Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity

Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

An illness-focused interactive booklet to optimise management and medication for childhood fever and infections in out-of-hours primary care: study protocol for a cluster randomised trial

Background Fever is the most common reason for a child to be taken to a general practitioner (GP), especially during out-of-hours care. It is mostly caused by self-limiting infections. However, antibiotic prescription rates remain high, especially during out-of-hours care. Anxiety and lack of knowledge among parents, and perceived pressure to prescribe antibiotics amongst GPs, are important determinants of excessive antibiotic prescriptions. An illness-focused interactive booklet has the potential to improve this by providing parents with information about fever self-management strategies. The aim of this study is to develop and determine the effectiveness of an interactive booklet on management of children presenting with fever at Dutch GP out-of-hours cooperatives. Methods/design We are conducting a cluster randomised controlled trial (RCT) with 20 GP out-of-hours cooperatives randomised to 1 of 2 arms: GP access to the illness-focused interactive booklet or care as usual. GPs working at intervention sites will have access to the booklet, which was developed in a multistage process. It consists of a traffic light system for parents on how to respond to fever-related symptoms, as well as information on natural course of infections, benefits and harms of (antibiotic) medications, self-management strategies and ‘safety net’ instructions. Children < 12 years of age with parent-reported or physician-measured fever are eligible for inclusion. The primary outcome is antibiotic prescribing during the initial consultation. Secondary outcomes are (intention to) (re)consult, antibiotic prescriptions during re-consultations, referrals, parental satisfaction and reassurance. In 6 months, 20,000 children will be recruited to find a difference in antibiotic prescribing rates of 25% in the control group and 19% in the intervention group. Statistical analysis will be performed using descriptive statistics and by fitting two-level (GP out-of-hours cooperative and patient) random intercept logistic regression models. Discussion This will be the first and largest cluster RCT evaluating the effectiveness of an illness-focused interactive booklet during GP out-of-hours consultations with febrile children receiving antibiotic prescriptions. It is hypothesised that use of the booklet will result in a reduced number of antibiotic prescriptions, improved parental satisfaction and reduced intention to re-consult

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

Complaints and complications associated with removal of the mandibular third molar. A prospective clinical study

OBJECTIVE: To study the incidence of complaints and complications after removal of the mandibular third molar and to evaluate the influence of this procedure on functioning of the patient during the first post-surgical week. DESIGN: Prospective, clinical. SETTING: Department of Oral and Maxillofacial Surgery, University Hospital Groningen. METHODS: Patients referred for removal of a mandibular third molar were asked to return one week after the procedure and to keep a daily record of the use of pain medication, duration of the pain and intensity of the pain. RESULTS: Removal of mandibular third molars resulted in an overall complication rate of 12%. Pain medication was used more frequently and for a longer period by patients with post-surgical complications. Due to complaints following the removal of the mandibular third molar, the mean absence from work was one and a half day. Work was generally resumed with decreased perceived efficiency. CONCLUSION: After this commonly performed procedure in dento-alveolar surgery most of the patients were hampered as a result of pain during the first four post-surgical days. Over 10% of the patients developed complications leading to more frequently and prolonged use of pain medication. Removal of the mandibular third molar gave rise to complaints which influenced the patients relatively strong in their daily functioning.</p

High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI

Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα-induced cytotoxicity

Drug-induced liver injury (DILI) is an important problem both in the clinic and in the development of new safer medicines. Two pivotal adaptation and survival responses to adverse drug reactions are oxidative stress and cytokine signaling based on the activation of the transcription factors Nrf2 and NF-κB, respectively. Here, we systematically investigated Nrf2 and NF-κB signaling upon DILI-related drug exposure. Transcriptomics analyses of 90 DILI compounds in primary human hepatocytes revealed that a strong Nrf2 activation is associated with a suppression of endogenous NF-κB activity. These responses were translated into quantitative high-content live-cell imaging of induction of a selective Nrf2 target, GFP-tagged Srxn1, and the altered nuclear translocation dynamics of a subunit of NF-κB, GFP-tagged p65, upon TNFR signaling induced by TNFα using HepG2 cells. Strong activation of GFP-Srxn1 expression by DILI compounds typically correlated with suppression of NF-κB nuclear translocation, yet reversely, activation of NF-κB by TNFα did not affect the Nrf2 response. DILI compounds that provided strong Nrf2 activation, including diclofenac, carbamazepine and ketoconazole, sensitized toward TNFα-mediated cytotoxicity. This was related to an adaptive primary protective response of Nrf2, since loss of Nrf2 enhanced this cytotoxic synergy with TNFα, while KEAP1 downregulation was cytoprotective. These data indicate that both Nrf2 and NF-κB signaling may be pivotal in the regulation of DILI. We propose that the NF-κB-inhibiting effects that coincide with a strong Nrf2 stress response likely sensitize liver cells to pro-apoptotic signaling cascades induced by intrinsic cytotoxic pro-inflammatory cytokines.Toxicolog