Diagnostic Delay in Hidradenitis Suppurativa: Still an Unsolved Problem.

Introduction: Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a diagnostic delay of 7-10 years. A later diagnosis is associated with development of local sequelae and systemic comorbidities, as well as a reduced response to medical treatment. The aim of this study was to analyze the time required for HS diagnosis and investigate factors associated with diagnostic delay. Method: A retrospective observational study was conducted based on clinical records from HS patients followed at a tertiary hospital, with diagnosis between January 2006 and December 2022. Results: A total of 285 patients were included. The mean diagnostic delay was 10.1 years, and there was no difference in time to diagnosis during the considered period. A diagnostic delay of more than 5 years was significantly associated with an earlier onset of symptoms, location on breasts and thighs, follicular phenotype, and with cardiovascular and psychiatric comorbidities. Smoking and body mass index ≥25 kg/m2 were also associated with a longer diagnostic delay. A personal history of acne and a greater disease severity were associated with an earlier diagnosis. Conclusions: This study reveals the lack of improvement in the diagnostic delay in HS and highlights its association with atypical clinical manifestations and systemic comorbidities, scarcely reported in literature.

Tuberculosis: Cliché or Outsider?

Tuberculosis is an infectious disease with the potential for multisystemic dissemination, including the central nervous system (CNS). It is difficult to diagnose when the central nervous system is involved. Brain biopsy is the diagnostic method par excellence for diagnostic confirmation; however, as it is an invasive method and therefore not free from risks, before carrying it out, extra-CNS sites should be privileged, whenever available, through mycobacteriological culture. Here, we present a case of a 34-year-old female with chronic onset of neurologic semiology, whose diagnostic evolution culminated in the diagnosis of cerebral tuberculomas and miliary tuberculosis. Rapid commencement of antibacillaty therapy led to the resolution of the neurologic deficits. Although we face a cliché clinical presentation, in the sense that is very common, the authors consider it outsider because such a presentation is rarely seen in Portugal.

Extracellular vesicle-based delivery of silencing sequences for the treatment of Machado-Joseph disease/spinocerebellar ataxia type 3.

Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.

[Guidelines for the Management of Patients with Hidradenitis Suppurativa]. / Recomendações na Abordagem do Doente com Hidradenite Supurativa.

Hidradenitis suppurativa is a chronic and recurrent inflammatory dermatosis characterized by the presence of inflammatory nodules and abscesses in the apocrine gland-rich areas that may progress to suppurative fistulas and scars. Despite being considered one of the dermatological conditions with the greatest impact on patient quality of life, it is often underdiagnosed. Hidradenitis suppurativa, especially in its severe forms, is associated with numerous comorbidities, so a holistic and multidisciplinary perspective is crucial for the management of these patients. The therapeutic approach is complex and challenging. The medical treatment options are diverse and must be adapted to clinical presentation and disease severity. Surgical therapy should be considered as an adjuvant to medical treatment, particularly in refractory cases and in the presence of scars or anatomical and/or functional mutilation. These recommendations reflect the main aspects of the management of the patient with hidradenitis suppurativa and are addressed to all healthcare professionals who take part in their follow-up.

A hidradenite supurativa é uma dermatose inflamatória crónica e recorrente que se caracteriza pela presença de nódulos inflamatórios e abcessos nas áreas ricas em glândulas apócrinas, que podem evoluir para fístulas supurativas e cicatrizes. Apesar de ser considerada uma das patologias dermatológicas com maior impacto na qualidade de vida dos doentes, é frequentemente subdiagnosticada. A hidradenite supurativa, sobretudo nas suas formas mais graves, associa-se a diversas comorbilidades, pelo que é fundamental adotar uma perspetiva holística e multidisciplinar na gestão destes doentes. A abordagem terapêutica é complexa e desafiante. A terapêutica médica é multifacetada e deve ser adaptada à apresentação clínica e gravidade da doença. A terapêutica cirúrgica deverá ser equacionada como adjuvante à terapêutica médica, em particular nos casos refratários e perante cicatrizes ou mutilação anatómica e/ou funcional. As presentes recomendações pretendem reunir os principais aspetos da abordagem ao doente com hidradenite supurativa e destinam-se a todos os profissionais de saúde envolvidos no seu acompanhamento.

Fast calculation of spectral optical properties and pigment content detection in human normal and pathological kidney.

A fast calculation method was used to obtain the spectral optical properties of human normal and pathological (chromophobe renal cell carcinoma) kidney tissues. Using total transmittance, total reflectance and collimated transmittance spectra acquired from ex vivo kidney samples, the spectral optical properties of both tissues, namely the absorption, the scattering and the reduced scattering coefficients, as well as the scattering anisotropy, dispersion and light penetration depth, were calculated between 200 and 1000 nm. Analysis of the mean absorption coefficient spectra of the kidney tissues showed that both contain melanin and lipofuscin, and that 83 % of the melanin in the normal kidney converts into lipofuscin in the pathological kidney.

ULK overexpression mitigates motor deficits and neuropathology in mouse models of Machado-Joseph disease.

Machado-Joseph disease (MJD) is a fatal neurodegenerative disorder clinically characterized by prominent ataxia. It is caused by an expansion of a CAG trinucleotide in ATXN3, translating into an expanded polyglutamine (polyQ) tract in the ATXN3 protein, that becomes prone to misfolding and aggregation. The pathogenesis of the disease has been associated with the dysfunction of several cellular mechanisms, including autophagy and transcription regulation. In this study, we investigated the transcriptional modifications of the autophagy pathway in models of MJD and assessed whether modulating the levels of the affected autophagy-associated transcripts (AATs) would alleviate MJD-associated pathology. Our results show that autophagy is impaired at the transcriptional level in MJD, affecting multiple AATs, including Unc-51 like autophagy activating kinase 1 and 2 (ULK1 and ULK2), two homologs involved in autophagy induction. Reinstating ULK1/2 levels by adeno-associated virus (AAV)-mediated gene transfer significantly improved motor performance while preventing neuropathology in two in vivo models of MJD. Moreover, in vitro studies showed that the observed positive effects may be mainly attributed to ULK1 activity. This study provides strong evidence of the beneficial effect of overexpression of ULK homologs, suggesting these as promising instruments for the treatment of MJD and other neurodegenerative disorders.

Redox-Oligomeric State of Peroxiredoxin-2 and Glyceraldehyde-3-Phosphate Dehydrogenase in Obstructive Sleep Apnea Red Blood Cells under Positive Airway Pressure Therapy.

In this study, we examined the effect of six months of positive airway pressure (PAP) therapy on Obstructive Sleep Apnea (OSA) red blood cell (RBC) proteome by two dimensional difference gel electrophoresis (2D-DIGE) - based proteomics followed by Western blotting (WB) validation. The discovered dysregulated proteins/proteoforms are associated with cell death, H2O2 catabolic/metabolic process, stress response, and protein oligomerization. Validation by nonreducing WB was performed for peroxiredoxin-2 (PRDX2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using antibodies against the sulfinylated/sulfonylated cysteine of these proteins to better evaluate their redox-oligomeric states under OSA and/or in response to PAP therapy. The results indicated that the redox-oligomeric state of GAPDH and PRDX2 involving overoxidation by sulfinic/sulfonic acids were differentially modulated in OSA RBC, which might be compromising RBC homeostasis. PAP therapy by restoring this modulation induced a higher oligomerization of overoxidized GAPDH and PRDX2 in some patients that could be associated with eryptosis and the chaperone "gain" of function, respectively. This varied response following PAP may result from the complex interplay between OSA and OSA metabolic comorbidity. Hence, information on the redox status of PRDX2 and GAPDH in RBC will help to better recognize OSA subtypes and predict the therapeutic response in these patients. GAPDH monomer combined with body mass index (BMI) and PRDX2 S-S dimer combined with homeostatic model assessment for insulin resistance (HOMA-IR) showed to be very promising biomarkers to predict OSA and OSA severity, respectively.

Antisense oligonucleotide therapeutics in neurodegenerative diseases: the case of polyglutamine disorders.

Polyglutamine (polyQ) disorders are a group of nine neurodegenerative diseases that share a common genetic cause, which is an expansion of CAG repeats in the coding region of the causative genes that are otherwise unrelated. The trinucleotide expansion encodes for an expanded polyQ tract in the respective proteins, resulting in toxic gain-of-function and eventually in neurodegeneration. Currently, no disease-modifying therapies are available for this group of disorders. Nevertheless, given their monogenic nature, polyQ disorders are ideal candidates for therapies that target specifically the gene transcripts. Antisense oligonucleotides (ASOs) have been under intense investigation over recent years as gene silencing tools. ASOs are small synthetic single-stranded chains of nucleic acids that target specific RNA transcripts through several mechanisms. ASOs can reduce the levels of mutant proteins by breaking down the targeted transcript, inhibit mRNA translation or alter the maturation of the pre-mRNA via splicing correction. Over the years, chemical optimization of ASO molecules has allowed significant improvement of their pharmacological properties, which has in turn made this class of therapeutics a very promising strategy to treat a variety of neurodegenerative diseases. Indeed, preclinical and clinical strategies have been developed in recent years for some polyQ disorders using ASO therapeutics. The success of ASOs in several animal models, as well as encouraging results in the clinic for Huntington's disease, points towards a promising future regarding the application of ASO-based therapies for polyQ disorders in humans, offering new opportunities to address unmet medical needs for this class of disorders. This review aims to present a brief overview of key chemical modifications, mechanisms of action and routes of administration that have been described for ASO-based therapies. Moreover, it presents a review of the most recent and relevant preclinical and clinical trials that have tested ASO therapeutics in polyQ disorders.

Serum vitamin D levels correlate with the presence and histological grading of colorectal adenomas in peri and postmenopausal women.

BACKGROUND & AIMS: Vitamin D is known to modulate immune function and proliferation. Higher vitamin D [25(OH)D3] serum levels have been reported to have protective effects on adenoma detection and colorectal cancer (CRC) development and survival. METHODS: This retrospective cohort study included 315 peri and post-menopausal women submitted to opportunistic colorectal and osteoporosis screening at the gynaecology outpatient clinic of a tertiary medical centre between 2004 and 2015. Colonoscopy findings were correlated with 25(OH)D3 and PTH serum levels, and subsequently adjusted in a multivariate logistic regression model. Confounding factors included demographic and colorectal risk factors, pharmacological therapies and bone densitometry metrics. RESULTS: A total of 77 lesions were identified in 66 patients. Vitamin D insufficiency (<30 ng/mL) and deficiency (<20 ng/mL) were identified in 79.4% and 35.2% of patients, respectively. In univariate analysis, lower levels of 25(OH)D3 were associated with polyp, adenoma and advanced adenoma detection. After adjusting for confounders, an association with polyps could not be observed, but a trend towards a negative correlation with adenoma detection was found (adjusted OR: 0.96; 95% CI 0.92-1.00; p = 0.083). Regarding advanced adenoma detection, 25(OH)D3 (adjusted OR: 0.86; 95% CI 0.77-0.97; p = 0.013) proved to be an independent predictive factor. No association was found between 25(OH)D3 levels and lesion detection site. CONCLUSION: The association of 25(OH)D3 serum levels with colorectal lesions seems to be restricted to adenomatous lesions and is influenced by histological grading. Vitamin D may be a valuable biomarker for optimization of risk stratification in group-specific CRC screening protocols.

The first-line antiepileptic drug carbamazepine: Reaction with biologically relevant free radicals.

Carbamazepine (CBZ) is one of the most widely used antiepileptic drugs by both adults and children. Despite its widespread use, CBZ is associated with central nervous system toxicity and severe hypersensitivity reactions, which raise concerns about its chronic use. While the precise mechanisms of CBZ-induced adverse events are still unclear, metabolic activation to the epoxide (CBZ-EP) has been thought to play a significant role. This work reports first-hand evidence that CBZ reacts readily with biologically relevant thiyl radicals with no need for bioactivation. Using liquid chromatography coupled with high resolution mass spectrometry, multiple products from direct reaction of CBZ with glutathione (GSH) and N-acetyl-L-cysteine (NAC) were unequivocally identified, including the same product obtained upon ring-opening of CBZ-EP. The product profile is complex and consistent with radical-mediated mechanisms. Importantly, side products and adducts compatible with this non-enzymatic pathway were identified in liver extracts from CBZ-treated Wistar rats. The reaction of CBZ with GSH and NAC is more extensive in the presence of oxygen. Taking into consideration that GSH conjugation is, in general, a detoxification pathway, these results suggest that under hyperoxia/oxidative stress conditions the bioavailability of the parent drug may be compromised. Additionally, this non-enzymatic process can be anticipated to play, at least in part, a role in the onset of CBZ-induced adverse reactions due to the concomitant generation of reactive oxygen species. Therefore, the search for causal relationships between the formation of non-enzymatically-driven CBZ products and the occurrence of CBZ-induced adverse events in human patients merits further research, aiming the translation of basic mechanistic findings into a clinical context that may ultimately lead to a safer CBZ prescription.

High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches.

Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk. Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent. In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy. We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.

Karyopherin α-3 is a key protein in the pathogenesis of spinocerebellar ataxia type 3 controlling the nuclear localization of ataxin-3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by a CAG expansion in the ATXN3 gene leading to a polyglutamine expansion in the ataxin-3 protein. The nuclear presence and aggregation of expanded ataxin-3 are critical steps in disease pathogenesis. To identify novel therapeutic targets, we investigated the nucleocytoplasmic transport system by screening a collection of importins and exportins that potentially modulate this nuclear localization. Using cell, Drosophila, and mouse models, we focused on three transport proteins, namely, CRM1, IPO13, KPNA3, and their respective Drosophila orthologs Emb, Cdm, and Kap-α3. While overexpression of CRM1/Emb demonstrated positive effects in Drosophila, KPNA3/Kap-α3 emerged as the most promising target, as knockdown via multiple RNAi lines demonstrated its ability to shuttle both truncated and full-length expanded ataxin-3, rescue neurodegeneration, restore photoreceptor formation, and reduce aggregation. Furthermore, KPNA3 knockout in SCA3 mice resulted in an amelioration of molecular and behavioral disturbances such as total activity, anxiety, and gait. Since KPNA3 is known to function as an import protein and recognize nuclear localization signals (NLSs), this work unites ataxin-3 structure to the nuclear pore machinery and provides a link between karyopherins, NLS signals, and polyglutamine disease, as well as demonstrates that KPNA3 is a key player in the pathogenesis of SCA3.

Mueller-Hillis maneuver and angle of progression: Are they correlated? / Manobra de Mueller-Hillis e ângulo de progressão: eles estão correlacionados?

Summary Objective: Mueller-Hillis maneuver (MHM) and angle of progression (AOP) measured by transperineal ultrasound have been used to assess fetal head descent during the second stage of labor. We aimed to assess whether AOP correlates with MHM in the second stage of labor. Method: A prospective observational study including women with singleton pregnancy in the second stage of labor was performed. The AOP was measured immediately after the Mueller-Hillis maneuver. A receiver-operating characteristics (ROC) curve analysis was performed to determine the best discriminatory AOP cut-off for the identification of a positive MHM. A p-value less than 0.05 was considered statistically significant. Results: One hundred and sixty-six (166) women were enrolled in the study and 81.3% (n=135) had a positive MHM. The median AOP was 143º (106º to 210º). The area under the curve for the prediction of a positive maneuver was 0.619 (p=0.040). Derived from the ROC curve, an AOP of 138.5º had the best diagnostic performance for the identification of a positive MHM (specificity of 65% and a sensitivity of 67%). Conclusion: An AOP of 138º seems to be associated with a positive MHM in the second stage of labor.

Resumo Objetivo: A manobra de Mueller-Hillis (MHM) e o ângulo de progressão da apresentação (AOP) medido através de ecografia transperineal têm sido utilizados para avaliar a descida do polo cefálico durante o segundo estágio do trabalho de parto. O objetivo do nosso trabalho foi avaliar se o AOP se correlaciona com a MHM no segundo estágio do trabalho de parto. Método: Conduzimos um estudo observacional e prospectivo. Incluímos mulheres com gravidez unifetal com feto em apresentação cefálica, no segundo estágio do trabalho de parto. O AOP foi medido imediatamente após a manobra de Mueller-Hillis. Foi construída uma curva ROC (receiver-operating characteristics) para determinar o melhor AOP para a identificação de uma manobra positiva. Um valor p inferior a 0,05 foi considerado estatisticamente significativo. Resultados: Cento e sessenta e seis mulheres (166) foram incluídas no estudo, e em 81,3% (n=135) a MHM foi positiva. A mediana do AOP foi de 143º (106º a 210º). A área abaixo da curva para a previsão de uma manobra positiva foi 0,619 (p=0,040). Derivado da curva ROC, um AOP de 138,5º teve o melhor desempenho diagnóstico para a identificação de uma MHM positiva (especificidade de 65% e sensibilidade de 67%). Conclusão: Um AOP de 138º parece estar associado com uma MHM positiva no segundo estágio de trabalho de parto.

Hemolysis, Elevated Liver Enzymes, Low Platelets Syndrome Superimposed on Hemolytic Uremic Syndrome / Síndrome HELLP sobreposta a síndrome hemolítica-urêmica

Abstract A pregnancy complicated by typical hemolytic uremic syndrome (HUS) and hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is reported. At 20 weeks of gestation, a case of HUS was diagnosed, with Shiga toxin-producing Escherichia coli identified. Plasmapheresis allowed continuation of the pregnancy for 5 weeks. Superimposed preeclampsia and HELLP syndrome were diagnosed after the establishment of nephrotic range proteinuria, hypertension and recurrence of hemolysis. This is a singular case, as it demonstrates that HELLP syndrome can superimpose upon HUS, a fact that can impact future research on reproductive immunology. It also reminds clinicians that the overlapping of clinical and laboratory findings in HELLP syndrome makes the diagnosis of other thrombotic microangiopathies during pregnancy a clinical challenge.

Resumo Descreve-se um caso clínico em que às 20 semanas de gestação é diagnosticada síndrome hemolítica-urêmica com isolamento de Escherichia coli produtora de toxina Shiga. A terapêutica com plasmaferese permitiu a manutenção da gravidez ao longo de 5 semanas. O surgimento de proteinúria nefrótica, hipertensão e recorrência de hemólise conduziu ao diagnóstico de sobreposição de pré-eclâmpsia e síndrome HELLP. Este caso é singular, pois demonstra que a síndrome HELLP pode se sobrepor a uma síndrome hemolítica-urêmica, um fato que pode influenciar futuras investigações no âmbito de imunologia reprodutiva. Reforça-se, ainda, que o diagnóstico de outras microangiopatias trombóticas durante a gestação é um desafio clínico ocasionado pela sobreposição de manifestações clínicas e laboratoriais com a síndrome HELLP.

Hepatocyte spheroids as a competent in vitro system for drug biotransformation studies: nevirapine as a bioactivation case study.

The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.

New insights into the molecular mechanisms of chemical carcinogenesis: In vivo adduction of histone H2B by a reactive metabolite of the chemical carcinogen furan.

Furan is a rodent hepatocarcinogen ubiquitously found in the environment and heat-processed foods. Furan undergoes cytochrome P450 2E1-catalyzed bioactivation to cis-2-butene-1,4-dial (BDA), which has been shown to form an electrophilic conjugate (GSH-BDA) with glutathione. Both BDA and GSH-BDA yield covalent adducts with lysine residues in proteins. Dose- and time-dependent epigenetic histone alterations have been observed in furan-treated rats. While the covalent modification of histones by chemical carcinogens has long been proposed, histone-carcinogen adducts have eluded detection in vivo. In this study, we investigated if the covalent modification of histones by furan may occur in vivo prior to epigenetic histone alterations. Using a "bottom-up" methodology, involving the analysis of tryptic peptides by liquid chromatography - high resolution mass spectrometry, we obtained evidence for a cross-link between GSH-BDA and lysine 107 of histone H2B isolated from the livers of male F344 rats treated with tumorigenic doses of furan. This cross-link was detected at the shortest treatment period (90 days) in the lowest dose group (0.92mg/kg body weight/day), prior to the identification of epigenetic changes, and occurred at a lysine residue that is a target for epigenetic modifications and crucial for nucleosome stability. Our results represent the first unequivocal proof of the occurrence of carcinogen-modified histones in vivo and suggest that such modification happens at the initial stages of furan-induced carcinogenesis. This type of alteration may be general in scope, opening new insights into the mechanisms of chemical carcinogenesis/toxicity and new opportunities for the development of early compound-specific biomarkers of exposure.