Achalasia is an esophageal motility disorder, with clinical presentation of dysphagia and regurgitation. This is a chronic condition with no cure. Current treatment options aim to reduce lower esophageal sphincter tone by pharmacological, endoscopic or surgical means, with the aim of improving patients’ symptoms. Peroral endoscopic myotomy (POEM) is an alternative endoscopic surgery to Heller cardiomyotomy, in which the procedure is performed orally, by endoscopy, offering efficacy comparable to surgical myotomy, with relative ease and minimal invasion, without external incisions.To study the safety of POEM by analyzing its results, adverse events and perioperative complications and the main ways to overcome them, in addition to evaluating the effectiveness of the procedure and the short-term postoperative quality of life.A qualitative and quantitative, observational and cross-sectional study that analyzed patients who underwent the POEM in a reference center, from December 2016 to December 2022, maintaining the technical standard of pre-, peri- and postoperative protocol.A total of 94 patients were included in the study, and only three had postoperative complications. The average early postoperative Eckardt score was 0.93 and the late 1.40, with a mean improvement of 7.1 in early results and 6.63 in late results (p<0.05).POEM can be reproduced with an excellent safety profile, significant relief of symptoms and improvement in esophageal emptying, and in quality of life. This condition is marked by the degeneration of the myenteric plexus, leading to a deficiency in the relaxation of the lower esophageal sphincter and disorganized peristalsis in the esophageal body. Achalasia is a chronic ailment for which there is currently no cure. The primary symptomatic manifestation of this motility disorder is the classic presentation of difficulty swallowing both solids and liquids, often accompanied by regurgitation of undigested food or saliva. While achalasia is considered rare, it is the most prevalent primary esophageal motility disorder, with an annual incidence of approximately 1.6 case per 100 thousand individuals and a prevalence rate of approximately 10.8 cases per 100 thousand individuals. It affects both sexes equally and typically presents around the age of 50. In addition to the idiopathic form of achalasia, there are other types of secondary achalasia, such as Chagas’ disease, which is caused by the parasite Trypanosoma cruzi.A chalasia is a motility disorder of the esophagus characterized by a lack of peristalsis within the body of the esophagus and reduced or absent relaxation of the lower esophageal sphincter (LES). It typically has an idiopathic origin.Barium swallow test plays a crucial role as a diagnostic test for assessing the esophagus’ morphology, including its diameter and alignment. When it comes to diagnosing achalasia, contrast examination reveals some characteristic findings, which include the absence of the gastric bubble, a tapering of the distal esophagus resulting in a “bird’s beak” configuration, and proximal organ dilation, occasionally accompanied by the presence of hydroaerial levels.The Rezende and Mascarenhas Classification (1982) utilizes an esophagogram to gauge the severity of achalasia, serving as one of the criteria for determining the appropriate treatment. This classification system categorizes achalasia into four grades: Grade I ; Grade II ; Grade III ; Grade IV .The Eckardt symptom score is a scoring system that takes into account the frequency of each of the four main symptoms of achalasia, including dysphagia, regurgitation, retrosternal pain and weight loss. Each symptom is scored on a scale of 0 to 3, resulting in total possible scores between 0 and 12, with higher scores indicating more symptomatic disease. Scores of 3 or less are considered a good response to treatment.High-resolution manometry is essential in the management of achalasia with endoscopic myotomy, since using the Chicago classification the extent of myotomy can be standardized according to type I, aperistalsis; type II, pan-pressurization; and type III, distal pressurization.Endoscopic examination is an integral part of achalasia investigation and can provide a diagnostic confirmation in cases of stasis and resistance at the lower sphincter. Care should be taken in cases of pseudo-achalasia, as it mimics the condition. It is also useful to rule out other diagnoses, such as neoplasms and esophagitis.Current treatment options for this condition aim to reduce the tone of the LES by pharmacological, endoscopic or surgical means. The goals in treating achalasia are to relieve the symptoms of dysphagia, to improve esophageal emptying and to prevent the organ from becoming more dilated over the years. These agents act by facilitating esophageal emptying, reducing tone, and causing relaxation of the LES. Oral agents, however, are among the least effective treatment options due to their mechanisms of action, providing only transient benefits and carrying a significant potential for side effects.Different oral pharmacological agents have been used in the management of achalasia, including calcium channel blockers (nifedipine), long-acting nitrates (isosorbide dinitrate), phosphodiesterase-5 inhibitors (sildenafil), anticholinergics (atropine and dicyclomine), and beta-adrenergic agonists . Calcium channel blockers and long-acting nitrates are the most commonly used. As a result, the overall effect of botulinum toxin injection is an approximate 50% reduction in LES tone. This reduction is usually sufficient to facilitate esophageal emptying and provides relief from symptoms in nearly two-thirds of patients.Another treatment option for achalasia involves injecting botulinum toxin directly into the LES using upper digestive endoscopy. Botulinum toxin causes presynaptic inhibition of acetylcholine release and blocks unopposed excitatory cholinergic stimulation of the LES, leading to paralysis of the sphincter due to neuronal inhibition, but with no effect on resting muscle tone, which is mainly driven by myogenic influence. For most patients, the initial dilation is conducted using a 3.0 cm balloon, followed by a symptomatic and objective assessment four to six weeks later. If patients continue to experience symptoms, further dilation with a balloon of a similar size can be considered. Studies have consistently demonstrated the effectiveness of pneumatic dilation, with some reporting that it provides good to excellent relief from symptoms in as many as 93% of patients.Pneumatic dilation involves the use of polyethylene balloons of varying sizes, which are gradually inflated to cause the rupture of the circular muscle fibers in the LES. Pneumatic dilation is currently the most widely used non-surgical option for treating achalasia. In addition, the risk of perforation may be lower in serial pneumatic dilatations, but they are less effective in young men under the age of 45 due to their thicker LES muscles.Pneumatic dilation has a risk of esophageal perforation with a median rate of 1.9% in the hands of experienced operators. Prolonged post-dilation chest pain, intramural hematoma and traumatic diverticula have also been described after pneumatic dilations.Additional complications associated with pneumatic dilation include the development of gastroesophageal reflux disease (GERD) in approximately 15–35% of patients. The success rate at two years, according to the Eckardt score, was 86% after pneumatic dilation and 90% after laparoscopic HM, with no statistically significant difference (p=0.46). In addition, there were no significant differences in post-procedure LES pressure, barium contrast column height or quality of life between the two groups. A long-term follow-up study showed similar efficacy, even at 5 years, although 25% of patients undergoing pneumatic dilatation required further dilatation.A European randomized controlled trial comparing the efficacy of pneumatic dilation versus laparoscopic HM showed no significant difference in the efficacy of the two modalities after two years of follow-up. In this procedure, myotomy is performed orally using an endoscope to create a submucosal tunnel. POEM offers efficacy comparable to surgical myotomy but with minimal invasiveness and relative ease, without requiring external incisions.POEM is an endoscopic surgical alternative to open or laparoscopic HM. This innovative approach involves creating a submucosal tunnel followed by myotomy of the esophageal circular layer, which differs from MH, where myotomy targets both the longitudinal and circular layers. POEM is considered a safe procedure, comparable to the safety profile of laparoscopic Heller cardiomyotomy.The concept of endoscopic myotomy for achalasia was initially reported in 1980, but there were no subsequent reports on the seven cases performed at that time. In 2009, Inoue et al. in Japan refined the technique and coined the term “POEM”This study evaluates the safety of POEM by analyzing its outcomes, adverse events, perioperative complications, and strategies for preventing them. Additionally, the study assesses the effectiveness of the procedure and its impact on postoperative quality of life.Sistema Único de Saúde (SUS), Brazil’s health system, in the state of Ceará, located in northeastern Brazil.This is a qualitative and quantitative, observational and cross-sectional study, with a review of medical records and carried out in a tertiary hospital belonging to the The study included all patients who received a confirmed diagnosis of esophageal achalasia based on the clinical presentation, digestive endoscopy, esophagogram and esophageal manometry and who underwent the POEM procedure, from December 2016 to December 2022, operated on by the same team, maintaining the same pre-, peri- and post-operative protocol, following these steps:Step 00 – Pasty diet for 48 hours, clear liquids without residue for 24 hours and fasting for 8 hours before the procedure.Step 1 – Test of the digestive endoscopy equipment, using Olympus Exera II CV-180 with CO2 system, 150 or 180 series endoscope.Step 2 – General anesthesia with orotracheal intubation and antibiotic prophylaxis with ciprofloxacin 400 mg and metronidazole 500 mg single dose.Step 3 – Esophageal assessment and lavage with distilled water using a distal attachment cap at the tip of the endoscope (endoscopes with larger working channels can be used in case of difficulty).Step 4 – Localization of the cardia by measuring the distance to the upper dental arch.Step 5 – Submucosal injection of saline solution with methylene blue into the right anterolateral wall, about 10 cm from the cardia, making a bubble in the submucosa .Step 06 – Excision of the mucosa measuring around 15 mm using a flush knife .Step 7 – Making the submucosal tunnel, detaching the submucosa from the circular muscle layer using a flush knife and injections of methylene blue solution .Step 8 – Endoscopic myotomy starting 2–3 cm below the excision of the mucosa up to 3 cm below the cardia .Step 9 – Review of hemostasis and closure of the mucosal excision .Step 10 – Endoscopic assessment of possible intraoperative events, with subsequent interventions according to medical criteria.Step 11 – Fasting was recommended for 24 hours, followed by a restricted liquid diet on the first postoperative day, full liquid on the second, pasty on the third, fourth and fifth. Pantoprazole 40 mg twice a day was prescribed for 60 days.Step 12 – Follow-up was carried out with a medical consultation and digestive endoscopy assessing symptoms, the Eckardt score and endoscopic findings at three, six, 12 months and annually after the procedure.Gender, age, prior achalasia treatments, including the type of treatment, Rezende’s radiological classification from preoperative barium esophagograms, preoperative Eckardt scores, the presence of adverse events, and intra- and peri-operative complications were assessed.Postoperative complications were classified according to the Clavien-Dindo criteria. Length of hospital stay, perioperative mortality and the need for readmissions were also assessed.Late follow-up was carried out through medical consultations assessing symptoms and the result of the most recent upper digestive endoscopy. The Eckardt scores were evaluated early after the procedure, at three to six months post-surgery, and, subsequently, after one year or more.Patients with incomplete medical records, under 15 years of age, who refused to take part in the study and patients in whom endoscopic surgery was not performed for technical reasons were excluded. Patients with a history of endoscopic dilation, botulinum toxin injection or a history of laparoscopic esophageal myotomy before POEM were not considered exclusion criteria.Data was collected retrospectively to compare pre- and post-procedure parameters and statistical analysis was performed. Data is presented as mean ± standard deviation. Student’s paired t-test was used for continuous variables and the proportion test for categorical variables. A p-value <0.05 was considered statistically significant. The study was approved by the Ethics Committee of Hospital Geral Doutor César Cals (nº 67147423.1.0000.5041).The study included 94 patients, with 55 females and 39 males, ranging in age from 17 to 90 years. The mean age was 51.05 years, with a median of 53 years. Rezende’s classification was applied to only 67 patients, of which 13 (19.3%) were grade I, 31 (46.3%) grade II, 18 (26.9%) grade III and five (7.5%) grade IV.2, which is rapidly absorbed by the tissues. Pneumothorax and pneumoperitoneum were treated by inserting a 14G needle into the fifth intercostal space of the affected hemithorax and into the left hypochondrium, respectively, keeping it open until the end of the procedure. Patients with tunnel mucosa rupture underwent treatment through endoscopic closure with a clip. Among those who experienced bleeding, three were managed through cauterization using the flush knife and/or a hemostatic forceps, and one patient needed treatment with a Sengstaken-Blakemore balloon.Adverse events were documented in 47 patients (50%) during the procedure. These events included subcutaneous emphysema in 29 patients (30.9%), pneumoperitoneum in nine (9.6%), bleeding in four (4.3%), pneumothorax in three (3.2%), and inadvertent injury to the tunnel mucosa in five (5.3%). Subcutaneous emphysema was self-limiting due to the use of COTwo patients required extended hospitalization, one for a total of nine days and the other for 14 days due to pneumothorax. Additionally, another patient needed readmission due to the development of pleural empyema, which was successfully treated through lung decortication.Out of the 94 patients included in the study, only three experienced post-operative complications: one patient was classified as Clavien-Dindo II (respiratory infection), one as Clavien-Dindo IIIa (pneumothorax), and one as Clavien-Dindo IIIb .The preoperative Eckardt score ranged from 4 to 12, with an average of 8.03.The early postoperative Eckardt score assessed in 42 patients had a mean of 0.93. The late postoperative Eckardt score assessed in 39 patients had a mean of 1.40, after 47 months of late follow-up. Therefore, there was an average improvement of 7.1 in the early results and 6.63 in the late results, with statistical significance (p<0.05) .It was possible to evaluate postoperative upper digestive endoscopy results in 46 patients, on average, 22.3 months after the surgery. Among them, 28 (60.9%) showed no esophagitis, seven (15.2%) had Los Angeles grade A esophagitis, seven (15.2%) had Los Angeles grade B esophagitis, three (6.5%) had Los Angeles grade C esophagitis, and one (2.1%) had Los Angeles grade D esophagitis. Additionally, 31 (67.4%) had no esophageal residue, seven (15.2%) had salivary esophageal residue, five (10.8%) had liquid food esophageal residue, and three (6.5%) had solid-pasty food esophageal residue.. In this study, the results of the procedure were evaluated with a description of the technique used for reproduction and comparison with other healthcare centers.Currently, there is no standard technique adopted by all health centers for the treatment of achalasia, with variations in periprocedural management between them. Despite the good results, excellent efficacy and safety of POEM are commonly reported, clinical success rates and adverse events vary between healthcare providers. An Eckardt score of 3 or less has been achieved in around 98% of cases, according to a meta-analysis. A cohort of more than 500 patients, with at least three years of follow-up, showed a success rate in maintaining symptom relief of 88.5%.Clinical remission after POEM ranges from 82 to 100%, with a reduction in the Eckardt score and a decrease in LES pressure.The guideline from the American College of Gastroenterology recommends and supports the use of POEM as a safe treatment option for achalasia patients, even those who have previously undergone Heller cardiomyotomy or pneumatic dilation. Some meta-analyses, involving over one thousand patients, have shown the short-term efficacy of POEM (within one year), including reductions in the Eckardt score and LES pressure. A meta-analysis comparing patients who underwent POEM versus laparoscopic HM revealed that neither group experienced serious adverse events. Minor adverse events were more common in the laparoscopic group, whereas major events were observed more frequently in the POEM group.In a review of laparoscopic HM, 0.6% of patients reported wound complications, 1.8% general complications and 2.4% major complications. The readmission rate was 3.1% and the reoperation rate was 2.3%. The 30-day mortality rate was 0.3%. The average operative time was just over 2 hours (141.6±63.4 min). Conservative treatment allows for rapid recovery, and such events have little influence on long-term prognosis. Another common adverse event is perforation, which in the case of the mucosa can be closed during the procedure using endoscopic clips.The main short-term adverse events of POEM include subcutaneous emphysema, pneumoperitoneum and pneumomediastinumThe present study demonstrates the excellent safety profile of POEM with significant symptom relief, improved esophageal emptying and a significant reduction in symptoms, offering an improvement in the patient’s quality of life. Common adverse events such as pneumoperitoneum and subcutaneous emphysema are easily managed if clinically significant, making POEM a safe procedure. Other factors such as operator experience and myotomy route may be additional factors responsible for the lower rate of these adverse events, which could be analyzed in further studies. Pneumothorax was the most serious complication and should be avoided. Among the various data analyzed in this study, one is very relevant: the mortality rate (postoperative complication Clavien-Dindo V) after POEM was zero, which is in line with other studies, as shown in the table below .This study has some limitations, such as the absence of some data and better stratification of the patient profile. Additional prospective and multicenter studies are needed to establish and standardize the technique.The POEM procedure can be reproduced safely, resulting in significant symptom relief, enhanced esophageal emptying and improved patient quality of life. However, additional prospective and multicenter studies are needed to establish and standardize the technique.


Many natural substances commonly found in healthy diets have been studied for their potential to reduce male infertility associated with varicocele. A positive role of selenium (Se) or lycopene alone was demonstrated in experimental varicocele, while no data are available on their association. One group of male Sprague–Dawley rats was sham operated and daily treated with Se , lycopene , or their association. A second group underwent surgery to induce varicocele. Sham and half of the varicocele animals were sacrificed after twenty-eight days, while the residual animals were treated for one more month and then sacrificed. In varicocele animals, testosterone levels and testes weight were reduced, Hypoxia Inducible Factor-1α (HIF-1α) expression was absent in the tubules and increased in Leydig cells, caspare-3 was increased, seminiferous epithelium showed evident structural changes, and many apoptotic germ cells were demonstrated with TUNEL assay. The treatment with lycopene or Se alone significantly increased testis weight and testosterone levels, reduced apoptosis and caspase-3 expression, improved the tubular organization, decreased HIF-1α positivity of Leydig cells, and restored its tubular positivity. Lycopene or Se association showed a better influence on all biochemical and morphological parameters. Therefore, the nutraceutical association of lycopene plus Se might be considered a possible therapeutic tool, together with surgery, in the treatment of male infertility. However, long-term experimental and clinical studies are necessary to evaluate sperm quantity and quality. A varicocele is characterized by the elongation and widening of the internal testicular vein and the spermatic pampiniform plexus, often leading to male infertility. It is caused either by an abnormal position of the left spermatic vein, by a dysfunction of venous valves, or by anomalies of the left renal vein, all inducing a hindered spermatic venous reflux .An analysis of the literature showed an overall prevalence of 15–20% in the healthy adult male population. In patients with male infertility, varicocele was demonstrated in 20–40% of subjects with primary infertility and in 75–81% of men with secondary infertility. Typically, left-sided varicoceles are present in 90% of cases and only 10% are bilateral [The hypotheses of varicocele contribution to infertility include higher temperature in scrotum, hypoxia, oxidative stress, accumulation of carbon dioxide, apoptosis, inflammation, autoimmunity, and damage to sperm cells, but no consensus theory is able to explain the effects of varicocele on infertility .While the full impact of varicocele on infertility remains unclear, oxidative stress and testicular apoptosis are believed to play significant roles. Under hypoxic conditions, an increased production of reactive oxygen species (ROS) occurs, inducing negative changes of the plasma membranes of spermatozoa and of the other cells of the germinal epithelium and stimulating testicular apoptosis ,4. This HIF-1, a nuclear transcription factor, is present either in normoxic conditions or under hypoxia . In partThe outcomes of different substances on the biochemical and morphological damages induced in testes by experimental varicocele were studied in the last years ,13,14,15Lycopene is considered the strongest anti-radical compound of the carotenoid family  and can Se, an essential trace element, plays significant roles in many biochemical and physiological processes, including the reproductive function in males . Its defInterestingly, the role of the association between lycopene and Se was not evaluated in experimental varicocele, even if our group has previously examined its positive effects in benign prostatic hypertrophy . As to tOn the basis of this background, the aim of the present work is to better elucidate the role of the nutraceutical association of lycopene and Se in an experimental model of varicocele in rats.All sham groups showed normal levels of testosterone; consequently, a single value is provided for sham. A sharp, significant decrease was observed in varicocele animals when compared to sham (−54%). Administration of lycopene or Se alone caused an increase in testosterone levels compared to varicocele rats ; however, the values were significantly lower than those of the sham groups. Only in varicocele rats treated with the association of lycopene plus Se were the testosterone levels close to sham  .MDA levels were normal in all sham groups; therefore, we provided a single value for the sake of simplification. Conversely, MDA levels were significantly augmented in varicocele rats. When treated with lycopene or Se alone, MDA levels were significantly decreased in both varicocele and contralateral testes, but they were significantly higher than sham. Only in varicocele rats treated with lycopene and Se was MDA reduced to close to normal levels in either operated or contralateral testes .A low expression of caspase-3 was present in all sham groups: therefore, a single value for sham is provided . ConversIn animals of all the sham groups, a normal morphology of the seminiferous tubules and of the extratubular compartment was observed with Hematoxylin and Eosin (HE) stain. Therefore, a single image is provided as representative of sham A and onlNo TUNEL-positive cells were demonstrated in the seminiferous tubules of all groups of sham rats; therefore, only one micrograph is shown as typical of all groups A. ConverAll sham groups showed seminiferous tubules with HIF-1α positive elongated spermatids and spermatozoa; HIF-1α positive Leydig cells were also present in the interstitial spaces. Consequently, a single micrograph is shown as typical of all shams A. On theVaricocele is considered to be a cause of male infertility, able to induce abnormal semen with decreased motility of spermatozoa, low sperm count, and abnormal structure of the seminiferous epithelium ,11,20. RAmong the above mechanisms, testicular hypoxia plays a leading role in varicocele pathophysiology. In particular, under hypoxic conditions, HIF-1α, the active subunit of HIF-1, a highly specific nuclear transcription factor, is expressed, showing a relationship with the degree of hypoxia ,21. Prev2 tension is higher than that of the luminal region, so that, under hypoxic conditions, HIF-1α produced by Leydig cells. It triggers apoptosis of spermatogonia, undergoing a self-renewal process [2 tension is normally lower, as indicated by the basal expression of HIF-1α in response to the reduced access of O2 [In a previous paper based on an experimental model of testicular ischemia/reperfusion in rats , we show process . On the ss of O2 . As a coAs to the reduction in testosterone levels observed in the varicocele group, this could be related to the increase in HIF-1α in Leydig cells, indicating a hypoxic suffering of these cells, which is able to interfere with steroid synthesis in the interstitial cells. This is probably related to the reduced production of the steroidogenic acute regulatory (StAR) protein, which has an important role in the first step of testosterone and other steroid hormones synthesis .Changes in testis weight are important parameters to analyze the effects of experimental procedures in laboratory animals. In our study, the varicocele group of rats showed reduced testicular weight when compared to the sham group, probably related to structural damage and to apoptosis of the germinal epithelium .Another mechanism involved in varicocele is oxidative stress, mainly caused by an increased production of ROS , which iThe main treatment of varicocele is varicocelectomy, which, particularly in adolescents, can ameliorate testicular growth and sperm characteristics. However, in the last two decades, in addition to surgery, the use of different substances, alone or in association, have been proposed in the management of the varicocele either in infertile men or in experimental animal models.Among these substances, vitamins  ,31,32,33In addition, the effects of two nutraceuticals, lycopene and Se, alone or in association with different substances, were studied.As to the lycopene, it is present in fruits and vegetables and is considered the most effective anti-free-radical of the carotenoid family . Its disAs to Se, this essential and widely distributed element plays fundamental roles in the testis, as it is necessary for its normal development, for spermatogenesis, and for the normal activities of spermatozoa . When adOur paper shows, for the first time, the effects of the association of lycopene and Se in an experimental model of murine varicocele. We demonstrated an improvement in all biochemical and morphological parameters after the administration of both substances, thus indicating a positive role in fertility. This could be related to the involvement of HIF-1α, suggesting an important role of this transcriptional factor in hypoxic conditions of the testis. Indeed, as reported in a previous review by our research group, since the damage to testicular function is linked to multiple physio-pathological events, including testicular hypoxia and hyperthermia, inflammation, oxidative stress, hormonal imbalance, and cellular death, all substances tested in experimental varicocele, acting on this molecular context, could show synergistic effects, thus representing a promising and safe approach in this multifaceted pathology . SpecifiMoreover, it appears consequential that healthy dietary habits, as plant-based diets (including the Mediterranean-style diet), which are particularly abundant in functional foods and/or nutraceuticals such as lycopene and Se, positively impact the above molecular targets deeply demodulated in varicocele .The examined nutraceutical association of lycopene and Se could be included in the natural and safe management of varicocele and male infertility-related disorders, in support to surgery, even if additional studies are needed to evaluate further molecular targets involved in experimental varicocele. Long-term experimental and clinical studies are necessary to evaluate sperm quantity and quality after treatment with lycopene and Se. However, the pathophysiological knowledge of varicocele appears fascinating, but also deeply complex. As previously stated , and on In the present work we followed the standards for the care and use of animals, as indicated in the ARRIVE  guidelines . Both thA total of 56 male Sprague–Dawley rats, aged 7 weeks and weighing 200–230 g, were purchased from Charles River Laboratories Italia srl . Animals were housed in controlled environmental conditions, under a cycle of 12-hour light/dark at approximately 23 °C, with food and water ad libitum. The animals were divided into 8 groups of 7 animals each. Four groups  were anesthetized and underwent a sham operation, as previously described . The anig. An HRP-conjugate and the specific antibody were added, followed by substrates and stop solution. The mean absorbance was calculated using a microplate reader at 450 nm and correlated with those from standard curves. Data were expressed in ng/mL.An ELISA kit was used for testosterone levels in serum, according to the protocol suggested by the manufacturer. In brief, blood, gained from cardiac puncture, was clotted, and serum was achieved by centrifugation for 10 min at 1000× Lipid peroxidation was evaluated through the MDA levels, indicating the results as nmol/g tissue .w/v nonfat dried milk, 0.1% Tween-20, and incubated overnight at 4 °C. Then, the membrane was incubated with a secondary anti-rabbit  for 1 h at room temperature. To establish the loading of blots with equal quantities of protein lysates, it was also incubated with the antibody against β-actin . Protein signal was analyzed by the enhanced chemiluminescence system  and quantified by a scanning densitometry system . Data were expressed as relative integrated intensity using β-actin  as a control for the equal loading of samples.Western blots were performed as previously described . SpecifiAll testes, after fixation in Bouin, were dehydrated in ethanol, cleared in xylene, and embedded in paraffin . Histological sections (5 µm) were mounted on silanized slides , and stained with HE. Photomicrographs were taken with a Nikon Ci-L  light microscope, using a digital camera Nikon DS-Ri2, saved as Tagged Image Format Files (TIFF), and blindly assessed by two trained histologists. Five microscopic fields from ten non-serial sections of all groups, each including two entire seminiferous tubules, were evaluated. The diameters of 100 separate seminiferous tubules, all showing a circular profile, were calculated to obtain the MTD, expressed in micrometers (µm). From the same tubules, the structural organization of the germinal epithelium was assessed, according to the Johnsen’s scoring system , as modi2O2 in methanol. Sections were treated with terminal deoxynucleotidyl transferase, biotin-labeled deoxynucleotides, streptavidin–horseradish peroxidase conjugate, and then with diaminobenzidine as chromogen. Micrographs were taken with a Nikon Ci-L light microscope using a digital camera Nikon Ds-Ri2. One hundred seminiferous tubules per group were blindly evaluated by two trained observers to determine the percentage of tubules with apoptotic cells (%TWAC) and the mean number of TUNEL-positive cells per tubule (apoptotic index) [A kit for the assay of apoptosis  was used; 5 µm sections, obtained from the same samples used for morphological evaluation, were cleared in xylene and rehydrated in ethanol. Permeabilization with proteinase K was performed, followed by endogenous peroxidase blocking with 3% Hc index) .2O2 in methanol. Primary antibody  was incubated overnight at 4 °C in a moisturized chamber the day after the secondary antibody  was added. The reaction was visualized with 3,3′-Diaminobenzidine. Counterstaining was performed in Mayer’s haematoxylin. Slides were photographed with a Nikon Ci-L light microscope.From the same blocks used for histological evaluation, 5 µm sections were mounted on silanized slides, cleared in xylene, and rehydrated in ethanol. Antigen retrieval was performed with pH 6.0 citrate buffer and endogenous peroxidase blocked with 0.3% Ht-test and one-way ANOVA with Tukey’s post-test for intergroup comparisons. Values are expressed as mean ± standard deviation (SD). A p value of ≤ 0.05 was considered statistically significant.The different experimental groups were analyzed by Student’s


Hexabromocyclododecane (HBCD) is a non-aromatic compound belonging to the bromine flame retardant family and is a known persistent organic pollutant (POP). This compound accumulates easily in the environment and has a high half-life in water. With a variety of uses, the HBCD is found in house dust, electronics, insulation, and construction. There are several isomers and the most studied are α-, β-, and γ-HBCD. Initially used as a substitute for other flame retardants, the polybrominated diphenyl ethers (PBDEs), the discovery of its role as a POP made HBCD use and manufacturing restricted in Europe and other countries. The adverse effects on the environment and human health have been piling, either as a result from its accumulation or considering its power as an endocrine disruptor (ED). Furthermore, it has also been proven that it has detrimental effects on the neuronal system, endocrine system, cardiovascular system, liver, and the reproductive system. HBCD has also been linked to cytokine production, DNA damage, increased cell apoptosis, increased oxidative stress, and reactive oxygen species (ROS) production. Therefore, this review aims to compile the most recent studies regarding the negative effects of this compound on the environment and human health, describing the possible mechanisms by which this compound acts and its possible toxic effects. Hexabromocyclododecane (HBCD)  is a parHBCD is highly lipophilic, with a low solubility in water, and aliphatic ,7 with aThrough its abundance in stereoisomers, HBCD can have several different isomer concentrations, depending on its final destination and its respective production process, since it can undergo rearrangement at temperatures above 160 °C ,10.From 1960 to 2009, the use of HBCD was mainly linked to the construction industry. In 2009, its production and use were restricted. By 2013, HBCD became part of the Stockholm Convention’s Annex A as a persistent organic pollutant . ConsequContinuous exposure to HBCD affects various systems, including increased liver weight and liver enzyme, and additional side-effects on the thyroid hormone. Furthermore, this exposure can affect the reproductive system and induce alterations in spontaneous behavior and memory defects. Studies in vitro show that HBCD has anti-androgen activity and can be an aromatase inhibitor, interacting with steroid hormone receptors. These effects show that HBCD can be an endocrine disruptor, causing neuronal changes in animals and affecting the hormonal system, along with several other systems ,14,15.This review summarizes the most important and relevant studies about the mode of action and effects of HBCD on the environment and human health. In addition, it intends to describe the effects on the various systems and metabolic pathways of plants, animals, and humans. This work highlights the importance of continued studies involving HBCD, focusing on its dangers, and stereoisomers. The main topics covered by this review are exposure routes; toxicity studies; and the effects on human health, animals, and plants. For this purpose, research was carried out on chemical properties and routes of exposure of the HBCD, and all the in vitro and in vivo studies performed in all the organisms , as to more rigorously assess safety and the benefits or hazards related with the usage of HBCD.This review summarized experimental studies that evaluated the effects of HBCD in plants (Topic 4), animal (Topic 5), and human models (Topic 6) after 2014. Furthermore, the HBCD’s effects in several body systems were also explored, mainly focusing on the cardiovascular, reproductive, endocrine, neuronal, and liver.A literature review was performed based on articles available in Scopus, Web of Science, PubMed, and ScienceDirect (Elsevier) databases. The research strategy was carried out using Boolean operators “AND”, “OR”, and “NOT” and a combination of terms relating to HBCD, such as “tHBCD”, “Hexabromocyclododecane”, “α-HBCD”, “β-HBCD”, and “γ-HBCD”. This article was conducted following the protocol used by Abrantes-Soares et al. . In addiHBCD was first found in the Swedish rivers in 1997 . After iConcerning the HBCD structure, Wu et al., 2012, and Heeb et al., 2007, describe that the technical HBCD (tHBCD), synthesized from the addition of bromine at 1,5,9-cyclododecatriene, consists of a mixture of three diastereomers: α-, β-, and γ-HBCD. In addition to these, other diastereomers, such as δ-HBCD, were found in abiotic  and biotWith the composition of abiotic matrices similar to the commercial mixture, and γ-HBCD as the most abundant diastereomer, Zhang et al., 2013, suggested the use of a ratio of γ- and α-HBCD in abiotic samples for the management of HBCD environmental contamination, since γ-HBCD tends to decline over time . HoweverIn agreement, in 2012, Du et al. exposed zebrafish to three diastereomers of HBCD , to determine their effects on the organism of these animals . The aut−14 Kg and 1.08 × 10−11 Kg [−18 mol/L and 9.51 × 10−16 mol/L) [−4 g/Kg and 0.032 g/Kg dry weight [Domestic dust, and indoor and outdoor airborne concentrations of HBCD have gathered special attention since they are the major human health influencers. Several scientists have led a series of studies using air and dust samples and hand wipes to determine the levels of HBCD contamination to which a human may be exposed without endangering human health. Harrad et al., 2010, reported high levels of HBCD exceeding 0.01 g/Kg in daycare centers and primary schools in the UK . Abdalla10−11 Kg . HBCD pr6 mol/L) , and dow6 mol/L)  and Spai6 mol/L) . Soils s−5 g/Kg lw and by 2007, this value increased to an average concentration of 0.00501 g/Kg lw [The occurrence of HBCD in sewage sludge ,45,46,47The presence of HBCD in vegetables has also been observed and can be correlated with the use of sewage sludge as fertilizer . Wu et aFish typically exhibit high HBCD levels, given their position in the food chain and their high exposure in aquatic environments . Higher Janak et al., 2005, first reported the presence of HBCD in marine mammals of the Western Scheldt estuary . Three y−6 g/Kg and 8.56 × 10−4 g/Kg lipids, respectively [−5 g/Kg [−6 g/Kg [−6 g/Kg [−7 g/Kg [Regarding human exposure to HBCD, dermal and oral are the main routes of exposure, but inhalation of vapor or particles was also an important source of HBCD . Industrectively . HBCD vaectively ,62. Foodectively ,63 and tectively . At the [−5 g/Kg , where t[−6 g/Kg  and 4.6 [−7 g/Kg  with α-HSince exposure to HBCD is high and comes from various pathways, it has already been found in adipose tissue  and humaIn summary, HBCD is still very prevalent in the environment, although its production and application are banned, which promotes significant human exposure. In addition, this compound presents a high bioaccumulation, promoting high biomagnification, along the entire trophic chain. Therefore, it is very important to understand the effects of this compound on plants, animals, and humans, and all the systems and mechanistic pathways that can affect the ecosystem.Regarding plant studies, as mentioned earlier, this literature review only looks at articles after 2014. Therefore, this review will address the four articles found and summarized in In 2016, Wu et al. analyzed the effect of HBCD on maize and discovered that the concentrations of this flame retardant were different for various parts of the plant. They then discovered that after the first HBCD uptake, the concentration of HBCD was higher in the roots than in the shoots. The highest concentrations, both in roots and shoots, of diastereoisomers were, in order, γ-HBCD, followed by β-HBCD and α-HBCD, which was the least concentrated. This shows that γ-HBCD is the most abundant diastereoisomer in plants samples. In addition, it has also been shown that exposure to this toxicant decreases the germination rate, root biomass, root elongation, biomass of shoots, and even the elongation of shoots. The HBCD also caused an increase in the level of hydroxy radical (OH·) in the roots and maize shoots, being higher in shoots. This can lead to oxidative stress and ROS formation and these levels are derived from HBCD exposure. They also found high values of γ-H2AX, suggesting that the damage to maize DNA would be very serious after exposure to HBCD .In the same year, Huang et al. studied the accumulation and biotransformation of HBCD in the roots of maize. All three HBCD enantiomers were found in maize roots and their accumulation was diastereomer- and enantiomer-specific, with γ-HBCD being the most concentrated. They found that isomerization occurred for all stereoisomers except (+) α-HBCD. Several metabolites of HBCD were found in the roots of maize, which did not appear in nutrient solutions or in the shoots and further demonstrates that its metabolization occurred. Debromination and hydroxylation were the two ways of metabolism for HBCD. All pairs of enantiomers could bind to various active cavities of maize enzymes CYP71C3v2 and glutathione s-transferase (GST) GST31. These findings suggest that the biotransformation of HBCD enantiomers may derive from their binding to different binding sites with maize enzymes, as (−) α-HBCD formed one halogen bond with residues of CYP71C3v2, such as Arg473 and Cys476, and (+) α-HBCD formed one halogen bond with Asn326 residue. With GST31 residues only (−)α-HBCD formed halogen bonds with the residue Ser14 .In 2018, Zhang et al. decided to use Pak Choi leaves to demonstrate the effects of HBCD. The analysis was performed on changes in the concentration of metabolites and showed that, in the presence of HBCD, several metabolic pathways were affected, such as the lipid, nucleotide, amino acid, and carbohydrates. The results also showed, for the first time, that HBCD can interfere with the biosynthesis of other secondary metabolites, with significant exchanges in eight metabolites: luteolin, rutin, caffeic acid, sinapic acid, conifer aldehyde, chlorogenic acid, and coniferyl. These secondary metabolites can direct and indirectly react. In addition, 13 metabolites with the power to be potential biomarkers, used to differentiate the presence of HBCD toxicity in plant leaves, were discovered . Even wiIn 2019, Huang et al. using maize microsomes analyzed if microsomal cytochrome P450 (CYPs) were involved in the degradation of HBCD through an in vitro and silico study. The findings described that after 120 min of incubation, HBCD levels were lower compared to the initial levels. Since they did not detect γ-HBCD-derive from α-HBCD, the authors suggested that there was no bio-isomerization of HBCD catalyzed by maize CYPs. The appearance of several HBCD metabolites confirmed that maize CYPs were involved in HBCD metabolism. They also showed that dependent on the diastereomers of HBCD, there were selective modifications in the protein content of CYP, and the activity of CYP. Thus, (−)/(+) α-HBCD significantly decreased the maize CYP protein level and enzyme activity, and (−)/(+) γ-HBCD were inducers of maize CYPs. These results suggest that HBCD has a disruptor power in the CYP system of maize, both at the protein content level and at the transcription level. Several CYP isoforms were altered after exposure to HBCD showing different catalytic activities which, alongside the presence of hydroxylated metabolites, suggests that these isoforms may be involved in HBCD metabolism. Despite these findings, the authors described that the mechanisms by which this biotransformation by CYPs occurs are not yet clear due to the absence of pure plant CYP isoforms, highlighting the need to verify how HBCD enantiomers enter the CYPs and how metabolic products are released .In conclusion, effects of HBCD in plants can be attributed to alterations in the activity of CYPs, suggesting that the CYPs can be involved in the biodegradation of HBCD, modifying the rate of germination of the plant, leading to oxidative stress, and affecting various metabolic pathways.−6, 5 × 10−6, and 1 × 10−5 mol/L) and analyzed the envisioned effects. They then discovered that HBCD caused a decrease in cellular levels of ATP, inhibited steroidogenesis at various levels, altered cAMP production, cholesterol transport to mitochondria (StAR), conversion of 22-OH cholesterol into pregnenolone (CYP11A1), and the conversion of androstenedione into testosterone (HSD17β) [Animal studies in this review, considering both in vitro and in vivo studies, are summarized in (HSD17β) .2+ (membrane network that acts as a reservoir for Ca2+ release) in H9C2 cells. Additionally, Ca2+-ATPase activity was also increased, suggesting that calcium activity is affected by HBCD. The expression of miRNA profiles was also altered, with miR-219, miR-145, miR-194b, and miR-221 significantly increased in a dose-dependent manner. MiR-1 and miR-146b were inhibited in zebrafish embryos. In addition, they also found that myocyte enhancer factor 2 (Mef2a) was upregulated in both zebrafish embryo samples and H9C2 cells. With these findings, the authors suggested that the cardiovascular system may be a target of HBCD [In the next year, 2015, several scientists performed animal studies in vitro and in vivo involving HBCD. Shi et al. used earthworms to discover the effects of HBCD in vitro and in vivo. The results showed that HBCD had no significant effects on the growth of earthworms, but internally, caused an up-regulation of superoxide dismutase (SOD) levels, suggesting that SOD transcription can be stimulated by oxidative stress. Regarding catalysis (CAT), exposure to HBCD showed no effect on its expression. They also conducted studies on heat shock proteins (Hsps), concluding that the compound significantly increased the expression of the Hsp70 gene . Wu et a of HBCD .In the same year, Genskow et al. performed in vitro and in vivo analyses to assess selective damage to dopaminergic transporters after exposure to HBCD. For the in vitro study, they used dopaminergic primary cultured ventral mesencephalic neurons. The data leads to the conclusion that tyrosine hydroxylase (TH+) dopamine neurons presented sensitivity to HBCD, with significant reductions in a set of neuronal metrics, such as length and arborization of neutrine outgrowths. In addition to the significant loss of TH+ neurons after HBCD treatment, it also caused a reduction in the length and complexity of TH+ neutrine outgrowths involved in communication with adjacent neurons. Experiments conducted in vivo focused on discovering the effects of HBCD on selected presynaptic dopaminergic proteins in the striatum. Exposure of 3-month-old rats to HBCD resulted in a precipitous reduction in the expression of dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2), and these two were integrated into homeostasis mediation and dopamine neurotransmission in the dopaminergic circuit. In contrast, no change in TH expression or dopamine levels in the striatum was observed. These changes occur before explicit damage or even before the loss of the dopaminergic terminal. A reduction in VMAT2 raises important issues over the effects of HBCD on dopamine in the cytosolic compartment of the dopaminergic terminal. The alterations observed by the authors in the striatum seemed to be specific to the dopamine circuit, and there were no alterations in the GABAergic or glutamatergic transporters . Du et aIn 2016, a group of scientists studied the effects of HBCD using a zebrafish embryo model. Exposure to the compound revealed body malformations and a decreased rate of spontaneous movement in a concentration-dependent manner. They also described an increase in GST activity compared to controls, possibly indicating an increase in oxidative stress. Despite these results, the authors demonstrated that further studies are needed to clarify the role of HBCD . Miller Additionally, in 2016, Bernhard et al. found it important to describe whether marine fatty acids aggravated the hepatotoxicity of α-HBCD in female rats BALB/c. The differences found in liver histology coincided with changes in liver levels of glutamic acid decarboxylase (GAD). Rats with diets based on marine fatty acids had livers with uniform appearances. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as markers of liver function and damage. AST levels increased overall in groups. The addition of marine fatty acids resulted in an increase in the expression of peroxisome proliferator-activated receptor α, thus showing that the addition of these fatty acids had an effective functionality in the liver. This addition, together with exposure to HBCD, demonstrated low levels of mRNA in the livers. These results indicate that exposure to α-HBCD has disruptive power in liver metabolism of lipids through β-oxidation leading to increased lipid and steatosis deposition. In addition, marine fatty acids increase the hepatotoxic effect of HBCD. This all shows that diet is a critical variable when we evaluate the effects of HBCD, showing that continued studies are needed to clarify this route of contamination . Miller 2+ and Zn2+ pathways were altered, and glutamatergic activity, apoptosis, and oxidative stress. Exposure to low concentrations of HBCD showed that the compound can affect neural signaling in mouse brains by deregulating Ca2+ and Zn2+ homeostasis [In 2018, Shi et al. investigated the expression of antioxidant genes and the metabolic responses of earthworms to HBCD. They found an upregulated SOD, but no changes in GST. After 14 days of exposure several metabolites, such as lactate, glycine, lysine, ATP, and vain, were increased. These results indicate that HBCD may induce anaerobic breathing. The visualized changes in osmotic pressure indicated damage to the membrane structure. Exposure to HBCD also induced oxidative stress . Reffatteostasis .Carassius carassius to discover antioxidant responses and enzyme biotransformation after exposure to HBCD. Scientists found HBCD very concentrated in fish liver. In addition, it induced the activity of antioxidant enzymes, and increased levels of lipid peroxidation. In conclusion, HBCD induced ecological stress in animals and low levels of biotransformation together with slow bio isomerization rates, suggesting a potential long-term toxic effect [Still, in 2018, Rasinger et al. studied the effects of HBCD on brain proteins and the expression of juvenile female BALB/c mice genes. HBCD demonstrated histopathological effects on the liver, thymus, and uterus. It also showed a decrease in oestradiol 17β (E2) concentrations and an increase in the testosterone/E2 ratio. Proteomic analysis of the brain provided support for the reduction in HBCD-induced E2 levels. The results of this study indicate that exposure to this compound is associated with subtle but relevant toxicological effects . Dong etc effect .−10, 2 × 10−9, 2 × 10−8, and 2 × 10−7 mol/L). After exposure, HBCD caused several side effects. They observed that toxicity was inherited. The expression of genes related to oxidative stress and apoptosis was altered. Exposure to the compound could also increase ROS production and cellular apoptosis. The findings of this study were important to evaluate the toxic potential of HBCD [In 2019, Farmahin et al. described the dose–response transcriptional hepatic analysis of male and female Fischer rats after exposure to HBCD. HBCD altered a considerable number of genes involved in xenobiotic metabolism, oxidative stress, immune response, and cell cycle, both in male and female rats. The study supported the idea that HBCD operates through constitutive and pregnane X receptors. Although HBCD demonstrates gender specificity, the pathways it affects are similar in both males and females . Chen et of HBCD . Guo et  of HBCD .In 2020, Xie et al. described the effects caused by HBCD on the adipogenesis of C57BL/6 rats. The results demonstrated that after exposure to HBCD, the adipogenic effect increased, showing that HBCD has a dominant role in the first moments of adipogenesis. The mechanism probably involves a specific blockade of the Wnt6 gene during the early stages of adipogenesis. These findings are important to relate HBCD to the development of obesity .In 2020, Bertucci et al. described the cytotoxic effects of HBCD on cultures of fathead minnow liver tissues. The results, involving analysis of genes linked to apoptosis, and genes related to antioxidant responses, revealed that cytotoxicity followed a dynamic pattern, being difficult to describe and characterize the cytotoxicity of this compound .In 2022, Park and Kwak analyzed p53-related apoptotic responses to HBCD in mud crabs. The levels of catalase were increased in the gill and hepatopancreas. The results made it clear that exposure to HBCD significantly increased the transcription levels of p53 in gills and hepatopancreas .M. balthica, which resulted in changes in nutrient recycling processes. Nevertheless, the authors suggest that it is important to continue to study the problem of chronic exposure of ecosystems to compounds, such as HBCD [Regarding the in vivo studies, in 2015, Heldur Hakk performed studies on rats doing tests on urine, blood and tissues. The authors orally exposed mice to 0.003 g/Kg and observed that this compound accumulated at the tissue level a maximum of 2.5% of the administered dose. The lipophilic tissues contained the highest concentrations, and the α-HBCD was the most favored. These results further support the hypothesis that α-HBCD is the most bio-accumulative and persistent diastereomer when compared to other diastereoisomers. At the excretion level, the author also demonstrated that almost half of the diastereoisomers administered in the animals were excreted in 4 days, with urine being the most important excretive pathway and α-HBCD being the diastereoisomer with less excreting power. Regarding HBCD metabolism, the analyses showed that all diastereoisomers were metabolized, where β-HBCD had the highest metabolized percentage . Bradsha as HBCD .In 2016, Wang et al. used the urine of CD-1 rats to metabolically investigate the effects of HBCD exposure. The results showed no symptoms of obvious toxicity or changes in body or body weight. HBCD, however, caused urinary metabolome disturbances after 28 days of continuous exposure and these alterations showed increased citrate and 2-ketoglutarate, along with a decrease in alanine, acetate, formate, trimethylamine (TMA), 3-hydroxybutyrate, and malonic acid. The increase in citrate and 2-ketoglutarate was of great relevance since these metabolites are associated with the tricarboxylic acid cycle (TCA), indicating that the activity of mitochondrial enzymes, related to the TCA cycle, can be activated by HBCD. In addition, they found that HBCD can influence the absorption and use of phenylalanine .In 2017, Ratel et al. studied liver volatolomics from laying hens to reveal their exposure to HBCD. The differences found between the control group and the HBCD-contaminated group show that the compound has the potential to alter metabolism in the liver. They were also able to identify some alkanes and branched alkanes as markers of HBCD contamination. The results showed that the liver volatolome is a good contamination meter through feeding . Szabo e−9, 3.12 × 10−8, and 3.12 × 10−7 mol/L), the total T3 and total T4 levels were decreased and the activity of acetylcholinesterase (AChE) in the brain was increased. This investigation shows that exposure to HBCD can cause adverse effects on the tested species [In 2018, Dong et al. analyze the bioconcentration and the effects of HBCD in crucian carp and the effect in the triode hormones concentrations. As a result of exposure , HBCD can induce oxidative stress and cell apoptosis, can affect the cardiovascular system, alter liver metabolism, affect thyroid hormone transport, metabolism, and formation, and increase adipogenic effect, indicating that it may have long-term toxic effects.Concerning the data in humans, summarized in In 2015, Koike et al. performed a study in vitro to analyze the effect of HBCD on human bronchial epithelial cells. After exposure, the normal human bronchial epithelial cell line (BEAS-2B cell lines) showed that at low HBCD concentrations, there was an increase in cell proliferation compared to controls. On the other hand, cell viability decreased significantly after exposure to this compound. There was also an increase in levels of the intercellular adhesion molecule (ICAM-1), and the cytokines, IL-6, and IL-8. Epidermal growth factor (EGF) production was also significantly increased. After 15 min of exposure, EGF receptor phosphorylation was increased, but the expression of total EGF receptor was not affected. The EGF receptor inhibitor, AG1478, blocked the increase in IL-6 and IL-8 induced by HBCD. Regarding transcriptional nuclear factors, HBCD caused increased activation of nuclear factor-nb (NFκB) p50, p65, and AP-1 c-Jun, but not in signal transducers and activators of transcription (Stat3). These results demonstrated that HBCD has effects on the expression of proinflammatory proteins and on cell viability and proliferation .In the same year, Van den Dungen and some other scientists wanted to study the effects of HBCD related to steroid hormones on H295R cells of adrenocortical carcinoma. After exposure, there was no change in cell viability. HBCD had no significant effects on hormone levels. These findings are in line with what other scientists have previously published, where HBCD has no significant effects on steroidogenesis . An et a−7, 10−6, and 10−5 M. The studies were conducted using three HBCD diastereoisomers and the authors observed that all the HBCD amylase had toxic effects on both cell lines, inhibiting cell viability, and causing effects on the redox state and DNA damage. The stability of the redox status of HepG2 cells performs an important role in inducing cell toxicity, so the change caused by HBCD would cause serious damage. In conclusion, the authors show that HBCD metabolic rate can influence the cytotoxic effects, and more studies are needed to better describe how the metabolism of this compound can be so harmful [In 2016, Almughamsi et al. performed studies referring to the changes in the secretion of interferon-gamma (IFN-γ) of human immune cells after exposure to HBCD. After the preparation of peripheral blood mononuclear cell cultures (PBMCs), human monocyte-depleted (MD), and natural killer cells (NK), and their exposure to HBCD, the authors noticed that there was no change in cell viability in any of the cell lines. On the other hand, HBCD caused increased secretion of IFN-γ in NK cells, and in MD-PMBCs and PBMCs. This compound also activated mitogen-activated protein kinases (MAPKs), more precisely the p44/42 pathway, known to regulate the secretion of IFN-γ. In this sense, the authors demonstrated that HBCD has harmful effects on IFN-γ secretion, which can cause inappropriate inflammation . Huang e harmful .In 2016, Erratico et al. described the metabolism of HBCD diastereoisomers using pooled human liver microsomes and CYP3A4. After incubation of microsomes with HBCD, they observed a varied formation of hydroxylated metabolites. In addition, they noticed that only CYP2B6 and CYP3A4 formed HBCD metabolites in findable quantities, with CYP3A4 being responsible for the formation of more than 80% of metabolites. Thus, the authors were able to conclude that HBCD diastereoisomers form specific and different metabolites, which may be useful for human studies, further suggesting that CYP3A4 performs a very important role in the metabolism of these diastereoisomers . KrivoshAdditionally, in 2016, Kim et al. describe the influence of HBCD on cell growth regulation, apoptosis and migration in LNCaP prostate cancer cells. The results led to an increase in cell viability after exposure, and an increase in cell proliferation. HBCD has been shown to decrease bax levels, but did not alter bcl-2 levels, indicating that HBCD has apoptotic potential in this cell line. Cell migration capacity was also increased, suggesting that HBCD promotes cell proliferation and migration. Thus, these results together demonstrate the potential of HBCD in the progression of prostate cancer . AnisuzzMoreover, in the same year, Canbaz et al. described the power of HBCD in activating human monocyte-derived dendritic cells. The results showed that exposure to HBCD led to the activation and production of cytokine, increasing the expression of human leukocyte antigen—DR isotype (HLA-DR), CD86, and IL-8. HBCD led to the maturation and activation of dendritic cells. Despite these findings, the authors also reported the need to conduct more studies to discover the contribution of HBCD in the development of immune allergic responses .In 2017, Li et al. described also in vitro studies of the genotoxicity of HBCD in cultured human breast cells through DNA damage. Using HBL-100 cells, the authors found that, with the increase in HBCD concentration, the rate of cell proliferation decreased, and cell viability. ROS production increased at high HBCD concentrations and lactate dehydrogenase (LDH) increased by a concentration-dependent trend, indicating that HBCD produced cytotoxic damage to cells damaging their membranes. The breast cancer gene 1 (BRCA1) expression was also promoted with HBCD increase, which showed a prognostic of breast cancer in the cell line. They were then able to conclude that HBCD induced oxidative stress by two-ways: the first by breaking DNA by ROS, and the second through cells with unrepaired DNA, which can lead to cell death. Despite these findings, the mechanisms of cell death and DNA repair cession need to further studies . In the In 2019, Xie et al. conducted an in vitro study on human preadipocytes from visceral adipose (HPA-V) to describe the promotion of adipogenesis after exposure to HBCD. The results showed that exposure to HBCD did not alter cell viability, but promoted the formation of lipid droplets, still increasing the lipid content. HBCD also elevated adipogenic genes, but without statistical significance. All these data together demonstrated that HBCD could promote adipogenesis in human preadipocytes . Shi et Additionally, in the same year, the impact of HBCD on lipid metabolism was studied. For this purpose, they used the murine cell line 3T3-L1 and the human cell line HepG2. The 3T3-L1 cell lines showed no changes in proliferation, but after exposure to HBCD showed a decrease in lipid accumulation. In HepG2, lipid accumulation increased. These results suggest that HBCD may have a major impact on lipid accumulation in hepatocytes .Recently, in 2022, Sousa et al. also performed an in vitro study and described the effect of HBCD on MCF-7 cells through the impact on the vitamin D pathway. The cell migration was decreased after exposure. After 4 weeks of exposure to HBCD, cells demonstrated a different response to calcitriol, associated with possible DNA damage. These results show that the response of the cell cycle to vitamin D changed, and may indicate DNA damage performed by HBCD, and further studies are needed to clarify the mechanisms of action .In 2019, Orgono et al. described, through an epidemiological study, exposure to HBCD and the risk of type 2 diabetes (T2D) in the French E3N cohort. The data showed that dietary exposure to HBCD was linearly associated with an increased risk of T2D, and the main food groups that contributed to greater exposure to HBCD were non-processed white meat and processed meat. This is the first epidemiological study to find a linear relationship between this compound and the risk of developing T2D, demonstrating the importance of better investigating the effects and risks of this compound for human health, and further studies are needed to confirm this discovery .−5 g/Kg, which were much higher than other Chinese cities, suggesting that Beijing has a very rich HBCD pollution. The authors also showed that HBCD values may continue to rise in human milk and in biotic and abiotic matrices [In the same year, Huang et al. also performed an epidemiological study in the population of Beijing, China, to detect the HBCD values in breast milk. The goal was also to discover the levels of contamination, temporal trends, and risk assessment. HBCD has been found in almost all the samples, with levels between the limit of detection (set at zero) and 3.63 × 10matrices .In 2021, Zainab et al. wanted to describe the role of HBCD in the progression of breast cancer and the identification of alpha estrogen receptor inhibitors through in silico studies. The results showed that HBCD may be involved in the prevailing of breast cancer and may be one of the major contributors to its appearance given its interaction with the Erα protein. Thus, the authors conclude that for these results to be validated, in vitro and in vivo studies will be necessary .In summary, the data present in this review demonstrate that this compound has harmful effects on human health . This flIn conclusion, HBCD and its substitutes can affect the entire environment in different ways. Despite the various restrictions already imposed on these compounds around the world, their prevalence in the food chain eventually affects plants, animals, and human systems. These compounds can be inherited and are accumulated in blood, urine, and breast milk. Several studies have found that these EDCs can affect several systems, such as the neuronal, cardiovascular, or endocrine system, and/or liver volatomics, by causing DNA damage, altered proteins, cytokine production, and inducing chronic inflammation. Currently, there is still only one epidemiological study with a positive correlation between high levels of HBCD and the risk of Type 2 diabetes. Despite this, to ascertain the pathways that HBCD substitutes use to cause toxicity, there is still a need for further epidemiological studies, in vivo, in vitro, and in silico. Thus, it is urgent to carry out more studies to evaluate the effect of these compounds on human health and the ecosystem, to prevent human pathologies and possible treatments for the most adverse effects of HBCD.


Pasteurellamultocida, Mycoplasmabovis, Mannheimia haemolytica, and Histophilus somni and the virus Bovine herpesvirus-1 (BHV-1), Bovine parainfluenza-3 virus (BPIV-3), Bovine respiratory syncytial virus (BRSV), Bovine adenovirus 3 (BAdV3), bovine coronavirus (BoCV) and Bovine viral diarrhea virus (BVDV) have commonly been identified in BRD cattle; however, no studies have investigated the fungal community and how it may also relate to BRD.Effective identification and treatment of bovine respiratory disease (BRD) is an ongoing health and economic issue for the dairy and beef cattle industries. Bacteria pathogens The objective of this study was to understand if the nasal mycobiome differs between a BRD-affected (n = 56) and visually healthy (n = 73) Holstein steers. Fungal nasal community was determined by using Internal Transcribed Spacer (ITS) sequencing.Trichosporon and Issatchenkia, were the most abundant among all animals, regardless of health status. We identified differences between healthy and BRD animals in abundance of Trichosporon and Issatchenkia orientalis at a sub-species level that could be a potential indicator of BRD. No differences were observed in the nasal fungal alpha and beta diversity between BRD and healthy animals. However, the fungal community structure was affected based on season, specifically when comparing samples collected in the summer to the winter season. We then performed a random forest model, based on the fungal community and abundance of the BRD-pathobionts , to classify healthy and BRD animals and determine the agreement with visual diagnosis. Classification of BRD or healthy animals using ITS sequencing was low and agreed with the visual diagnosis with an accuracy of 51.9%. A portion of the ITS-predicted BRD animals were not predicted based on the abundance of BRD pathobionts. Lastly, fungal and bacterial co-occurrence were more common in BRD animals than healthy animals.The phyla, Ascomycota and Basidiomycota, and the genera, The results from this novel study provide a baseline understanding of the fungal diversity and composition in the nasal cavity of BRD and healthy animals, upon which future interaction studies, including other nasal microbiome members to further understand and accurately diagnose BRD, can be designed. P. multocida, H. somni, M. haemolytica and M. bovis), viruses bovine herpesvirus-1 (BHV-1), bovine parainfluenza-3 virus (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine adenovirus 3 (BAdV3), bovine coronavirus (BoCV) and bovine viral diarrhea virus (BVDV) (Bovine respiratory disease (BRD) is a respiratory disease in which microbes infect the lung of dairy and beef cattle lungs causing morbidity and mortality , 2. Anims (BVDV) .M. haemolytica, P. multocida, H. somni, and M. bovis in the nasal cavity of healthy and BRD animals  and BRD-pathobiont abundance (model accuracy of 66%). These results indicate the potential of utilizing the nasal microbiome composition to classify healthy and BRD animals.In the cattle industry, detecting respiratory disease in cattle is often based on the observing animal behavior  and appearance  . Differe animals . We showCandida species have been related with mastitis, abortion, otitis extrema, gastrointestinal infection, disseminated candidiasis, bronchopneumonia in cattle , Bovine parainfluenza-3 virus (BPIV-3), Bovine respiratory syncytial virus (BRSV), Bovine adenovirus 3 (BAdV3), bovine coronavirus (BoCV) and Bovine viral diarrhea virus (BVDV) . These vn cattle . In addiabortion , 25.Candida krusei was determined to be the cause of a bronchopneumonia case in a heifer; fungi isolated from the patient nasal discharge . A total of 133 Holstein steers approximately 6–7 months old, housed in the same environment were sampled from July to December 2020 at a feedlot . Each animal was sampled once throughout the study.Animals with BRD clinical signs were identified following the DART method that uses depression, appetite loss, respiratory character change and rectal temperature  as cliniAfter identifying the healthy and BRD animals, two nasal swabs were collected from each animal. From the nasal swabs, the total DNA was extracted using the DNeasy Blood & Tissue Kit  following the protocol described in Holman et al. . All theThe raw sequence data obtained from the ITS gene amplification were analyzed using Quantitative Insight Into Microbial Ecology (QIIME2) v.2020.2. Using DADA2 (v. 2022.11.1) , the forp < 0.05 were selected as differentially abundant ASV between BRD and healthy animals. In addition, based on the DESEq results, we were able to observe specific Trichosporon and Issatchenkia orientalis ASVs that were increase on both BRD and healthy group. Therefore, we then determined if the Issatchenkia and Trischosporon enriched in either in the BRD or healthy group formed subspecies clades. To achieve this, all amplicon sequences assigned to Issatchenkia and Trischosporon, as well as three reference sequences identified as Issatchenkia or Trichosporon were obtained from NCBI, were selected to create a phylogenetic tree. Using BLAST, we found that our sequences matched more closely to Pichia kudriavzevii sequences. Therefore, for the tree, we selected the ITS region of Pichia kudriavzevii ATCC 6258  with weighted sum-of-pairs (WSP) and consistency score with 1,000 iterations  were selected as the outgroup and for Trichosporon, the genera Vanrija  were selected.DESeq  was usedTCC 6258 , Pichia late 3Y3 , and Picain VN9Y . For Tri ovoides , 42, Trie mYJh51 , and Trisp. Bio4  were selerations . Two ASVThe raw ITS gene sequencing data from the 10 known fungal strains  and PCR grade water were used as a PCR negative control were analyzed separately using QIIME v.2020.2. as described above. Using DADA2 , the forp < 0.05. We square-transformed dependent variables when assumptions were not met. Only the dependent variables Observed ASV and Chao1 were square-transformed. The F values were reported with their corresponding degrees of freedom associated with the model. In the case of Faith’s PD and Pielou, these dependent variables did not meet normality assumptions after transformation . Thus, a non-parametric test, Mann–Whitney’s test, using the ‘wilcox.test’ function in R  were analyzed with a Generalized Linear Mixed Model with a random factor structure specified to only include random slopes using the afex package . In the ion in R , 49 was n = 24 and healthy, n = 30), September–October , and November–December  and season. Group was included as an independent factor in the model. In addition, a dispersion test, using as input the distance matrices determined by Bray–Curtis dissimilarity and Weighted UniFrac, was performed to analyze the average distance of the samples from the two groups  using the function ‘betadisper’ from the vegan package  between BRD and healthy animals as well as based on the date of sampling, a permutational multivariate analysis and variance test (PERMANOVA) of the Bray–Curtis dissimilarity and Weighted UniFrac distances was performed using the ‘adonis’ function from the vegan package . To dete package  followedn = 79, containing 33 BRD samples and 46 Healthy samples) and testing set . The RF model was built from the training set using 17 trees; with this number, it was possible to achieve the lowest error rate  using the package randomForest in R v.40.0.3 was performed using the unrarefied fungal ASV table . As a first step, the fungal ASV table was filtered to only include ASVs with an abundance >0.0001 and rarified to 40,420 reads per sample. After applying the filtering step, a total of 1,109 ASVs were used to build the RF model. Following this step, the samples were divided into training , present in more than 60% of the samples  and with a probability >0.75 that the two ASVs  will co-occur in the same sample. In the case of the negative associations, it was required that the ASV pairs be significant (p < 0.05) with a probability of <0.05 that the two ASVs  would co-occur in the same sample.A co-occurrence analysis was performed to identify fungal and bacterial ASVs that co-occur in the same sample. In this analysis, it was possible to identify fungal and bacteria ASVs that coexist in the same sample (positive associations) or do not co-exist (negative associations). Both ASV tables, fungal  and bacterial [, 18,010 H. somni, P. multocida, M. haemolytica, and M. bovis) were selected with a significance of p < 0.05. This allowed the identification of fungi associated with BRD-pathobionts in the nasal cavity of healthy and BRD animals.In addition, any positive fungal ASV pair combination with BRD-pathobionts (Cyberlindnera jadinni and Oidiodendron truncatum (>50% of community composition) whereas Water2, was composed of Fusarium sporotrichioides  in the fungal community used in the study. After comparing the fungal mock community with the 10 known fungal strains DNA, 9 out of 10 fungal strains were identified. In the case of the PCR negative control (water), a total of 105,882 sequences were identified and classified into 21 ASVs. Interestingly, both water samples presented a different fungal composition. Water1 was mostly composed of n = 73). From these results, the fungal phyla with the higher relative abundance were Ascomycota (~54.36%), and Basidiomycota (38.81%), followed by unclassified fungi (3.71%). No other phyla had a relative abundance >1% (Trichosporon (25.93%) and Issatchenkia (14.15%), followed by unidentified fungi (7.14%) and Alternaria (5.26%). Any other fungal genus identified had a relative abundance of <5%  and 6,092,893 after denoising from healthy and BRD cattle nasal swabs. A total of 7,050 fungal ASVs from 11 different phyla were observed in the nasal cavities of healthy animals (ance >1% . At the e of <5% .n = 56). A total of 11 phyla fungal were identified in the study, from which the phyla Ascomycota (58.34%), and Basidiomycota (36.49%) followed by unidentified fungi (4.72%) were the most abundant in BRD animal swabs. Any other phylum group observed in the study had a relative abundance <1% (Issatchenkia (19.59%), and Trichosporon (18.88%) were the most abundant followed by unidentified fungi (11.0%) and Alternaria (4.50%). All other genera had an average relative abundance of <4% . Meanwhile, in the healthy group, Trichosporon-ASV1 and Issatchenkia orientalis-ASV27 had the highest log2 fold change . A total of 12 ASVs classified as Issatchenkia orientalis, 6 classified as Trichosporon, and 2 classified as Apiotrichum, were significantly increased in one of the groups. No other genera had ASVs that were significantly increased or decreased in both groups. Differentially abundant fungi in the healthy group were assigned to more genera than those in the BRD group. Of the 25 differentially abundant fungal ASVs in the healthy group, 12 were not classified as Issatchenkia or Trichosporon: Mortierella indohii, Hydropisphaera fungicola, unclassified fungi, Pseudopithomyces rosae, two ASVs classified as Capnodiales, Alternaria metachromatica, Leucosporidium creatinivorum, Oidiodendron truncatum, unclassified Cladosporiaceae, Aspergillus sydowii, and Wicherhamomyces anomalus. On the other hand, only 2 out of 9 differentially abundant fungi in the BRD group were not classified as Issatchenkia or Trichosporon: Mycosphaerella tassiana and Fusarium sporotrichioides , which all had a high abundance in the healthy group .In this study, the fungal richness estimated by Observed ASVs and Chao1, phylogenetic diversity by Faith’s PD, and fungal community evenness by Pielou were not different between BRD and healthy animals nor by sample collection date . Nasal swabs were grouped by date of sampling from July to December 2020 , September–October , and November–December . This grouping allowed us to identify if season affects the nasal fungal community structure. Interestingly, the pairwise testing between the three seasonal groups demonstrated that the fungal community was different between the July–August and November–December groups . Additionally, no difference was observed in the fungal nasal community as predicted by Weighted UniFrac based on the season or between the two groups. In addition, no difference in the dispersion of the samples as predicted by Bray–Curtis dissimilarity and Weighted UniFrac was observed between the season group centroids and between BRD animals and the healthy group (p > 0.05).The nasal fungal community structure or distance between the BRD and healthy animals (beta diversity) as determined by Bray–Curtis dissimilarity  were utilized to train the random forest model and 40% of the samples (n = 54) were used for the testing set. In the testing set, 29 animals were identified as healthy based on the BRD clinical signs and 25 as BRD. Once the RF analysis using the fungal community data was applied to the testing set, 12 animals were correctly predicted as BRD and 15 as healthy animals with an accuracy of 51.9%, a sensitivity of 48%, and a specificity of 55% (Random forest (RF) analysis was used to predict animal health status based on (1) nasal fungal community and (2) abundance of BRD-pathobionts. To create the model, 60% of the samples (y of 55% . When thy of 55% .Mycophaerella (ASV14), Kluyveromyces (ASV6), Alternaria (ASV2), and at the family level Cladosporiaceae (ASV4) were positively associated with bacteria in both groups. On the other hand, a total of 733 negative associations were observed in the BRD group and 1,543 in the healthy group. In the BRD group, one fungal ASV presented the most negative associations (>30 negative co-occurrences) with bacteria: Rhodotorula kratochvilovae. In the case of the healthy group, 7 fungal species presented the most negative associations with bacteria ASVs (>30 co-occurrence): Ophiosphaerella aquatica, Curvibasidium cyaneicollum, Aspergillus caesiellus, Penicillium melanoconidium, Wickerhamomyces anomalus, Leucosporidium creatinivorum, Cyverlindnera culbertsonii, and Cyberlindnera rhodanensis. Additionally, we sought to identify fungal ASVs that were associated with BRD-pathogens: Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni in the nasal cavity of BRD and healthy animals. However, no significant positive co-occurrences between fungi and BRD-pathobionts were identified (p > 0.05).A total of 36 positive co-occurrence associations were observed in the BRD group , and 11 Pasteurella multocida, Histophilus somni, Mannheimia haemolytica, and Mycoplasma bovis are strongly related with BRD , but their relative abundance in the nasal cavity of healthy animals was lower than 1% of the community. Interestingly, the genus Trichosporon has been linked to mucosal or cutaneous infections in the immunocompromised host  in the BRD animals but reflected a relative abundance of 1%. After comparing the fungal genus profile between the BRD and healthy animals, it was surprising to detect that the Issatcheknia genus was more abundant in the BRD animals and the Trichosporon genus were more numerically abundant in the healthy animals. From these results, we expect to set a starting point in understanding and identifying the “commensal” respiratory mycobiome composition specifically in cattle, and how it can be disturbed when disease occurs. In addition, it was observed that the succession in the healthy mycobiome seems more predictable than in BRD animals.In our study, we observed that the nasal fungal community in BRD animals had a similar composition at the phylum level as the healthy animals, with database  , there are serotypes or subgroups with more pathogenic capacity than other serotypes. Studies revealed that M. haemolytica serotypes A1 and A6 are related to bovine pneumonic pasteurellosis . In previous studies, it was identified that BRD animals had a significant increase in the relative abundance of the bacterial member elopment , 69, 70.l cavity . These rd cattle , 65, 66.rellosis , 72 and RD cases , 74. TheFusarium sporotrichioides was increased in the BRD group, while Mortierella indohii and Hydropisphaera fungicola were decreased in the BRD group. Fusarium species including F. sporotrichioides, which was increased in the BRD group, are considered toxic fungi due to the capacity of producing mycotoxins in animal feedstuffs  compared to those collected in Winter (November–December), regardless of the disease status. It has been observed that meteorological parameters, such as differences in season mediated by temperature change, can influence the abundance of fungal aerosols . Interesvia secreted molecules  and bacterial species has been used as a means to understand their interactions in the soil environment  database under Bioproject PRJNA906497, BioSamples SAMN31926246 - SAMN31926380.The original contributions presented in the study are publicly available. All the data and fungal statistical analysis were performed in R v.4.0.3 and the codes are available at The animal study was reviewed and approved by Purdue University Animal Care and Use Committee (protocol #1906001911). Written informed consent was obtained from the owners for the participation of their animals in this study.REC-M, MSV, and TAJ conceived the project. REC-M, SM, JD, JPS, and AA the acquired the nasal swab samples, REC-M analyzed the samples. REC-M and TAJ interpreted the data. REC-M, MSV, and TAJ wrote or substantively revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Agriculture and Food Research Initiative Competitive Grants Program Award 2020-68014-31302 from the U.S. Department of Agriculture National Institute of Food and Agriculture. This work is also supported by Purdue University’s Colleges of Agriculture and Engineering Collaborative Projects Program 2018, and an Agricultural Science and Extension for Economic Development (AgSEED) grant. The funding organizations played no role in the design of the study: collection, analysis, and interpretation of data, or in writing the manuscript.MSV has interests in Krishi, Inc., a company interested commercializing on farm diagnostics technology. The work performed here was not funded by Krishi, Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.