Xiaojianzhong decoction attenuates aspirin-induced gastric mucosal injury via the PI3K/AKT/mTOR/ULK1 and AMPK/ULK1 pathways.

CONTEXT: Xiaojianzhong decoction (XJZD), classically prescribed in Chinese medicine, has protective and healing effects on gastric mucosal injury. However, the exact mechanism behind this effect remains unclear. OBJECTIVE: To investigate the effect of XJZD on gastric mucosal injury and explore its underlying mechanisms. MATERIALS AND METHODS: C57BL/6 mice were randomized into six groups (n = 10): the control group receiving sterile water, the model (aspirin 300 mg/kg), the XJZD high-dose (12 g/kg), XJZD medium-dose (6 g/kg), XJZD low-dose (3 g/kg) and omeprazole (20 mg/kg) groups, by gavage daily for 14 days. The area of gastric mucosal injury, mucosal injury index and degree of histopathological damage were analysed. Gastric mucosal epithelial cell apoptosis was detected. Epithelial cell autophagy was observed. The expression levels of tight junction proteins and proteins related to apoptosis, autophagy and the pentose phosphate pathway were analysed. RESULTS: The results showed that after treatment with XJZD (12, 6 and 3 g/kg), the mucosal injury area was reduced (83.4%, 22.6% and 11.3%), the expression level of ZO-1 and occludin was up-regulated, the apoptosis rate of epithelial cells was reduced (40.8%, 25.4% and 8.7%), the expression of autophagy-related proteins LC3 and Beclin1 was decreased and the expression of p62 was increased, the PI3K/AKT/mTOR/ULK1(ser757) signalling pathway was activated, and the AMPK/ULK1(ser317) signalling pathway was inhibited. In addition, XJZD can antagonize the imbalance of redox homeostasis caused by aspirin and protect the gastric mucosa. DISCUSSION AND CONCLUSIONS: XJZD protects against aspirin-induced gastric mucosal injury, implying it to be a potential therapeutic agent.

Ecotoxicological impacts of environmentally relevant concentrations of aspirin in the liver of Labeo rohita: Biochemical and histopathological investigation.

Aspirin is one of the emerging pharmaceutical contaminants in the aquatic environment and thus it could impart toxicity to non-target organisms including fish. The present study aims to investigate the biochemical and histopathological alterations in the liver of the fish, Labeo rohita exposed to environmentally relevant concentrations of aspirin (1, 10, and 100 µg/L) for 7, 14, 21, and 28 days. The biochemical investigation revealed a significant (p < 0.05) decrease in the activity of antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase; and reduced glutathione content in a concentration and duration dependent manner. Further, the decrease in the activity of superoxide dismutase was in a dose dependent manner. The activity of glutathione-s-transferase, however, increased significantly (p < 0.05) in a dose dependent manner. The lipid peroxidation and total nitrate content showed a significant (p < 0.05) increase in a dose and duration dependent manner. The metabolic enzymes such as acid phosphatase, alkaline phosphatase, and lactate dehydrogenase showed a significant (p < 0.05) increase in all three exposure concentrations and durations. The histopathological alterations in the liver such as vacuolization, hypertrophy of the hepatocytes, nuclear degenerative changes, and bile stagnosis increased in a dose and duration dependent manner. Hence, the present study concludes aspirin has a toxic impact on fish, which is evidenced by its profound effect on biochemical parameters and histopathological analysis. These can be employed as potential indicators of pharmaceutical toxicity in the field of environmental biomonitoring.

C-Phycocyanin and Lycium barbarum Polysaccharides Protect against Aspirin-Induced Inflammation and Apoptosis in Gastric RGM-1 Cells.

Aspirin causes gastrotoxicity and damaged epithelial defense via cyclooxygenase inhibition. C-phycocyanin (CPC) and Lycium barbarum polysaccharides (LBP), an active ingredient of Spirulina platensis and wolfberry, respectively, exerted antioxidation, anti-inflammation, and/or immunoregulation. The actions of CPC and/or LBP on gastric damage induced by aspirin were explored in rat gastric mucosal RGM-1 cells. Gastric injury was performed by 21 mM aspirin for 3 h after the pretreatment of CPC and/or LBP (100-500 µg/mL) for 24 h in RGM-1 cells. Proinflammatory, anti-inflammatory, and apoptotic markers were examined by ELISA or gel electrophoresis and Western blotting. Cell viability and interleukin 10 (IL-10) were reduced by aspirin. Increased proinflammatory markers, caspase 3 activity, and Bax protein were observed in RGM-1 cells with aspirin treatment. Aspirin elevated nuclear factor-κB (NF-κB), extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) activation, while CPC and/or LBP increased IL-10, and attenuated proinflammatory markers, Bax protein, NF-κB, and the activation of ERK and JNK. Therefore, CPC and/or LBP possess anti-inflammation by restraining the activation of the ERK signaling pathway, and LBP decreases apoptosis by suppressing the JNK signaling pathway activation in gastric RGM-1 cells with aspirin-induced epithelial damage.

Aspirin inhibits stem cell proliferation during freshwater Dugesia japonica regeneration by STAT3/SOX2/OCT4 signaling pathway.

As a widely used drug in clinical practice, aspirin has a large number of residual drugs and metabolites discharged into the environment during the pharmaceutical process or after taking the drug. Aspirin content and its metabolite, salicylic acid, have been reported and detected in several river water samples and municipal wastewaters. However, little is known about the toxicity mechanisms of this drug in aquatic invertebrates. In this study, we examine the toxic effect and investigate the toxicity mechanism of aspirin in planarian, which own the excellent regeneration and sensitive toxicity detection ability. Planarian is treated with 0.7 mM aspirin for 6 h, 48 h, 3 d and 5 d, and the mRNA and protein expression levels of the stem cells markers, in parallel with the target genes of the signaling pathway are analyzed by RT-qPCR, whole-mount immunofluorescence, and Western blot. The results show that aspirin strongly inhibits stem cell proliferation and causes retarded blastemas growth in planarians. Furthermore, the mRNA and protein expression levels of stem cells markers and the target genes dramatically decrease after the aspirin treatment. Meanwhile, the expression level of apoptotic cells also shows a downward trend. Their significant and coincident downregulations after the aspirin treatment suggest that aspirin regulates planarian regeneration via STAT3/SOX2/OCT4 signaling pathway. Our work reveals the toxicological effect and the mechanism of aspirin to the planarian, and provides basic data for therapeutic applications of aspirin in regeneration and warns about the ecological damage of aspirin abuse.

Spirulina supplements: an approach moderating aspirin persuaded histological and ultra-structural alterations in albino rats gastric mucosa.

Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most used drugs. The pathogenesis of aspirin-induced gastric ulceration includes blocking the activities of the cyclooxygenase enzymes (COX-1 and COX-2) leading to reduced mucus and bicarbonate secretion. Spirulina contains many functional bioactive ingredients with antioxidant and anti-inflammatory activities, including phenolic phytochemicals and phycobiliprotein C-phycocyanin. To investigate the possible gastroprotective role of spirulina against aspirin-induced gastric mucosal insults. Forty adult male albino rats were randomly divided into four experimental groups. Group I (Control) and group II (Spirulina control) were given spirulina for 3 days, group III (Ulcer model) were given single dose of acetyl salicylic acid to induce ulcer and group IV (Treatment) were given spirulina for 3 days after induction of ulcer formation. Animals were sacrificed, stomachs were collected and processed for examination of light and scanning electron microscope histopathological examination. Statistical difference mucosal mucin area percentage among groups was determined and data were analyzed. Histological examination of the H&E-stained and combined Alcian-blue-PAS-stained sections of Group III rats illustrated severe destruction of the mucosal architecture and reduction of the mucin surface area while those examined for group IV illustrated minor affection of the gastric mucosa and mucin protective layer. Oxidant antioxidant markers: Nitric oxide (NO) is elevated, Glutathione (GSH) and superoxide dismutase (SOD) are reduced in aspirin treated group. The use of Spirulina restores the normal balance between the oxidant antioxidant system. Spirulina has a great potential in protecting the gastric mucosa against harmful effect of NSAID.

Acetylsalicylic acid improves cognitive performance in sleep deprived adult Zebrafish (Danio rerio) model.

Sleep deprivation (SD) is commonly associated with decreased attention, reduced responsiveness to external stimuli, and impaired locomotor and cognitive performances. Strong evidence indicates that SD disrupts neuro-immuno-endocrine system which is also linked to cognitive function. Recently Zebrafish have emerged as a powerful model sharing organizational and functional characteristics with other vertebrates, providing great translational relevance with rapid and reliable screening results. In the current study, we examined the effects of acetylsalicylic acid (aspirin) on cognitive and locomotor activity in sleep deprived Zebrafish model. Learning and memory were assessed by T-maze and locomotor activity was assessed by partition preference and swimming time in spinning tasks. Furthermore, brain bioavailability of aspirin was determined by high performance liquid chromatography. Following drug exposure and tasks, histopathology of the brain was performed. It was observed that three-day SD significantly reduces learning and memory and locomotion in the Zebrafish. Aspirin was found to restore SD induced cognitive decline and improve the locomotor functions. Neuro-inflammation and impaired functional network connectivity is linked to cognitive defects, which implicate the possible benefits of immunotherapeutics. In the present study, aspirin decreased neutrophil infiltration, and increased spine density in dentate gyrus granular and shrinkage and basophil in the CA1 neurons of hippocampus. This hints the benefit of aspirin on neuroimmune functions in sleep deprived fish and warrants more studies to establish the clear molecular mechanism behind this protective effect.

Inhibition of desmoglein-1 by aspirin leads to synthetic lethality of keratinocytes in Shuanghuanglian-induced cutaneous eruption response.

Cutaneous eruptions caused by the combination of Chinese and Western medicine have attracted widespread attention; however, the underlying mechanism remains unclear. This study aimed to evaluate the potential mechanism of cutaneous eruptions in vivo and in vitro using the combination of Shuanghuanglian injection powder (SHL) and aspirin (ASA) as an example. ASA and SHL co-administration induced inflammatory responses in HaCat cells, as evidenced by marked increases in the expression of IL-4 and TNF-α, and the level of apoptosis. Additionally, histopathological investigation of mice skin tissues showed local inflammatory cell infiltration. Western boltting was used to detect the effects of ASA on desmoglein-1 (DSG1) expression; we found that DSG1 expression was down-regulated in vivo and in vitro. Finally, the key components of SHL were administered to HaCat cells with down-regulated DSG1; it was seen that neochlorogenic acid and rutin have a significant effect on HaCat cell apoptosis. These results demonstrate that DSG1 deficiency is a potential cause of cutaneous eruptions caused by the combination of SHL and ASA, and neochlorogenic acid and rutin are the main allergenic components. This study provides a new research strategy for the safety evaluation of integrated traditional Chinese and Western medicine.

Non-selective COX inhibitors impair memory formation and short-term but not long-term synaptic plasticity.

Cyclooxygenase (COX) plays a critical role in synaptic plasticity. Therefore, long-term administration of acetylsalicylic acid (ASA) and its main metabolite, salicylate, as a COX inhibitor may impair synaptic plasticity and subsequently memory formation. Although different studies have tried to explain the effects of ASA and sodium salicylate (SS) on learning and memory, the results are contradictory and the mechanisms are not exactly known. The present study was designed to investigate the effects of long-term low-dose (equivalent to prophylactic dose) and short-term high-dose (equivalent to analgesic dose) administration of ASA and SS respectively, on spatial learning and memory and hippocampal synaptic plasticity. Animals were treated with a low dose of ASA (2 mg/ml solvated in drinking water, 6 weeks) or a high dose of SS, a metabolite of ASA, (300 mg/kg, 3 days, twice-daily, i.p). Spatial memory and synaptic plasticity were assessed by water maze performance and in vivo field potential recording from CA1, respectively. Animals treated with ASA but not SS showed a significant increase in escape latency and distance moved. Furthermore, in the probe test, animals treated with both drugs spent less time in the target quadrant zone. The paired-pulse ratio (PPR) at 20-ms inter-pulse intervals (IPI) as an index of short-term plasticity in both treated groups was significantly higher than of the control group. Interestingly, none of the administered drugs affected long-term potentiation (LTP). These data suggested that long-term inhibition of COX disrupted memory acquisition and retrieval. Interestingly, cognitive impairments happened along with short-term but not long-term synaptic plasticity disturbance.

Food antigens exacerbate intestinal damage and inflammation following the disruption of the mucosal barrier.

Food antigens are closely related to progression of inflammatory bowel disease; however, details of how they induce intestinal immune responses and causes intestinal inflammation is not yet clear. The present study aimed to examine the effects of oral collagen on the intestinal mucosa, and elucidate the mechanism of food antigen-induced enteritis. Here, we provide evidence that Aspirin (a mucosal-damaging agent) and type II collagen (CII; a food antigen) acted synergistically to disrupt the intestinal mucosal barrier, and increase intestinal permeability, which resulted in a large amount of CII entered into the lamina propria, where it interacted with the intestinal immune system, promoted intestinal inflammation, and shaped innate and adaptive immune reactions into Th1-dominant. The underlying mechanism of the CII-induced intestinal inflammation may associate with higher levels of Th1, TLR2 and TLR4, and lower levels of Th2 in the intestine of Aspirin + CII treated rats. The study indicate that compromised integrity of the intestinal barrier appears to be a prerequisite for CII-induced intestinal inflammation. The synergistic effect of food antigens and mucosal barrier injury is an important cause of intestinal inflammation. This new understanding the role of food antigen on intestinal inflammation will provide us with a new strategy for treatment and prevention of intestinal inflammation.

Treatment effects of Shilajit on aspirin-induced gastric lesions in rats.

The present study investigated the effects of Shilajit extract on aspirin-induced gastric lesions in rats. We evaluated macroscopic and histopathological lesions in the stomach, measured the activity of oxidative stress enzymes in gastric tissue homogenates, and assessed serum electrolytes and parameters of kidney and liver function. Forty-five male rats were allocated to five groups: Normal control, positive control, omeprazole treatment, Shilajit treatment, and Shilajit control. The treatment period lasted for four consecutive days. The size and number of gastric lesions were significantly reduced in the Shilajit and omeprazole groups compared to the positive control group, indicating a reduction in mucosal damage and the severity of edema and leukocyte infiltration in tissue sections. A significant increase was observed in the levels of all oxidative stress parameters, except malondialdehyde, in rats treated with Shilajit and omeprazole compared to those in the positive control group. The effect of the aqueous extract of Shilajit was comparable to that of omeprazole. These results indicated the protective effects of Shilajit against aspirin-induced gastric lesions.

Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 µM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 µM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.

Human ES and iPS cells display less drug resistance than differentiated cells, and naïve-state induction further decreases drug resistance.

Pluripotent stem cells (PSCs) possess unique characteristics that distinguish them from other cell types. Human embryonic stem (ES) cells are recently gaining attention as a powerful tool for human toxicity assessment without the use of experimental animals, and an embryonic stem cell test (EST) was introduced for this purpose. However, human PSCs have not been thoroughly investigated in terms of drug resistance or compared with other cell types or cell states, such as naïve state, to date. Aiming to close this gap in research knowledge, we assessed and compared several human PSC lines for their resistance to drug exposure. Firstly, we report that RIKEN-2A human induced pluripotent stem (iPS) cells possessed approximately the same sensitivity to selected drugs as KhES-3 human ES cells. Secondly, both ES and iPS cells were several times less resistant to drug exposure than other non-pluripotent cell types. Finally, we showed that iPS cells subjected to naïve-state induction procedures exhibited a sharp increase in drug sensitivity. Upon passage of these naïve-like cells in non-naïve PSC culture medium, their sensitivity to drug exposure decreased. We thus revealed differences in sensitivity to drug exposure among different types or states of PSCs and, importantly, indicated that naïve-state induction could increase this sensitivity.

Mitigative Effects Of Nigella Sativa On The Histology Of Kidneys Against Aspirin-Induced Toxicity.

BACKGROUND: Aspirin is one of the popular, economical, easily accessible and commonly used drugs all over the world. Injudicious use of this over-the-counter available drug is a common cause of nephrotoxicity. The aim of the present study is to assess the protective effects of Nigella Sativa (NS) on the histology of kidneys against aspirin-induced toxicity. It was an experimental study that included comparative analysis of control and experimental groups, conducted in the department of Anatomy, University of Health Sciences, Lahore, from October 2016 to December 2016. METHODS: The study included thirty-two female albino rats which were equally distributed into 4 groups. Group A was run as control and given single oral dose of 1% methyl-cellulose (10mg/100gm body weight of rat). Group B and C were treated with a single oral dose of aspirin (1000mg/kg body weight) dissolved in 1% methyl-cellulose (10mg/100gm body weight). Group C animals were left untreated for 7 days. Group D was pre-treated on day 1 with oral dose of Nigella Sativa (NS) extract (250mg/kg body weight) followed one hour later by a single oral dose of aspirin (1000mg/kg body weight), subsequently NS extract was administered till day 7. Rats of group B were euthanized and dissected on 2nd day of experiment while those of groups A, C and D on 8th day. Kidneys were dissected out, weighed and fixed in 10% formalin. 5µm thick sections were yielded after tissue processing and stained with haematoxylin, eosin (H&E staining) and periodic acid Schiff's reagent (PAS staining). Histological parameters of distal convoluted tubules (DCT) were observed. RESULTS: All histological parameters were normal in group A. Group B showed marked increase in epithelial necrosis, intraluminal protein casts and broken basement membranes of distal convoluted tubules. Group C showed no self-recovery. Statistically significant improvement was observed in the histology of distal convoluted tubules with treatment of Nigella Sativa extract in aspirin-ingested rats in group D. CONCLUSIONS: Nigella Sativa extract has shown protective effects on kidneys against aspirin-induced damage as shown by improvement in the histological parameters of distal convoluted tubules.

Antioxidant prevents clearance of hemostatically competent platelets after long-term cold storage.

BACKGROUND: Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. STUDY DESIGN AND METHODS: Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. RESULTS: Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. CONCLUSIONS: Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

Cashew apple byproduct: Gastroprotective effects of standardized extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of gastric mucosa lesions in the adult population has increased mainly due to the continued use of nonsteroidal anti-inflammatory drugs (NSAIDs). The cashew (Anacardium occidentale L.) is a tropical tree, cultivated in several countries, whose barks, leaves and pseudofruit (cashew apple) are popularly used in traditional medicine for the treatment of many diseases, including gastric ulcer. AIM: Our study evaluated the potential gastroprotective effect of the carotenoid and anacardic acids-enriched aqueous extract (CAE), prepared from cashew apple pomace, in the dose-repeated acetylsalicylic acid (ASA)-induced gastric lesions model in rats. MATERIAL AND METHODS: After randomly distribution into five group (G1 - G5, n = 8 animals/group), male Wistar rats were daily treated with ASA solution (200 mg/kg, 5 ml/kg, G2 - G5) or potable water (Satellite group, G1) during 14 days. From 8th to 14th experimental day, rats in G3 - G5 groups were orally treated with CAE (50, 100 and 500 mg/kg, 5 ml/kg, respectively). Body weight was measured on 0, 7th and 14th day. On the 14th experimental day, all surviving animals were euthanized for macroscopic evaluation of the inner organs and stomach removal. After weighting, each stomach was properly prepared for biochemical analysis [myeloperoxidase activity (MPO), reduced glutathione analysis (GSH), IL-1ß, CXCL2/MIP-2, TNF-α and IL-10 levels]. RESULTS: At the most efficient dose (100 mg/kg, p.o.), CAE-treated animals showed a slight improvement in the macroscopic aspect of gastric mucosa associated with significant (p < 0.05) reduced levels of IL-1ß, CXCL2/MIP-2, and MPO activity besides increased levels of GSH (partially), and IL-10 in stomach tissues. CONCLUSIONS: The present study demonstrated that the carotenoid and anacardic acids-enriched extract obtained from cashew apple pomace is a promising raw material for the development of herbal medicine and/or functional food supplements for the adjuvant treatment of NSAIDs-induced gastric ulcers.

Long-term aspirin intervention can inhibit TIGIT, regulating T cells to reverse damage to intestines.

Aspirin is one of the most commonly prescribed medications. Evidence shows that it can even treat and prevent intestinal tumors. However, it has also caused a great deal of controversy due to its intestinal side effects. We therefore explored whether aspirin was beneficial or harmful to the intestines. We used aspirin continuously interfered with C57BL/6 J mice for 48 weeks, examining their intestinal tissues at 13, 26 and 48 weeks to determine the drug's effect on the intestines. In addition, we used flow cytometry (FCM) used to detect T cells and expression of T-cell immunoreceptor with immunoglobulin (Ig)- and tyrosine-based inhibitory motif (ITIM) domain (TIGIT) on their surfaces to determine aspirin's immunomodulatory effects. The results showed that long-term aspirin intervention could reverse damage to the intestines, an effect related to the drug's significant inhibitory effect on TIGIT. The change in TIGIT level could regulate T-cell subsets, so that counts of Cluster of Differentiation 4 (CD4)+/chemokine (C-X3-C motif) receptor 3 (CXCR3)+ T-helper 1 (Th1) cells and CD4+/interleukin-4 (IL-4)+ Th2 cells increased, while those of CD4+/C-C chemokine receptor type 6 (CCR6)+ Th17 cells and CD4+/CD25+ regulatory T cells (Tregs) decreased. In summary, we demonstrated that long-term aspirin intervention could inhibit TIGIT, regulating T cells to reverse damage to the intestines. Furthermore, aspirin is a potential therapy for diseases related to an increase in TIGIT.

Effects of common pharmaceutical drugs (paracetamol and acetylsalicylic acid) short term exposure on biomarkers of the mussel Mytilus spp.

Pharmaceutical drugs in the wild may pose significant risks to non-target exposed organisms. This situation is even more troublesome for coastal marine or estuarine environments, located in the vicinity of large human conglomerates, for which the putative number of pollutants is extremely high, and the regime by which wild organisms are exposed is continuous. In addition, the number of studies addressing this issue is still scarce, despite evidences that show the potential contamination profiles and adverse biological effects in organisms from such areas. In this study, the ecotoxicity of common pharmaceutical drugs (namely paracetamol and acetylsalicylic acid) was assessed, by studying the susceptibility of the mussel species Mytilus spp to oxidative stress after being exposed for 96 h to increasing but ecologically relevant concentrations of the two mentioned pharmaceuticals (paracetamol: 0, 0.5, 5, 50, and 500 µg/L; acetylsalicylic acid: 0, 0.1, 1, 10, and 100 µg/L). The oxidative status in exposed organisms was analyzed by measuring oxidative stress biomarkers, namely catalase (CAT), glutathione-S-transferases (GSTs), and lipoperoxidation (LPO) levels, whose alteration was indicative of chemical exposure, in both digestive gland and gills of the organisms. In addition, the food uptake and the nutritional reserve status of exposed organisms were also assessed, by measuring the consumption of ingested food, and levels of tissue reserves of glycogen in gills and digestive gland. No significant alterations were observed in the assessed oxidative stress parameters so it was possible to hypothesize that the studied drugs may have probably exerted a limited alteration of antioxidant defenses and damage, which was reverted by the activation of defensive adaptive mechanisms. This set of data evidenced that the pro-oxidative metabolism that was already described for both drugs in other animal models, was not fully established in the exposed mussels. On the contrary, glycogen reserves were substantially changed after exposure to both toxicants, being possible to observe opposite responses caused by both drugs. Food uptake was not altered following exposure to the drugs. Further evaluations are thus required to conclude about both drugs ecotoxicity and other parameters, namely seasonality, which should be considered when performing ecotoxicology tests, especially with the selected species.

Does hepatotoxicity interfere with endocrine activity in zebrafish (Danio rerio)?

Vitellogenin (VTG), a well-established biomarker for the diagnosis of endocrine activity in fish, is used in multiple OECD test guidelines (TG) to identify activities of chemicals on hormonal pathways. However, the synthesis of VTG may not only be modified by typical endocrine-related pathways, but also through non-endocrine-mediated processes. In particular, hepatotoxicity, i.e. toxicant-induced impairment of liver structure and function, might influence VTG as a biomarker, since VTG is synthesized in hepatocytes. An intimate understanding of the interplay between endocrine-related and non-endocrine-related pathways influencing VTG production is crucial for the avoidance of erroneous diagnoses in hazard assessment for regulatory purposes of chemical compounds. In order to investigate whether hepatotoxicity may interfere with hepatic VTG synthesis, adult zebrafish (Danio rerio) were exposed to three well-known hepatotoxicants, acetaminophen, isoniazid and acetylsalicylic acid, according to OECD TG 230. Various hepatotoxicity- and endocrine system-related endpoints were recorded: mRNA expression of selected endocrine- and hepatotoxicity-related marker genes in the liver; VTG levels in head/tail homogenates; and liver histopathology. All three test compounds induced significant, but mild single cell necrosis of hepatocytes and transcriptional changes of hepatotoxicity-related marker genes, thus confirming hepatotoxic effects. A positive correlation between hepatotoxicity and reduced hepatic VTG synthesis was not observed, with the single exception of a weak increase in female zebrafish exposed to APAP. This suggests that - in studies conducted according to OECD TG 229 or 230 - it is unlikely that hepatotoxic chemicals will interfere with the hepatic capacity for VTG synthesis.

Implementation of an in vitro methodology for phototoxicity evaluation in a human keratinocyte cell line.

To assess photoxicity, several in vitro methods using different cellular models have been developed for preclinical testing. Over prediction of the in vivo photosafety hazard has been however appointed. Herein, we describe the implementation and validation of an in vitro methodology for phototoxicity evaluation based on the 3T3 neutral red uptake phototoxicity test using the HaCaT human keratinocyte cell line, and UVA/UVB radiation. Known positive (5-methoxypsoralen, chlorpromazine, and quinine) and negative (acetyl salicylic acid, hexachlorophene, and sodium lauryl sulphate) controls were tested together with a set of chemical currently used in cosmetic/pharmaceutical formulations. Apart from the advantage of using a cell line of human origin, these cells were generally more resistant to the cytotoxic effects of the test substances relative to the 3T3 mouse fibroblasts when exposed to an UVA irradiation dose of 1.7 mW/cm2. Therefore, this HaCaT NRU assay provides a more realistic experimental model that overcomes the over/high sensitivity frequently noted with the 3T3 NRU assay and that is more consistent with the human in vivo situation. Using a more representative method can prevent time-consuming and expensive in vivo testing in both animal models and humans that can significantly delay the clinical development of new chemicals.