Mingwei Xing

Neurotoxicity induced by arsenic in Gallus Gallus: Regulation of oxidative stress and heat shock protein response

Arsenic, a naturally occurring heavy metal pollutant, is one of the functioning risk factors for neurological toxicity in humans. However, little is known about the effects of arsenic on the nervous system of Gallus Gallus. To investigate whether arsenic induce neurotoxicity and influence the oxidative stress and heat shock proteins (Hsps) response in chickens, seventy-two 1-day-old male Hy-line chickens were treated with different doses of arsenic trioxide (As2O3). The histological changes, antioxidant enzyme activity, and the expressions of Hsps were detected. Results showed slightly histology changes were obvious in the brain tissues exposure to arsenic. The activities of Glutathione peroxidase (GSH-Px) and catalase (CAT) were decreased compared to the control, whereas the malondialdehyde (MDA) content was increased gradually along with increase in diet-arsenic. The mRNA levels of Hsps and protein expressions of Hsp60 and Hsp70 were up-regulated. These results suggested that sub-chronic exposure to arsenic induced neurotoxicity in chickens. Arsenic exposure disturbed the balance of oxidants and antioxidants. Increased heat shock response tried to protect chicken brain tissues from tissues damage caused by oxidative stress. The mechanisms of neurotoxicity induced by arsenic include oxidative stress and heat shock protein response in chicken brain tissues.

Effects of Arsenic Trioxide Exposure on Heat Shock Protein Response in the Immune Organs of Chickens.

Arsenic trioxide (As2O3), a kind of pentavalent arsenic, has recently been linked to disrupted immune function. Heat shock proteins (Hsps), a group of highly conserved proteins, are rapidly synthesised when living organisms are exposed to various stress conditions. The objective of this study is to determine the effects of As2O3 on the expression level of Hsps (Hsp90, Hsp70, Hsp60, Hsp40 and Hsp27) in the immune organs (spleen, thymus and bursa of Fabricius (BF)) of chickens. A total of 72 1-day-old male Hy-line chickens were randomly divided into four groups, including the low-As group (L group), middle-As group (M group), high-As group (H group) and control group (C group). Immune organs were collected, and levels of Hsp messenger RNA (mRNA) and protein were examined on days 30, 60 and 90. The results showed that the levels of Hsp mRNA (Hsp90, Hsp70, Hsp60, Hsp40 and Hsp27) and protein (Hsp70 and Hsp60) expression were significantly increased (p < 0.05 or p < 0.01) in the As2O3 treatment groups compared with the corresponding control groups. Taken together, these results suggest that As2O3 influences the level of Hsps in immune organs.

Subchronic arsenism-induced oxidative stress and inflammation contribute to apoptosis through mitochondrial and death receptor dependent pathways in chicken immune organs

In many organ dysfunctions, arsenic and its compounds are well known to induce apoptosis by the mitochondria and death receptor apoptotic pathways in liver and airway. However, it is less reported that which signaling pathways contribute to excessive apoptosis of chicken immune organs, a major target of toxic metals biotransformation, which suffer from subchronic arsenism. In this study, we investigated whether the mitochondria or death receptor apoptotic pathways activated in the immune organs (spleen, thymus and bursa of Fabricius) of one-day-old male Hy-line chickens exposed to arsenic trioxide (As2O3), which were fed on diets supplemented with 0, 0.625, 1.25 and 2.5 mg/kg BW of As2O3 for 30, 60 and 90 days. We found that (1) Oxidative damage and inflammatory response were confirmed in the immune organs of chickens fed on As2O3 diet. (2) Subchronic arsenism induced typical apoptotic changes in ultrastructure. (3) TdT-mediated dUTP Nick-End Labeling (TUNEL) showed that the number of apoptotic cells significantly increased under subchronic arsenism. (4) As2O3-induced apoptosis of immune organs involved in mitochondrial pathway (decrease of B-cell lymphoma-2 (Bcl-2) and increase of protein 53 (p53), Bcl-2 Associated X Protein (Bax), caspase-9, caspase-3) and death receptor pathway (increase of factor associated suicide (Fas) and caspase-8). In conclusion, this work is the first to demonstrate that the activation of mitochondria and death receptor apoptosis pathways can lead to excessive apoptosis in immune organs of chickens, which suffer from subchronic arsenism, meanwhile, oxidative stress as well as subsequent inflammatory is a crucial driver of apoptosis.

Synergistic effect of copper and arsenic upon oxidative stress, inflammation and autophagy alterations in brain tissues of Gallus gallus

Arsenic or copper is one of the most highly toxic pollution that can cause dysfunction to brains, however, the exact mechanism remains unclear. The aim of the study is to investigate the mechanisms of arsenic or/and copper-induced oxidative stress, inflammation and autophagy in chicken brains and elucidate the interactions between arsenic and copper. A total of 72 1-day-old Hy-line chickens were divided into four groups (18 chickens per group) treated with 30mg/kg arsenic trioxide (As2O3) or/and 300mg/kg copper sulfate (CuSO4) for 12weeks. Histological signs of inflammation were found in the cerebrum, cerebellum and brainstem exposure to arsenic or/and copper. The malondialdehyde (MDA) content were up-regulation, whereas oxidative damage parameters total antioxidant capacity (T-AOC), glutathione (GSH), the inhibition ability of hydroxyl radical (OH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were significantly decreased (P<0.05). The mRNA levels and protein expressions of inflammation markers, such as nuclear factor kappa B (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PTGEs) were significantly increased (P<0.05). The mRNA levels and protein expressions of autophagy markers including phosphatidylinositol 3-kinase (PI3K), Akt, autophagy-related gene 5 (ATG5), microtubule-associated protein light chains 3 (LC3), ATG4B, and Becline1 in different regions of brains were up-regulation (P<0.05), except the mammalian target of rapamycin complex (mTORC). In conclusion, we speculated that arsenic or copper could induce oxidative stress, inflammation and autophagy in chicken brains, and there may have a synergistic effect between copper and arsenic.

Copper (II) and/or arsenite-induced oxidative stress cascades apoptosis and autophagy in the skeletal muscles of chicken

Arsenic (As) is a ubiquitous environmental toxin and robust inducer of oxidative stress (OxS). Copper (Cu) is an essential microelement, which participates in OxS as a cofactor for certain enzymes, with narrow optimal range between essential and toxic concentrations. However, their effects are rarely studied in chicken skeletal muscles, which have soaring per capita consumption andare susceptible to oxidative damage. In the present study, we demonstrated that the administration of copper sulfate (300 mg kg-1) or arsenite (30 mg kg-1) individually or their co-administration leads to varying degrees of OxS in the skeletal muscles of chickens. Corresponding to the protein expression pattern, the mRNA levels of caspase, B-cell lymphoma-2 (Bcl-2) families, and autophagy-related genes were also compromised in the experimental groups, indicating the involvement of both apoptotic and autophagic cell death. Additionally, rampant mitochondrial fission caused the vicious cycle between imbalanced mitochondrial dynamics and OxS, thus tethering intracellular homeostasis. The abovementioned muscle damage and index anomalies were time dependent, and more deteriorated effects were observed in Cu2+ and arsenite co-administered groups than those in groups administered Cu2+ and arsenite alone. Intriguingly, in the studied skeletal muscles, namely wing biceps brachii and leg gastrocnemius, there were conspicuous differences in oxidative toxicity susceptibility, which needs further study. The present study showed that Cu and/or As induce oxidative damage in chicken skeletal muscles and discussed its mechanism in terms of apoptosis, autophagy, and mitochondrial dynamics, thus voicing concerns about poultry breeding areas cross-contaminated with Cu2+ and arsenite.

Copper and arsenic-induced oxidative stress and immune imbalance are associated with activation of heat shock proteins in chicken intestines

ABSTRACT Arsenic and copper, two ubiquitous pollutants, can be oxidative stress inducers when organisms are heavy or chronically exposed, causing adverse effects on digestion and absorption function, resulting in potential losses to poultry husbandry. The present study examined the effects of arsenic trioxide (30mg/kg)‐ and copper sulfate (300mg/kg)‐mixed foods, administered alone or in combination for 12weeks, on various biochemical indices of oxidative stress and immunity in the small intestines of Hy‐line chickens. The results showed that for the first four weeks of exposure, both the redox and immune systems were unaffected. Subsequently, exposure to arsenic or copper significantly increased the level of lipid peroxidation (malondialdehyde and ability of anti‐hydroxy radical) concomitant with a collapse of the antioxidant system (catalase and glutathione peroxidase), in a time‐dependent manner. An increase in the mRNA and protein levels of pro‐inflammatory indicators (nuclear factor kappa B, cyclooxygenases‐2, tumor necrosis factor‐&agr; and prostaglandin E2 synthases) with a definite tendency toward Th1 (Th, helper T cell) cytokines was observed in both arsenic and copper treated chickens. Histologically, the destruction of the biofilm structure and inflammatory infiltrates was observed. Thus, in the intestine, heat shock proteins play protective roles against tissue damage. In some cases, we observed that the tissues of the small intestine were more sensitive to arsenic than to copper. Moreover, co‐exposure induced more serious intestinal toxicity than single treatment group, and this mechanism needs further exploration. HIGHLIGHTSCu and/or As induce intestinal toxicity by destructing antioxidant and immune system.Intestinal toxicity is more pronounced in co‐exposed groups than in individual.Duodenum seems more vulnerable to oxidative damage compared to jejunum and ileum.Heat shock proteins play intestinal protective function against tissues damage.

Copper or/and arsenic induce oxidative stress-cascaded, nuclear factor kappa B-dependent inflammation and immune imbalance, trigging heat shock response in the kidney of chicken

Excessive amount of copper (Cu) and inorganic arsenic (iAs) coexists in drinking water in many regions, this is associated with high risk of nephropathy, defined as chronic structural and functional disorders of the kidney. However, the underlying mechanisms are not well understood. In this study, a total of 72 day-old Hy-line chickens were exposed to 300 mg/kg copper sulphate or/and 30 mg/kg arsenic trioxide for 12 weeks. Indicators of oxidative stress, inflammation and heat shock proteins (HSPs) production were analyzed in kidney. The results showed that, when the toxicant was administrated alone, there is an antagonism between redox homeostasis during the first 4 weeks, which follows a collapse of antioxidant system manifested by damaged biomembrane structure. Whats worse, oxidative damage-cascaded histopathological lesions were accompanied by increases of proinflammatory mediators and an imbalance of “Th1/Th2 drift” (Th, helper T cell) regulated by nuclear factor kappa B (NF-κB). Simultaneously, intense heat shock response went with the organism. The above-mentioned renal lesions and indicators changes were time-dependent, more complex and deteriorated effects were observed in Cu/iAs combined groups compared with the others. This study supports Cu and iAs have a synergistic type on the nephro-toxicological process additively. In conclusion, oxidative stress and inflammatory induced by Cu or/and iAs are potential mechanisms in their nephrotoxicity, increased heat shock response may play a renoprotection function in tissues damage.

Arsenic Trioxide Attenuates NF-κB and Cytokine mRNA Levels in the Livers of Cocks

Arsenic (As) is a trace element widely found in nature. It exists in several forms, including organic arsenic, inorganic arsenic, and trivalent arsenic, the most toxic. Arsenic trioxide (As2O3) is widespread in nature. This form tends to accumulate in animals and humans and therefore has a potential harm for them. Cytokines play essential roles in the immune response and inflammation. Although the importance of cytokines in the responses to arsenic exposure has been demonstrated in many types of mammals, the function of these in poultry, especially in chickens, remains unclear. The purpose of the present study was to examine the effect of As2O3 exposure on cytokines in cock livers. In this study, 72 1-day-old male Hy-line cocks were randomly divided into four groups including the control group, low-As group, middle-As group, and high-As group. The livers were collected on days 30, 60, and 90 of the experiment. The levels of nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-12 beta (IL-12β), and interleukin-1 beta (IL-1β) mRNA in the livers of the cocks were measured using real-time PCR. The results showed that the expression levels of IL-6, IL-8, TNF-α, and NF-κB which seemed to be a critical mediator in the inflammatory response tended to increase in the birds chronically treated with As2O3. However, the mRNA expression levels of IL-4, IL-12β, and IL-1β were decreased in the experiment. The information regarding the effects of As2O3 on cytokine mRNA expression generated in this study will be important information for arsenic toxicology evaluation.

Arsenic-induced testicular toxicity in Gallus gallus: Expressions of inflammatory cytokines and heat shock proteins

ABSTRACT The aim of the study is to investigate the effects of sub‐chronic poisoning with arsenic on the testes of chickens treated. Seventy‐two 1‐day‐old chickens were randomly divided into 4 groups and provided food with different doses arsenic. The histological changes were examined. The mRNA levels of inflammatory factors, including transcription factor nuclear factor‐&kgr;B (NF‐&kgr;B), tumor necrosis factor‐&agr; (TNF‐&agr;), inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2), prostaglandin E synthase (PTGEs), and heat shock proteins (Hsps), including Hsp70, Hsp90, Hsp60, Hsp40, and Hsp27 were assessed by quantitative real‐time PCR in the testes of chickens. The protein expressions of iNOS, Hsp60, and Hsp70 were detected by western blot. Increased mRNA and protein levels of inflammatory factors and Hsps with testicular damage showed that arsenic‐induced testicular toxicity includes inflammatory and heat shock response in chickens, and that increased Hsps levels may play a protective role in inflammation damage induced by arsenic on the testes of chickens.

Regulation of autophagy factors by oxidative stress and cardiac enzymes imbalance during arsenic or/and copper induced cardiotoxicity in Gallus gallus

Basal autophagy has an indispensable role in the functioning and maintenance of cardiac geometry under physiological conditions. Recently, increasing evidence has demonstrated that arsenic (As)/copper (Cu) play important roles in the autophagy of the heart. The current study was to evaluate whether oxidative damage by As or/and Cu was correlated with autophagy through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway in the heart of birds. Arsenic trioxide (30mg/kg) or/and cupric sulfate (300mg/kg) were administered in a basal diet to male Hy-line chickens (one-day-old) for 12 weeks. The results showed that heart weight/body weight ratio decreased in the As + Cu group only at 4, 8 and 12 weeks. Moreover, we observed that As or/and Cu decreased high-density lipoprotein cholesterol (HDL-C) concentrations, increased total cholesterol (T-CHO) concentrations and cardiac enzymes activities in the serum. On the other hand, As or/and Cu significantly reduced the activities of total antioxidant (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px)) along with decreased nonenzymic antioxidant (glutathione (GSH)) concentrations and increased malondialdehyde (MDA) concentrations in the heart. Furthermore, As or/and Cu could induce autophagy in the heart of chickens through decreased mRNA levels of TORC1, TORC2, microtubule associated light chains 3-I (LC3-I) and increased PI3K, AKT1, Beclin1, autophagy associated gene 4B (Atg4B), microtubule associated light chains 3-II (LC3-II), autophagy associated gene 5 (Atg5) and Dynein. Meanwhile, ultrastructural examinations showed that As/Cu could result in the appearance of autolygosomes, autophagic vacuoles and double-membrane structures in the heart. In conclusion, As or/and Cu induced cardiac damage and autophagy via elevating cardiac enzymes activities, inducing oxidative stress and activating the PI3K/AKT/mTORC pathway in heart of chickens. Moreover, As and Cu had a possible synergistic relationship in the heart of chickens.