and validated for the quantification of 57 pharmaceuticals and illicit drugs in plasma samples. Sample pre-treatment consists of protein precipitation of 50 µL of the sample with 240 µL of organic solvent mixture (MeOH:ACN, 3:1, v/v), centrifugation, and injection into the LC-MS/MS, with a chromatographic run time of 7 min. The method was validated considering lower limit of quantification (LLOQ), interferences, linearity, precision, accuracy, dilution integrity, carryover, and matrix effect. The LLOQs ranged from 5 to 20 ng/mL and all analytes were linear (r2>0.99) in the tested concentration ranges. The method proved to be precise and accurate, presenting QC concentrations for all analytes within acceptable limits by the guideline used (CV % ≤20 % and bias ± 20 %). The developed method was successfully applied in 470 plasma samples of real cases of poisoning. A total of 80 % of the samples were positive for at least one substance, with acetaminophen (32.1 %), diazepam (25.1 %), and lidocaine (18.9 %) being the most detected. The most prevalent exposure circumstance among the cases was suicide attempt. The most frequent age groups were young adults between 20 and 29 years old and children under 5 years old. The methodology developed proved to be efficient in the simultaneous determination of 57 substances of toxicological interest, contributing to a correct diagnosis and, consequently, to the most appropriate management and treatment of the intoxicated patient. Furthermore, it is possible to observe the most commonly involved toxic agents in the Rio Grande do Sul, southern Brazil, helping to trace a profile of the poisoning patient, important in toxicovigilance actions.

The potential of Brazilian native plant species used in the therapy for snakebites: A literature review.

Snakebite envenoming is a potentially fatal disease categorized as a neglected public health issue for not receiving the appropriate attention from national and international health authorities. The most affected people by this problem usually live in poor rural communities, where medical resources are often sparse and, in some instances, there is even a scarcity of serum therapy. The administration of the appropriate antivenom is the only specific treatment available, however it has limited efficacy against venom-induced local effects. In this scenario, various plant species are used as local first aid for the treatment of snakebite accidents in Brazil, and some of them can effectively inhibit lethality, neurotoxicity, hemorrhage, and venom enzymes activities. This review compiles a list of plants used in the treatment of snakebites in Brazil, focusing on the native Brazilian species registered in the databases Pubmed, Scielo, Scopus and Google Scholar. All these searches were limited to peer-reviewed journals written in English, with the exception of a few articles written in Portuguese. The most cited native plant species were Casearia sylvestris Sw., Eclipta prostrata (L.) L., Mikania glomerata Spreng., Schizolobium parahyba (Vell.) S.F.Blake and Dipteryx alata Vogel, all used to decrease the severity of toxic signs, inhibit proteolytic and hemorrhagic activities, thus increasing survival time and neutralizing myotoxicity effects. Different active compounds showing important activity against the snake venoms and their toxins include flavonoids, alkaloids and tannins. Although some limitations to the experimental studies with medicinal plants were observed, including lack of comparison with control drugs and unknown active extracts compounds, species with anti-venom characteristics are effective and considered as candidates for the development of adjuvants in the treatment of snake envenomation. Further studies on the chemistry and pharmacology of traditionally used plant species will help to understand the role that snakebite herbal remedies may display in local medical health systems. It might also contribute to the development of alternative or complementary treatments to reduce the number of severe disabilities and deaths.

Imidacloprid-based commercial pesticide causes behavioral, biochemical, and hematological impairments in Wistar rats.

Imidacloprid (IMI) is a neonicotinoid insecticide employed worldwide for crop protection. IMI's mode of action occurs through the agonism of postsynaptic nicotinic acetylcholine receptors (nAChRs), with high specificity for insect nAChRs although there are reports of mammals' toxicity. Studies on IMI's neurotoxicity are not conclusive; therefore, the aim of this study was to evaluate the subchronic toxic effects of an IMI based commercial pesticide on rats. Adult male Wistar rats received an IMI suspension via the oral route at doses of 1.5, 5, and 15 mg/kg for 45 consecutive days. IMI caused an increase in rearing and time spent at the periphery in the locomotor activity test and a decrease in time spent to finish the OX maze task (p < 0.05; ANOVA/Bonferroni). In blood, there was a decrease in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (p < 0.05; ANOVA/Bonferroni) and an increase in serum butyrylcholinesterase activity (p < 0.001; ANOVA/Bonferroni). Therefore, subchronic administration of an IMI-based-pesticide caused behavioral and systemic impairments in rats.

Toxicity of pesticides widely applied on soybean cultivation: Synergistic effects of fipronil, glyphosate and imidacloprid in HepG2 cells.

The transgenic soy monoculture demands supplementation with pesticides. The aim of this study was to evaluate the individual and mixture effects of fipronil, glyphosate and imidacloprid in human HepG2 cells. Cytotoxicity was evaluated after 48-h incubations through MTT reduction and neutral red uptake assays. Free radicals production, mitochondrial membrane potential, DNA damage, and release of liver enzymes were also evaluated. Data obtained for individual agents were used to compute the additivity expectations for two mixtures of definite composition (one equipotent mixture, based in the EC50 values achieved in the MTT assay; the other one based in the acceptable daily intake of each pesticide), using the models of concentration addition and independent action. The EC50 values for fipronil, glyphosate and imidacloprid were 37.59, 41.13, and 663.66 mg/L, respectively. The mixtures of pesticides elicited significant synergistic effects (p < 0.05), which were greater than the expected by both addictive predictions. Decreased in mitochondrial membrane potential and increased in the transaminases enzymatic activities were observed. As they occur simultaneously, interactions between pesticides, even at non-effective single levels, can reverberate in significant deleterious effects, justifying the need for a more realistic approach in safety evaluations to better predict the effects to human health.

Toxic effects of pesticides on cellular and humoral immunity: an overview.

People are exposed to pesticides through food, drinking water, and the environment. These compounds are associated with several disorders, such as inflammatory diseases, rheumatoid arthritis, cancer, and a condition related to metabolic syndrome. The immunotoxicants or immunotoxic compounds can cause a wide variety of effects on immune function, altering humoral immunity and cell-mediated immunity, resulting in adverse effects to the body. Here, immune system disorders are highlighted because they are closely linked to multiple organs, including the nervous, endocrine, reproductive, cardiovascular, and respiratory systems, leading to transient or permanent changes. Therefore, this study reviewed the mechanisms involved in the immunotoxicity of fungicides, herbicides, and insecticides in cells, animals, and humans in the past 11 years. According to the studies analyzed, the pesticides interfere with innate and adaptive immune functions, but the effects observed mainly on cellular and humoral immunity were highlighted. These compounds affected specific immune cells, causing apoptosis, changes in factor nuclear kappa B (NF-κB) expression, pro-inflammatory factors interleukin 6 (IL-6), interleukin 8 (IL-8), interferon-gamma (IFN-γ), chemokines (CXCL-c1c), and anti-inflammatory factor, such as interleukin 10 (IL-10). To verify the threats of these compounds, new evaluations with immunotoxicological biomarkers are necessary. HighlightsPesticides interfere with the innate and adaptive immune response.Cells, animals and human studies demonstrate the immunotoxicity of pesticides in the cellular and humoral immune response.Fungicides, herbicides, and insecticides alter the immune system by various mechanisms, such as pro-inflammatory and anti-inflammatory factors.

Hepatoprotective Native Plants Documented in Brazilian Traditional Medicine Literature: Current Knowledge and Prospects.

The native flora of Brazil covers a large biodiversity of plants, some of which have important pharmacological properties. Ethnobotanical studies have established that the folk use of plants for hepatobiliary disorders is very common in Brazil. This review aimed to compile, describe and discuss the main native Brazilian medicinal plants used as hepatoprotective agents listed on 25 books by authors from various regions in the country. Based on the 153 species found, a comprehensive analysis of the most relevant scientific studies is presented, including Piper umbellatum, Echinodorus macrophyllus, Fevillea trilobata and Achyrocline satureioides. Some medicinal plants share both a large number of citations and scientific evidence to corroborate their hepatoprotective effects, including Solanum paniculatum, Baccharis crispa and Phyllanthus niruri, which could be of interest to develop new phytomedicines. Additionally, some active ingredients are indicated as being responsible for the liver protective effects described, particularly phenolic compounds (flavonoids, coumestans and lignans).

Levamisole, a cocaine cutting agent, induces acute and subchronic systemic alterations in Wistar rats.

The use of the anthelmintic levamisole as a cocaine adulterant has been increasing worldwide. Complications caused by this association include systemic vasculitis, agranulocytosis, neutropenia, tissue necrosis, pulmonary hemorrhage, and renal injury. Data about toxicity of levamisole are scarce, therefore the aim of this study was to evaluate the acute and subchronic toxic effects of levamisole in rats. Male Wistar rats received saline or levamisole by intraperitoneal route at the doses of 12, 24 and 36 mg/kg in the acute toxicity test; and at 3, 6 and 12 mg/kg in the subchronic toxicity test. Toxicity was evaluated using behavioral, cognitive, renal, hematological, biochemical and histopathological parameters. Acute administration of levamisole caused behavioral and histopathological alterations. Subchronic administration caused behavioral, cognitive and hematological alterations (p < 0.0001 and p < 0.05, respectively), impairment of liver and kidney functions (p < 0.05), and changes of antioxidant defenses (p ≤ 0.0001). Both administrations produced toxic effects of clinical relevance, which make levamisole a dangerous cutting agent. Furthermore, the knowledge of these effects can contribute to the correct diagnosis and treatment of cocaine dependents with unusual systemic alterations.

Plants with Anti-Addictive Potential.

Drug addiction is prevalent among individuals of modern society, being a major cause of disability and premature loss of life. Although the drug addiction have profound social, economical and health impact in the world population, its management remains a challenge as available pharmacological treatments remains ineffective for most people. The limited efficacy and adverse effects have led to a search for alternative therapies to treat drug addiction. In this context, natural products are an important source for new chemical substances with a potential therapeutic applicability. Therefore, this chapter will present data obtained after an extensive literature search regarding the use of medicinal plants as a pharmacological alternative for drug addiction treatment.

Sintocalmy, a Passiflora incarnata Based Herbal, Attenuates Morphine Withdrawal in Mice.

Chronic opioid use changes brain chemistry in areas related to reward processes, memory, decision-making, and addiction. Both neurons and astrocytes are affected, ultimately leading to dependence. Passiflora incarnata L. (Passifloraceae) is the basis of frequently used herbals to manage anxiety and insomnia, with proven central nervous system depressant effects. Anti-addiction properties of P. incarnata have been reported. The aim of this study was to investigate the effect of a commercial extract of Passiflora incarnata (Sintocalmy®, Aché Laboratory) in the naloxone-induced jumping mice model of morphine withdrawal. In addition, glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B) levels were assessed in the frontal cortex and hippocampus, and DNA damage was verified on blood cells. In order to improve solubilization a Sintocalmy methanol extract (SME) was used. SME is mainly composed by flavonoids isovitexin and vitexin. The effects of SME 50, 100 and 200 mg/kg (i.p.) were evaluated in the naloxone-induced withdrawal syndrome in mice. SME 50 and SME 100 mg/kg decreased naloxone-induced jumping in morphine-dependent mice without reducing locomotor activity. No alterations were found in GFAP levels, however SME 50 mg/kg prevented the S100B increase in the frontal cortex and DNA damage. This study shows anti-addiction effects for a commercial standardized extract of P. incarnata and suggests the relevance of proper clinical assessment.

Biochemical, hematological and immunological parameters and relationship with occupational exposure to pesticides and metals.

This study aimed to evaluate biomarkers of exposure to cholinesterase inhibitors insecticides (AChE and BuChE activities) and metals (As, Cd, Cr, Mn, Ni, and Pb blood levels) and their associations with biochemical, hematological, and immunological parameters in farmers from Southern Brazil. One hundred and sixteen individuals were divided into two groups: 62 farmers (exposed group) and 54 subjects non-occupationally exposed (NOE) to agrochemicals. Erythrocyte (AChE) and serum (BuChE) cholinesterases activities were significantly reduced as well as blood Cd and Pb levels were increased in farmers when compared to NOE group (p < 0.05). Farmers presented increased glucose and urea levels compared to NOE group, which were inversely associated with AChE and positively correlated with Cd (p < 0.05), respectively. In addition, Cd was inversely associated with the hematological cells counts, which were significantly reduced in farmers (p < 0.05). C3 complement was higher in farmers and was positively associated with blood Pb (p < 0.05). Surface protein expression analysis revealed a downregulation of LFA-1 and ICAM-1 in farmers. Inverse associations were found between LFA-1 and blood As, Cr, and Ni levels (p < 0.05). Taken together, our results pointed to a relationship between agrochemicals and metals exposure and biochemical, hematological, and immunological disorders that can lead to several chronic conditions.

Piperazine designer drugs elicit toxicity in the alternative in vivo model Caenorhabditis elegans.

Piperazine designer drugs are a group of synthetic drugs of abuse that have appeared on the illicit market since the second half of the 1990s. The most common derivatives are 1-benzylpiperazine (BZP), 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(3,4-methylenedioxybenzyl)piperazine (MDBP). They can be consumed as capsules, tablets, but also in powder or liquid forms. Generally, although less potent than amphetamines, piperazines have dopaminergic and serotonergic activities. The aim of this work was to evaluate the toxic effects of BZP, MeOPP and MDBP using Caenorhabditis elegans as in vivo model for acute toxicity, development, reproduction and behavior testing. The LC50 for BZP, MeOPP and MDBP were 52.21, 5.72 and 1.22 mm, respectively. All concentrations induced a significant decrease in the body surface of the worms, indicating developmental alterations, and decrease in the brood size. Worms exposed to piperazine designer drugs also presented a decrease in locomotor activity and mechanical sensitivity, suggesting the possible dysfunction of the nervous system. Neuronal damage was confirmed through the decrease in fluorescence of BY200 strains, indicating loss of dopaminergic transporters. In conclusion, we suggest that piperazine designer drugs lead to neuronal damage, which might be the underlying cause of the altered behavior observed in humans.

In vitro toxic evaluation of two gliptins and their main impurities of synthesis.

BACKGROUND: The presence of impurities in some drugs may compromise the safety and efficacy of the patient's treatment. Therefore, establishing of the biological safety of the impurities is essential. Diabetic patients are predisposed to tissue damage due to an increased oxidative stress process; and drug impurities may contribute to these toxic effects. In this context, the aim of this work was to study the toxicity, in 3 T3 cells, of the antidiabetic agents sitagliptin, vildagliptin, and their two main impurities of synthesis (S1 and S2; V1 and V2, respectively). METHODS: MTT reduction and neutral red uptake assays were performed in cytotoxicity tests. In addition, DNA damage (measured by comet assay), intracellular free radicals (by DCF), NO production, and mitochondrial membrane potential (ΔψM) were evaluated. RESULTS: Cytotoxicity was observed for impurity V2. Free radicals generation was found at 1000 µM of sitagliptin and 10 µM of both vildagliptin impurities (V1 and V2). A decrease in NO production was observed for all vildagliptin concentrations. No alterations were observed in ΔψM or DNA damage at the tested concentrations. CONCLUSIONS: This study demonstrated that the presence of impurities might increase the cytotoxicity and oxidative stress of the pharmaceutical formulations at the concentrations studied.

In vitro cardiotoxicity evaluation of graphene oxide.

Graphene is a two-dimensional (2D) monolayer of carbon atoms, tightly packed, forming a honey comb crystal lattice, with physical, chemical, and mechanical properties greatly used for energy storage, electrochemical devices, and in nanomedicine. Many studies showed that nanomaterials have side-effects on health. At present, there is a lack of information regarding graphene and its derivatives including their cardiotoxic properties. The aim of the present study was to evaluate the toxicity of nano-graphene oxide (nano-GO) in the rat cardiomyoblast cell line H9c2 and the involvement of oxidative processes. The cell viability was evaluated with the fluorescein diacetate (FDA)/propidium iodide (PI) and in the trypan blue exclusion assay, furthermore mitochondrial membrane potential and production of free radicals were measured. Genotoxicity was evaluated in comet assay and low molecular weight DNA experiment. Reduction of cell viability with 20, 40, 60, 80, and 100 µg/mL nano-GO was observed after 24 h incubation. Besides, nano-GO induced a mitochondrial hyperpolarization and a significant increase of free radicals production in the same concentrations. DNA breaks were observed at 40, 60, 80, and 100 µg/mL. This DNA damage was accompanied by a significant increase in LMW DNA only at 40 µg/mL. In conclusion, the nano-GO caused cardiotoxicity in our in vitro model, with mitochondrial disturbances, generation of reactive species and interactions with DNA, indicating the importance of the further evaluation of the safety of nanomaterials.