Red Orange and Lemon Extract Ameliorates the Renal Oxidative Stress and Inflammation Induced by Ochratoxin A through the Modulation of Nrf2.

BACKGROUND: The presence of ochratoxin A (OTA) in food and feed is a public health concern. OTA intoxication is caused by several mechanisms, one of which consists of the alteration of the antioxidant activity of the cell due to the oxidative stress (OS). In this context, the use of natural antioxidant substances could be a potential biological decontamination method of mitigating the negative outcomes induced by OTA. METHODS: we aimed to investigate how a red orange and lemon extract (RLE), rich in anthocyanins, would affect OTA-treated rats. The current work sought to clarify the renal protective efficacy of RLE in an OTA-treated rat model (RLE (90 mg/kg b.w.); OTA (0.5 mg/kg b.w.)) by investigating, thorough Western blot analysis, the involvement of the Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. The OS parameters and inflammatory status were evaluated by spectrophotometry. The inflammatory infiltrates in the kidney were evaluated by immunohistochemical assays. RESULTS AND CONCLUSION: Our findings showed a significant increase in oxidative and inflammatory parameters after OTA exposure, while the OTA + RLE co-treatment counteracted both the inflammatory and OS damage through the modulation of the Nrf2 pathway.

Protective Effects of a Red Orange and Lemon Extract (RLE) on the Hepatotoxicity Induced by Ochratoxin A in Rats.

Ochratoxin A (OTA) is a highly potent mycotoxin that contaminates many kinds of food and feed sources. Its significant impact on human health and animal productivity makes it a topic of particular concern. The role of specific bioactive compounds used as dietary antioxidants is believed to be substantial due to their capacity to act as free radical scavengers. Because of the well-known oxidative stress induced by OTA, the primary objective of this work was to evaluate the antioxidant effects of a standardized powder extract recovered from citrus processing waste, red orange and lemon extract (RLE), on liver damage induced by OTA in a rat model. This study aimed to examine the impact of oral administration of RLE (90 mg/kg b.w.) on hepatic function and oxidative balance in Sprague-Dawley rats (n = 6/group) treated with OTA (0.5 mg/kg b.w.) over a period of 14 days. The administration of OTA alone resulted in both biochemical changes and an imbalance in redox status in the liver. However, the use of RLE alleviated the activity of antioxidant enzymes and dramatically decreased the serum levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and ALP (alkaline phosphatase), providing evidence of its protective benefits. Based on the findings from liver histology tests, the administration of RLE resulted in mitigation of lymphoplasmacytic inflammation, steatosis, and necrosis in the OTA group. These results indicate that the novel phytoextract RLE holds potential for application in the field of nutraceuticals.

Apoptosis is mediated by FeHV-1 through the intrinsic pathway and interacts with the autophagic process.

BACKGROUND: Although FeHV-1 is a primary feline pathogen, little is known about its interactions with host cells. Its relationship with several cellular pathways has recently been described, whereas its interplay with the apoptotic process, unlike other herpesviruses, has not yet been clarified. The aim of this work was to evaluate whether FeHV-1 induces apoptosis in its permissive cells, as well as the pathway involved and the effects of induction and inhibition of apoptosis on viral replication. METHODS: Monolayers of CRFK cells were infected at different times with different viral doses. A cytofluorimetric approach allowed the quantification of cells in early and late apoptosis. All infections and related controls were also subjected to Western blot analysis to assess the expression of apoptotic markers (caspase 3-8-9, Bcl-2, Bcl-xL, NF-κB). An inhibitor (Z-VAD-FMK) and an inducer (ionomycin) were used to evaluate the role of apoptosis in viral replication. Finally, the expression of autophagy markers during the apoptosis inhibition/induction and the expression of apoptosis markers during autophagy inhibition/induction were evaluated to highlight any crosstalk between the two pathways. RESULTS: FeHV-1 triggered apoptosis in a time- and dose-dependent manner. Caspase 3 cleavage was evident 48 h after infection, indicating the completeness of the process at this stage. While caspase 8 was not involved, caspase 9 cleavage started 24 h post-infection. The expression of other mitochondrial damage markers also changed, suggesting that apoptosis was induced via the intrinsic pathway. NF- κB was up-regulated at 12 h, followed by a gradual decrease in levels up to 72 h. The effects of apoptosis inhibitors and inducers on viral replication and autophagy were also investigated. Inhibition of caspases resulted in an increase in viral glycoprotein expression, higher titers, and enhanced autophagy, whereas induction of apoptosis resulted in a decrease in viral protein expression, lower viral titer, and attenuated autophagy. On the other hand, the induction of autophagy reduced the cleavage of caspase 3. CONCLUSIONS: In this study, we established how FeHV-1 induces the apoptotic process, contributing to the understanding of the relationship between FeHV-1 and this pathway.

Oxidative Status and Histological Evaluation of Wild Boars' Tissues Positive for Zearalenone Contamination in the Campania Region, Southern Italy.

Zearalenone (ZEN) is a mycotoxin produced by fungi belonging to the genera Fusarium spp. and commonly found in feed and food. It is frequently related to reproductive disorders in farm animals and, occasionally, to hyperestrogenic syndromes in humans. Nowadays, knowledge about ZEN effects on wild boars (Sus scrofa) is extremely scarce, despite the fact that they represent one of the most hunted game species in Italy. The aim of this study was to investigate how ZEN affects the liver, kidney, and muscle oxidative status and morphology of wild boars hunted in various locations throughout the province of Avellino, Campania Region, Southern Italy, during the 2021-2022 hunting season. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, as well as the malondialdehyde (MDA) levels, were assessed by colorimetric assays; tissue morphology was evaluated by hematoxylin-eosin and Masson's stains. Our data showed that ZEN contamination might result in oxidative stress (OS) and some histopathological alterations in wild boars' livers and kidneys rather than in muscles, emphasizing the importance of developing a wildlife monitoring and management strategy for dealing not only with the problem of ZEN but the surveillance of mycotoxins in general.

Autophagy up-regulation upon FeHV-1 infection on permissive cells.

FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.

Modifications of the PI3K/Akt/mTOR axis during FeHV-1 infection in permissive cells.

FeHV-1 is the causative agent of infectious rhinotracheitis in cats. The relationship between viral infection and the PI3K/Akt/mTOR pathway, as well as its function in crucial physiological processes like as autophagy, apoptosis or the IFN induction cascade is known for other varicelloviruses. However, there is no information on whether autophagy is activated during FeHV-1 infection nor on how this infection modifies PI3K/Akt/mTOR pathway. In this work, we aim to elucidate the involvement of this pathway during cytolytic infection by FeHV-1 in permissive cell lines. Using a phenotypic approach, the expression of proteins involved in the PI3K/Akt/mTOR pathway was examined by Western blot analysis. The findings demonstrated the lack of modifications in relation to viral dose (except for phospho-mTOR), whereas there were changes in the expression of several markers in relation to time as well as a mismatch in the time of activation of this axis. These results suggest that FeHV-1 may interact independently with different autophagic signaling pathways. In addition, we found an early phosphorylation of Akt, approximately 3 h after infection, without a concomitant decrease in constitutive Akt. This result suggests a possible role for this axis in viral entry. In a second phase, the use of early autophagy inhibitors was examined for viral yield, cytotoxic effects, viral glycoprotein expression, and autophagy markers and resulted in inefficient inhibition of viral replication (12 h post-infection for LY294002 and 48 h post-infection for 3-methyladenine). The same markers were examined during Akt knockdown, and we observed no differences in viral replication. This result could be explained by the presence of a protein kinase in the FeHV-1 genome (encoded by the Us3 gene) that can phosphorylate various Akt substrates as an Akt surrogate, as has already been demonstrated in genetically related viruses (HSV-1, PRV, etc.). For the same reasons, the use of LY294002 at the beginning of infection did not affect FeHV-1-mediated Akt phosphorylation. Our findings highlight changes in the PI3K/Akt/mTOR pathway during FeHV-1 infection, although further research is needed to understand the importance of these changes and how they affect cellular processes and viral propagation.

Zearalenone (ZEN) and Its Metabolite Levels in Tissues of Wild Boar (Sus scrofa) from Southern Italy: A Pilot Study.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by the fungi of the Fusarium genera, and is a contaminant of cereals and plant products. ZEN and its metabolites are considered endocrine disruptors, and could have various toxic effects on animals and humans. In recent years, there has been a significant demographic increase in wild boar (Sus scrofa) in many mountainous and hilly areas of Italy, including the Campania region, mainly due to global climate change. The wild boar can be defined as a generalist and omnivorous species capable of varying its diet; therefore, it can play a role as an environmental bioindicator towards contaminants such as mycotoxins. This study was conducted to evaluate, for the first time, the concentrations of ZEN and its metabolites in the liver, kidney, and muscle of 82 wild boars shot in their habitat by hunters with hunting permits in different localities of Avellino province (Campania region, Southern Italy) from 2021 to 2022. The samples were collected and analyzed with an SPE clean-up and high-pressure liquid chromatography method with fluorescence detection. The results indicated that ZEN and α-Zearalenol were present in most of the samples, suggesting that a plan to monitor these mycoestrogens is essential to achieve the goals of "One Health".

The Microalga Skeletonema marinoi Induces Apoptosis and DNA Damage in K562 Cell Line by Modulating NADPH Oxidase.

Chronic myeloid leukemia (CML) is a myeloproliferative disease that activates multiple signaling pathways, causing cells to produce higher levels of reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major generator of ROS in leukemia, and marine natural products have shown promising activities for the treatment of hematopoietic malignancies. In the present study, we investigated the effect of the marine microalga Skeletonema marinoi (S.M.), a ubiquitous diatom that forms massive blooms in the oceans, on the human leukemia cell line K562. The effects of S.M. extract on cell viability, production of ROS, nitric oxide (NO), and apoptosis were examined. In this preliminary work, S.M. was able to decrease cell viability (p < 0.05) and increase apoptosis levels (p < 0.05) in K562 cells after 48 h of treatment. In addition, the levels of NOX, NO, and malondialdehyde (MDA) were reduced in K562-treated cells (p < 0.05), whereas the levels of SOD, CAT, and GPx increased during treatment (p < 0.05). Finally, analyzing Bax and Bcl-2 expression, we found a significant increase in the proapoptotic protein Bax and a sustained decrease in the antiapoptotic protein Bcl-2 (p < 0.05) in the K562-treated cells.

Retrospective Serosurvey of Three Porcine Coronaviruses among the Wild Boar (Sus scrofa) Population in the Campania Region of Italy.

The growing interest in porcine coronaviruses (CoVs) is due to both their negative effect on the swine industry and their propensity to mutate and overcome host barriers. Since information on CoVs in wild boar (Sus scrofa) is limited, especially in Italy, a serosurvey was conducted to assess the epidemiologic situation in the Campania region and to clarify the role of wild boar as reservoirs for enteric (porcine epidemic diarrhea virus [PEDV], transmissible gastroenteritis virus [TGEV]) and respiratory (respiratory coronavirus [PRCV]) swine CoVs. During the 2016-17 hunting season, serum samples were collected from 444 wild boars and tested for antibodies to enteric (PEDV, TGEV) and respiratory (PRCV) porcine CoVs by enzyme-linked immunosorbent assay. The highest seroprevalence in wild boars was for PEDV, with a positivity of 3.83% (95% confidence interval [CI] 2.05-5.6), whereas very low seroprevalences were found for TGEV and PRCV (0.67% positivity; 95% CI 0-1.44 in both cases). There was no statistical association between seropositivity to CoVs, sex, and location, whereas the prevalence of seropositive animals was positively correlated with young age (0-12 mo old). Our data confirm the presence of CoVs in wild boars in the Campania region. Our data are in agreement with the results of similar studies from other European countries, which attribute a minor role to wild boar in the transmission of these infections to domestic pigs. Our results suggest that continuous serologic surveys are necessary to monitor wild animals and detect emerging threats to livestock and humans.

Ochratoxin A and Kidney Oxidative Stress: The Role of Nutraceuticals in Veterinary Medicine-A Review.

The problem of residues of toxic contaminants in food products has assumed considerable importance in terms of food safety. Naturally occurring contaminants, such as mycotoxins, are monitored routinely in the agricultural and food industries. Unfortunately, the consequences of the presence of mycotoxins in foodstuffs are evident in livestock farms, where both subacute and chronic effects on animal health are observed and could have non-negligible effects on human health. Ochratoxin A (OTA) is a common mycotoxin that contaminates food and feeds. Due to its thermal stability, the eradication of OTA from the food chain is very difficult. Consequently, humans and animals are frequently exposed to OTA in daily life. In this review article, we will devote time to highlighting the redox-based nephrotoxicity that occurs during OTA intoxication. In the past few decades, the literature has improved on the main molecules and enzymes involved in the redox signaling pathway as well as on some new antioxidant compounds as therapeutic strategies to counteract oxidative stress. The knowledge shown in this work will address the use of nutraceutical substances as dietary supplements, which would in turn improve the prophylactic and pharmacological treatment of redox-associated kidney diseases during OTA exposure, and will attempt to promote animal feed supplementation.

Aujeszky's Disease in South-Italian Wild Boars (Sus scrofa): A Serological Survey.

Aujeszky's disease (AD, pseudorabies) is a viral disease of suids caused by Suid Herpesvirus 1 (SHV-1) also referred as Aujeszky's disease virus (ADV) or Pseudorabies virus (ADV). Domestic pig and Wild boar (Sus scrofa) are the natural host, but many species can be infected with ADV. The aim of our study was to evaluate seroprevalence of AD in wild boar hunted in the Campania Region, during the 2016-2017 hunting season. A total of 503 serum samples from wild boars hunted in the provinces of Campania Region (Southern Italy) were collected and were tested for antibody against ADV using an AD, blocking ELISA assay. A Seroprevalence of 23.85% (120/503, 95% Confidence Interval (CI): 20.15-27.55) was found. Gender was not significantly associated with of ADV seropositivity (p > 0.05), while the presence of ADV antibodies was statistically associated with age (>36-month, p < 0.0001) and location (Avellino, p = 0.0161). Our prevalence values are like those obtained in 2010 in our laboratory (30.7%), demonstrating a constant circulation of ADV in the area.

Curcumin Modulates Nitrosative Stress, Inflammation, and DNA Damage and Protects against Ochratoxin A-Induced Hepatotoxicity and Nephrotoxicity in Rats.

Ochratoxin A (OTA) is a fungal toxin of critical concern for food safety both for human health and several animal species, also representing a cancer threat to humans. Curcumin (CURC) is a natural polyphenol that has anti-apoptotic, anti-inflammatory, and antioxidant effects. The aim of this study was to investigate the cytoprotective effect of CURC against OTA-induced nephrotoxicity and hepatotoxicity through the study of the nitrosative stress, pro-inflammatory cytokines, and deoxyribonucleic acid (DNA) damage. Sprague Dawley rats were daily treated with CURC (100 mg/kg b.w.), OTA (0.5 mg/kg b.w), or CURC with OTA by oral gavage for 14 days. Our results demonstrated that OTA exposure was associated with significant increase of pro-inflammatory and DNA oxidative-damage biomarkers. Moreover, OTA induced the inducible nitric oxide synthase, (iNOS) resulting in increased nitric oxide (NO) levels both in kidney and liver. The co-treatment OTA + CURC counteracted the harmful effects of chronic OTA treatment by regulating inflammation, reducing NO levels and oxidative DNA damage in kidney and liver tissues. Histology revealed that OTA + CURC treatment determinates mainly an Iba1+ macrophagic infiltration with fewer CD3+ T-lymphocytes in the tissues. In conclusion, we evidenced that CURC exerted cytoprotective and antioxidant activities against OTA-induced toxicity in rats.

Antioxidative Effects of Curcumin on the Hepatotoxicity Induced by Ochratoxin A in Rats.

Ochratoxin A (OTA) is a powerful mycotoxin found in various foods and feedstuff, responsible for subchronic and chronic toxicity, such as nephrotoxicity, hepatotoxicity, teratogenicity, and immunotoxicity to both humans and several animal species. The severity of the liver damage caused depends on both dose and duration of exposure. Several studies have suggested that oxidative stress might contribute to increasing the hepatotoxicity of OTA, and several antioxidants, including curcumin (CURC), have been tested to counteract the toxic hepatic action of OTA in various classes of animals. Therefore, the present study was designed to evaluate the protective effect of CURC, a bioactive compound with different therapeutic properties on hepatic injuries caused by OTA in rat animal models. CURC effects were examined in Sprague Dawley rats treated with CURC (100 mg/kg), alone or in combination with OTA (0.5 mg/kg), by gavage daily for 14 days. At the end of the experiment, rats treated with OTA showed alterations in biochemical parameters and oxidative stress in the liver. CURC dosing significantly attenuated oxidative stress and lipid peroxidation versus the OTA group. Furthermore, liver histological tests showed that CURC reduced the multifocal lymphoplasmacellular hepatitis, the periportal fibrosis, and the necrosis observed in the OTA group. This study provides evidence that CURC can preserve OTA-induced oxidative damage in the liver of rats.

Curcumin Supplementation Protects Broiler Chickens Against the Renal Oxidative Stress Induced by the Dietary Exposure to Low Levels of Aflatoxin B1.

Aflatoxin B1 (AFB1) causes hepatotoxicity, immunotoxicity, and kidney damage, and it is included in group I of human carcinogens. The European Commission has established maximum limits of AFB1 in feed, ranging from 5 to 20 µg/kg. Chicken is moderately sensitive to AFB1, which results in reduced growth performance and economic losses. Oxidative stress triggered by AFB1 plays a crucial role in kidney damage and the antioxidant activity of Curcumin (CURC) could help in preventing such adverse effect. Twenty-days-old broilers were treated for 10 days with AFB1 (0.02 mg/kg feed), alone or in combination with CURC (400 mg/kg feed), to explore the effects on the renal tissue. Animals exposed to AFB1 alone displayed alterations of the oxidative stress parameters compared with controls: serum antioxidant capacity, and enzymatic activity of kidney superoxide dismutase, catalase and glutathione peroxidase were decreased, while renal malondialdehyde levels and NADPH oxidase complex expression were increased. The administration of CURC attenuates all the oxidative stress parameters modified by AFB1 in the chicken kidney, opening new perspectives in the management of aflatoxicosis.

Potential Approaches Versus Approved or Developing Chronic Myeloid Leukemia Therapy.

Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of patients with chronic myeloid leukemia (CML). However, continued use of these inhibitors has contributed to the increase in clinical resistance and the persistence of resistant leukemic stem cells (LSCs). So, there is an urgent need to introduce additional targeted and selective therapies to eradicate quiescent LSCs, and to avoid the relapse and disease progression. Here, we focused on emerging BCR-ABL targeted and non-BCR-ABL targeted drugs employed in clinical trials and on alternative CML treatments, including antioxidants, oncolytic virus, engineered exosomes, and natural products obtained from marine organisms that could pave the way for new therapeutic approaches for CML patients.

Effects of a Red Orange and Lemon Extract in Obese Diabetic Zucker Rats: Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase.

Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, worldwide, and oxidative stress has been recognized as a key factor in the pathogenesis and progression of DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has the most important contribution to reactive oxygen species generation during the development of DN. Bioactive compound use has emerged as a potential approach to reduce chronic renal failure. Therefore, a red orange and lemon extract (RLE) rich in anthocyanins was chosen in our study, to reduce the toxic renal effects during the development of DN in Zucker diabetic fatty rat (ZDF). RLE effects were examined daily for 24 weeks, through gavage, in ZDF rats treated with RLE (90 mg/kg). At the end of the experiment, ZDF rats treated with RLE showed a reduction of the diabetes-associated up-regulation of both NOX4 and the p47-phox and p22-phox subunits, and restored the BAX/BCL-2 ratio respect to ZDF rats. Furthermore, RLE was able to reduce the oxidative DNA damage measured in urine samples in ZDF rats. This study showed that RLE could prevent the renal damage induced by DN through its capacity to inhibit NOX4 and apoptosis mechanisms.

Effects of Curcumin on the Renal Toxicity Induced by Ochratoxin A in Rats.

Ochratoxin A (OTA) is a powerful nephrotoxin and the severity of its damage to kidneys depends on both the dose and duration of exposure. According to the scientific data currently available, the mechanism of action still is not completely clarified, but it is supposed that oxidative stress is responsible for OTA-induced nephrotoxicity. Bioactive compound use has emerged as a potential approach to reduce chronic renal failure. Therefore, curcumin (CURC), due to its therapeutic effects, has been chosen for our study to reduce the toxic renal effects induced by OTA. CURC effects are examined in Sprague Dawley rats treated with CURC (100 mg/kg), alone or in combination with OTA (0.5 mg/kg), by gavage daily for 14 days. The end result of the experiment finds rats treated with OTA show alterations in biochemical and oxidative stress parameters in the kidney, related to a decrease in the Glomerular Filtration Rate (GFR). Conversely, the administration of CURC attenuates oxidative stress and prevents glomerular hyperfiltration versus the OTA group. Furthermore, kidney histological tests show a reduction in glomerular and tubular damage, inflammation and tubulointerstitial fibrosis. This study shows that CURC can mitigate OTA-induced oxidative damage in the kidneys of rats.