Antioxidant Systems in Poultry Biology: Heat Shock Proteins

Commercial poultry production is associated with various stresses decreasing productive and reproductive performance of birds. A growing body of evidence indicates that most of stresses in poultry production at the cellular level are associated with oxidative stress due to excess of free radical production or inadequate antioxidant protection. Recently, a concept of the cellular antioxidant defence has been revised with special attention paid to cell signalling. Antioxidant systems of the living cell are based on three major levels of defence and include several options [1]: Decrease localized oxygen concentration; decrease activity of pro-oxidant enzymes and improve efficiency of electron chain in the mitochondria and decreasing electron leakage leading to superoxide production; prevention of chain initiation by scavenging initial radicals due to inducing various transcription factors (e.g., Nrf2, NF-κB and others) and ARE-related synthesis of AO enzymes (SOD, GSH-Px, CAT, GR, GST, etc.); binding metal ions (metal-binding proteins) and metal chelating; decomposition of peroxides by converting them to non-radical, nontoxic products (SeGSH-Px); chain breaking by scavenging intermediate radicals such as peroxyl and alkoxyl radicals (vitamins E, C, GSH, uric acid, ubiquinol, bilirubin, etc.); repair and removal of damaged molecules (methionine sulfoxide reductase, DNA-repair enzymes, chaperons, etc.) and vitagene activation and synthesis and increased expression of protective molecules (GSH, Thioredoxins, SOD, HSPs, sirtuins, etc.)

Indeed, understanding roles of vitagenes in stress resistance of poultry as a background for the development of effective strategies to deal with stresses is an emerging topic of research [1-5]. It is known that vitagenes are responsible for synthesis of various protective molecules and HSP synthesis is under vitagene control. However, by the time of writing no comprehensive review on the roles and effects of HSPs in poultry biology has appeared. Therefore, the aim of this review is a critical analysis of the role of HSPs in poultry biology with special emphasis to their functions as an essential part of the vitagene network, responsible for adaptive ability of the cells or whole organisms to various stress conditions.

 

The heat shock response (HSR) is one of the main adaptive stress responses of the cell, restoring cellular homeostasis upon exposure to proteotoxic stress, including heat shock, cold, oxidative stress, hypoxia, toxins, chemicals, pathogen, etc. (Table 1) [6-8]. In fact, cooperative interactions between the transcription factors and various homeostatic mechanisms are responsible for effective adaptation to stressful conditions [9-11]. Indeed, to maintain vital life function it is imperative that organisms preserve the integrity of their proteins. Therefore, HSR in vertebrates is characterized by the induction of HSPs and related elements, such as the ubiquitin–proteasome system [7]. Because HSPs act as molecular chaperones that facilitate protein folding and suppress protein aggregation, this response plays a major role in maintaining protein homeostasis. Generally, HSR is regulated mainly at the level of transcription by four heat shock transcription factors (HSFs), including HSF1, HSF2, HSF3, and HSF4, which bind to HSE [9], thus resulting in stimulation of HSPs expression.

Among other heat shock factors, HSF1 has received tremendous attention as the main factor governing the HSR by coordinating stress-induced transcription [12]. Although originally discovered as a response to thermal stress, HSR can be triggered by a variety of stress conditions that interfere with protein folding and result in accumulation of misfolded or aggregated proteins [13]. HSF1 activation is a multistep process that is negatively regulated by chaperones, including HSP90 and HSP70 [14], which sets the stage for rapid induction of gene expression within minutes of cellular stress [15]. In physiological conditions the majority of HSFs form a complex with HSP70 or HSP90 interacting with the HSF1 activation domain. In stress conditions, HSP70 and HSP90 form complexes with denatured proteins, which releases HSFs [16]. Furthermore, in unstressed state, HSF1 is present in the cytoplasm as a latent monomeric molecule. Upon heat shock, monomeric HSF1 is hyperphosphorylated and converts to a trimer with the capacity to bind DNA that accumulates in the nucleus and subsequently binds to the heat shock element within the promoter region of HSP genes. In addition, extensive posttranslational modifications such as phosphorylation, acetylation, and sumoylation are thought to fine-tune HSF1 activity [8, 11]. The increased expression of HSPs continues until the amount of HSP70 and HSP90 reaches the level sufficient to block the activation domain of the HSFs [16].

 

Avian cells express at least three HSFs (HSFs 1-3). Initially, three avian HSF genes corresponding to a novel factor, HSF3, and the avian homologs of mammalian HSF1 and HSF2 have been cloned [17[. The predicted amino acid sequence of HSF3 is approximately 40% related to the sequence of HSF1 and HSF2. Similar to HSF1 and HSF2, the HSF3 message, is coexpressed during developmen

 

Table 1. Chemical and physical inducers with their target genes in the stress response pathway (Adapted from [3, 63, 334])

 

In most tissues, which suggests a general role for the regulatory pathway involving HSF3 [17]. It was shown that the regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3 [18]. Indeed, the regulatory domain was found to be functionally homologous between chicken and human HSF1. In fact, HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock [19]. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1 and heat shock leads to the translocation of HSF3 to the nucleus [19]. It has been shown that HSF1 is rapidly activated by even mild heat shock, while HSF3 is activated only by severe heat shock. In contrast, HSF2 is not activated by heat stress and has been speculated to have developmental functions [20]. Indeed, cHSF3 (chick HSF3) was activated at higher temperatures than the cHSF1. In fact, at a mild heat shock, such as 41oC, only cHSF1 was activated, whereas both cHSF1 and cHSF3 were activated following a severe heat shock at 45oC. Similarly, cHSF3 was activated by treating cells with higher concentrations of sodium arsenite compared to cHSF1. Furthermore, the DNA binding activity of cHSF3 by severe heat shock lasted for a longer period than that of cHSF1. In addition, the total amount of cHSF3 increased only upon severe heat shock, whereas that of HSF1 decreased. Indeed, cHSF3 is involved in the persistent and burst activation of stress genes upon severe stress in chicken cells [20]. It seems likely that denaturation of nascent polypeptides could be the first trigger for the activation of cHSF1 and cHSF3 [20]. It has been suggested that HSF3 has a dominant role in the regulation of the heat shock response and directly influences HSF1 activity. Thus, disruption of the HSF3 gene results in the severe reduction of heat shock gene expression and loss of thermotolerance [21]. In addition, null cells lacking HSF3, yet expressing normal levels of HSF1, exhibited a severe reduction in the heat shock response, as measured by inducible expression of heat shock genes, and did not exhibit thermotolerance.

Important information related to HSFs in avian species has been obtained in experiments with chick embryos. In fact, it was shown that HSF3 was almost constantly expressed in various tissues during early to late chicken embryonic development [22]. The expression of HSF1 was equally high in most tissues early in development and thereafter declined to different levels in a tissue-dependent manner and HSF3 became the dominant heat-responsive factor mediating stress signals to heat shock gene expression in the chicken. Furthermore, the high-level and ubiquitous expression of HSF2 as well as HSF1 and HSF3 in early embryogenesis suggest the involvement of these factors in all developmental processes [22]. It is interesting to note that in avian, HSF1 and HSF3 are maintained in a cryptic monomer and dimer form, respectively, in the cytoplasm in the absence of stress. Upon heat stress, they undergo conformational change associated with the formation of a trimer and nuclear translocation and the nuclear localization signal acts positively on the trimer formation of cHSF3 upon stress conditions [23]. Indeed, avian cells express two redundant heat-shock responsive factors, HSF1 and HSF3, which differ in their activation kinetics and threshold induction temperature. For example, in birds, HSF1 only slightly induces HSP70 expression during heat shock and indeed HSF3 is a master regulator of the heat shock genes in avian cells, as is HSF1 in mammalian cells [24]. Avian cells lacking two heatinducible HSFs, HSF1 and HSF3 were generated [25]. In addition to complete loss of activation of heat shock genes under stress conditions, these cells exhibited a marked reduction in HSP90α expression under normal growth conditions. Reduction in HSP90α expression caused instability of a cyclin-dependent kinase, Cdc2, and cell cycle progression was blocked mainly at the G2 phase, but also at G1 phase even at mild heat shock temperatures. Restoration of HSP90α expression rescued the temperature sensitivity without induction of HSPs [25].

Whereas HSF1 mediates transcriptional activity only in the brain upon severe heat shock, HSF3 is exclusively activated in blood cells upon light, moderate, and severe heat shock, promoting induction of heat-shock genes [26]. Although not activated, HSF1 is expressed in blood cell nuclei in a granular appearance, suggesting regulation of genes other than heat-shock genes. It was shown that HSF1 and HSF3 mediate transcriptional activity of adult tissues and differentiated cells in a nonredundant manner. Instead, an exclusive, tissuespecific activation is observed, implying that redundancy may be developmentally related [26].

The heat shock response regulated by the HSF family is shown to consist of the induction of classical as well as of nonclassical heat shock genes, both of which might be required to maintain protein homeostasis [9]. Recently, additional information on the roles of HSF2 has been obtained. In particular it has been shown that vertebrate HSF2 is activated during heat shock in the physiological range [27].

HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wildtype cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression [27]. In fact, HSF2 was identified as a major regulator of proteostasis capacity against febrile-range thermal stress [27]. It was also shown that chicken HSF3, but not chicken HSF1, induces the expression of the major avian pyrogenic cytokine IL-6 during heat shock [28].

In general, important roles of HSFs in adaptation of poultry to various stress conditions are difficult to overestimate. However, recent genome-wide studies have revealed that HSF1 is capable of reprogramming transcription more extensively than previously assumed; it is also involved in a multitude of processes in stressed and non-stressed cells [29].

 

Heat shock proteins (HSPs) are highly conserved families of proteins discovered in 1962 [30]. Later, it has been realized that most HSPs have strong cytoprotective effects and are molecular chaperones for other cellular proteins. Taking into account current knowledge of the mode of action of HSPs, the name of ‘‘stress proteins’’ would be more appropriate for them but due to historical reasons they are still called HSPs. Indeed, in the case of oxidative stress, HSP network participates in detecting intracellular changes, protecting against protein misfolding and preventing activation of downstream events related to inflammation and apoptosis (Figure 1)[31]. Since oxidative stress plays a major role in a number of diseases and disease mechanisms in human [31] and decreases productive and reproductive performance in farm animals [32], it is likely that any medication/treatment that is able to reduce levels of oxidative stress will make a significant impact on human health and animal performance. Some HSPs are constitutively expressed, whereas others are strictly stress-inducible. Under physiologic conditions, HSPs play an important role as molecular chaperones by promoting the correct protein folding and participating in the transportation of proteins across intracellular membranes and repair of denatured proteins. Therefore, HSPs participate in the regulation of essential cell functions, such as protein translocation, refolding, assembly and the recognition, prevention of protein aggregation, renaturation of misfolded proteins, degradation of unstable proteins, etc. [33]. It should be mentioned that the events of cell stress and cell death are linked and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis [34-35]. A key feature of HSPs is their ability to provide cytoprotection. Synthesis of these proteins under stress conditions is a highly conserved mechanism of the cell response and adaptation is common among all living organisms (Figure 2). In fact, HSPs are synthesized in response to a great variety of cellular stresses, including heat stress, hypoxia, ischemia, hypothermia, virus infections as well as the effects of various toxicants, including mycotoxins [7]. It is important to note that upregulation of the synthesis of HSPs is considered an endogenous adaptive phenomenon leading to improved tolerance to various stress conditions/factors. In mammals and birds the HSP superfamily includes five broadly conserved families of proteins (Table 2). Among them HSP70, HSP90 and HSP32 (HO-1) are considered as vitagenes.

Among the HSPs, HSP70 is one of the most conserved and important protein families and has been extensively reviewed [36-39] and will be briefly dealt with.

 

Figure 1. Functions of HSP under physiological and stress conditions (Adapted from [337])

 

Figure 2. Activation of the HSP by specific stimuli and their protective effects (Adapted from [33])

 

Table 2. Mammalian heat shock proteins (Adapted from [335-336])

 

Here. In fact, HSP70 refers to a family of 70 kDa chaperone proteins. Some of the important house-keeping functions attributed to HSP70 include [40]: import of proteins into cellular compartments; folding of proteins in the cytosol, endoplasmic reticulum and mitochondria; degradation of unstable proteins; dissolution of protein complexes; control of regulatory proteins; refolding of misfolded proteins and translocation of precursor proteins into mitochondria. These molecular chaperones are implicated in a wide variety of cellular processes, including protein biogenesis, protection of the proteome from negative consequences of stress, recovery of proteins from aggregates, facilitation of protein translocation across membranes, as well as disassembly of particular protein complexes and cell signaling for growth, differentiation, and apoptosis [41]. In particular, HSP70 can inhibit apoptosis by interfering with target proteins [42]. In eukaryotic cells, HSP70s are subject to a large number of post-translational modifications [36]. These ATP-dependent chaperones represent central components of the cellular protein surveillance network and are involved in a large variety of protein-folding processes. In fact, they effectively interact with practically all proteins in their unfolded, misfolded, or aggregated states but do not interact with their folded counterparts [36]. A number of eukaryotic proteins are regulated through transient association with HSP70, including steroid hormone receptors, kinases and transcription factors. Eight different and unique HSP70 have been identified in eukaryote cells being distributed in different subcellular compartments, including cytosol, nucleus, endoplasmic reticulum, and mitochondria [43- 44]. The two most important members of the HSP70 family are the constitutively expressed 73 kDa heat shock cognate (HSC73, HSC70, HSPA8) and stress-inducible 72 kDa heat shock protein (HSP72, HSP1A) [45]. Indeed, under normal conditions HSP70 proteins function as ATP-dependent molecular chaperones maintaining important cell functions related to proteostasis [36]. Under various stress conditions additional synthesis of stress-inducible HSP70 enhances the ability of stressed cells to deal with increased concentrations of unfolded or denatured proteins [41]. HSP70 expression is associated with a reduction in JNK1 phosphorylation and/or an increase in oxidative capacity consequential of improvements in mitochondrial homeostasis [46]. It seems likely that HSP70s do not work alone but with a team of cochaperones. Recently it has been found that the organelle distribution of HSP70 is determined by their specific lipid compositions. In particular, HSP70 attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with HSP70 in the extended conformation with acyl chains inserting into hydrophobic crevices within HSP70, and other chains remaining in the bilayer [44]. It seems likely that this could represent an important connection between HSPs and lipid quality control in the cell and the HSP90/HSP70-based chaperone machinery may function as a comprehensive protein management system for quality control of damaged proteins. Actually, in a recently developed model, it was proposed that the heat shock protein HSP90/HSP70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation [47].

In 1978 it was shown that the pattern of proteins synthesized by chicken embryo fibroblasts changes dramatically after heat treatment (45oC for a few hours). In fact, three proteins (Mr = 22,000, 76,000, and 95,000) accounted for almost 50% of the cell's protein synthetic capacity immediately after the heat-shock [48]. The university of the heat shock response and conservation of proteins induced by this type of stress was proven in different experimental conditions. In particular, antibodies to chicken HSPs, cHSP89 and cHSP70, cross-reacted with proteins of similar molecular weights in embryonic and adult chicken tissues and in extracts from widely different organisms ranging from yeast to mammals [49]. Heat-shock polypeptides of identical sizes of 85,000, 70,000, and 25,000 Da were synthesized predominantly in chicken embryo fibroblasts and in many different organs of 18-day-old embryos at 42.5-44°C [50]. Effects of heat treatments on chick embryo fibroblasts, Drosophila embryonic cells, and human lymphoblastoid cells have been compared [51]. Cells from all three species synthesize large HSPs with Mr = 70,000 and 84,000- 85,000. Different small HSPs with Mr between 22,000 and 27,000 are made at high rates in heat-treated chicken and Drosophila cells but could not be observed in human cells. It was found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (HSP89, HSP70, HSP23 and HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2-3 °C) and blocked by preincubation with actinomycin D [52]. Furthermore, incubation of chicken reticulocytes at elevated temperatures (43-45°C) resulted in a rapid change in the pattern of protein synthesis, characterized by the decreased synthesis of normal proteins, e.g., alpha and beta globin, and the preferential and increased synthesis of HSP70 [53]. Indeed, the rapid 20-fold increase in the synthesis of HSP70 was observed after heat shock and preincubation of reticulocytes with the transcription inhibitor actinomycin D or 5,6-dichloro- 1-beta-D-ribofuranosylbenzimidazole blocked the heat shock-induced synthesis of HSP70.

In 1986 Morimoto and co-authors studied organization, nucleotide sequence, and transcription of the chicken HSP70 gene. They isolated a gene encoding a 70,000-Da heat shock protein (HSP70) from a chicken genomic library and showed that the order and spacing of the sequences share many features in common with the promoter for the human HSP70 gene [54]. The expression of HSP70 during maturation of avian erythroid cells was also studied [55]. It was shown that definitive red cells respond to heat shock by a 10- to 20-fold increase in HSP70 protein synthesis with little change in HSP70 mRNA levels. Therefore, the increased expression of HSP70 in cells was due to increased translatability of HSP70 mRNA. Furthermore, the authors showed that HSP70 expression in erythroid cells is lineage specific and although HSP70 was constitutively expressed, neither HSP70 synthesis nor HSP70 mRNA levels were heat shock inducible in primitive red cells.

HSP70 was shown to constitutively expressed in the embryonic chicken lens. In fact, HSP70 mRNA in the embryonic chicken lens was associated primarily with cells in the early stages of fiber formation, and increased transcription of this gene was part of the differentiation process [56]. It was shown that the heat induced increase in HSP70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals [57]. An increase in the amount of HSP70 was detected from the first up to the fifth hour of acute heat exposure (35°C for 5 h), while an increase in HSP70 mRNA peaked at 3 h. It seems likely that heat shock response in avian species is related to temperatures above 41°C. For example, the spatial expression of HSP70 transcripts was detected in chicken embryos under normal incubation conditions and moderate heat stress (41°C) did not induce enhancements on HSP70 mRNA levels [58]. At the same time, acute exposure to severe heat stress (44°C) for one hour resulted in a fifteen-fold increase in HSP70 mRNA levels. It is interesting to note that the return of stressed embryos to normal incubation temperature resulted in increased HSP70 mRNA levels for three hours which was normalised after six hours. The increased expression of HSP70 in broiler chicken embryos was shown to be affected not only by heat (40°C) but also by cold (32°C) stress, and is tissue- and age-dependent [59]. In fact, HSP70 was detected in the liver, heart, breast muscle, and lungs and the brain contained 2- to 5-times more HSP70 protein compared to the other embryonic tissues. These data are in agreement with our observations indicating low level of vitamin E and high levels of PUFAs in chicken embryonic brain [60]. Therefore, increased HSP70 expression is an adaptive mechanism of increasing antioxidant defences. Younger embryos had higher HSP70 synthesis than older embryos, irrespective of the type of thermal stressor [59]. Again, these data confirm our finding about maturation of the antioxidant defences during chicken embryonic development [61].

It was shown that HSP70 expression in postnatal chickens is tissue- and allele-dependent [62]. Indeed, the expression of HSP70 gene in the liver was significantly (more than 2-fold) higher than that in the muscle under normal growth conditions. This could reflect an importance of HSP70 chaperone functions, since the liver is the major site of synthesis of many important proteins. However, during acute heat stress (44oC for 4 hours) the expression of HSP70 gene in the brain was the highest being significantly different from those in the liver and muscle. This adaptive response by HSP70 also is an important mechanism to compensate for relatively low levels of antioxidants in the brain tissue of the chicken [63]. Long-term, moderate heat stress (30-32oC) was associated with significantly increased HSP70 levels in mononuclear blood cells of laying hens [64]. However, the age-dependent responses of different genotypes were not uniform. HSP70 gene expression was genderdependent with significantly higher levels in male than in female chickens [65] and tissue-dependent heat induction of HSP70 expression may correlate with DNA methylation pattern in the HSP70 promoter [66]. During the exposure to heat stress (37±1 oC), the heart, liver and kidney of broiler chickens exhibited increased amounts of HSP70 protein and mRNA. The expression of HSP70 mRNA in the heart, liver and kidney of heat-stressed broilers increased significantly and attained the highest level after a 2-h exposure to elevated temperatures. Significant elevations in HSP70 protein occurred after 2, 5, and 3 h of heat stressing, respectively, indicating that the stress-induced responses vary among different tissues [67]. Furthermore, the expression of HSF3 and HSP70 mRNA in Lingshan chickens (LSC) and White Recessive Rock (WRR) exhibited species-specific and tissuespecific differences during heat treatment [68]. For example, after 2 h of heat treatment, HSP70 expression was significantly higher in the liver and leg muscle of WRR compared to LSC. Recent analysis of genetic diversity of the HSP70 gene in 8 native Chinese chicken breeds indicates presence of 36 variations, which included 34 single nucleotide polymorphisms and 2 indel mutations [69]. Furthermore, 57 haplotypes were observed, of which, 43 were breed-specific and 14 were shared.

HSP expression in the gut could be considered as an important mechanism of the antioxidant protection [5]. However, there were no effects of HSP70 overexpression on intestinal morphology under heat stress, but there was a strong correlation between HSP70 expression and the digestive enzyme activity in broilers [70]. In another study from the same department, HSP70 induction was shown to protect the intestinal mucosa from heat-stress injury by improving antioxidant capacity of broilers and inhibiting the lipid peroxidation production [71]. In fact, HSP70 significantly protected the integrity of the intestinal mucosa from heat stress (36 ± 1°C) by significantly elevating antioxidant enzyme activities (SOD, GSH-Px and total antioxidant capacity) and inhibiting lipid peroxidation to relieve intestinal mucosal oxidative injury.

To investigate the alterations introduced by domestication and selective breeding in heat stress response, two experiments were conducted using Red Jungle Fowl (RJF), village fowl (VF), and commercial broilers (CB). Birds of similar age (30 d old) or common body weight (930 ± 15 g) were exposed to 36 ± 1°C for 3 h [72]. The RJF at a common age and common BW showed significantly higher levels of basal HSP70 and cortisone compared with VF and CB. Heat treatment was shown to significantly increase body temperature, heterophil: lymphocyte ratio, and plasma corticosterone concentration in CB but not in VF and RJF. Irrespective of stage of heat treatment, RJF showed lower heterophil:lymphocyte ratio and higher plasma corticosterone concentration than VF and CB. It was concluded that domestication and selective breeding are leading to individuals that are more susceptible to stress rather than resistant [72]. Furthermore, laying hens exposed to HS (32.6°C) showed higher concentrations of HSP70 in the liver [73]. In addition, kind gentle hens (a line of group-selected hens for high productivity and survivability) had higher concentrations of HSP70 than DeKalb XL hens (commercial line of individually selected hens for high egg production) regardless of treatment.

HSP70 is also shown to be expressed in other avian species. Notably, quail HSP70 showed 98% homology with HSP70 stress protein in Gallus gallus and 99% homology with Numida meleageris [74]. Duck HSP70 gene was also identified and characterised (GenBank: EU678246) and shown to contain no introns [75]. Fifteen variations were identified within the open reading frame. The expression of duck HSP70 gene was tissue-specific and the highest expression level was seen in pectoral muscle [75]. To sum up, the results from the aforementioned studies consistently demonstrate that increased HSP70 expression in chicken tissues is one of the most important protective responses to prevent or deal with, detrimental changes in protein structure and functions due to various stresses. However, there is a need for further research to understand molecular mechanisms of HSP70 regulation in avian species.

Hsp90, the major soluble protein of the cell, has recently received great attention and a range of reviews described its structure, functions and regulation [76-79]. In fact, in the cell, HSP90 is known to comprise 1-2% of total proteins under non-stress conditions and it is further upregulated under stress [80]. For example, heat shock (37–42°C) has been reported to induce HSP90 levels by as much as twofold [81]. Furthermore, fish naturally living in a hot spring with relatively high water temperature (34.4±0.6°C) is characterised by increased levels of all the studied HSPs (HSP70, HSP60, HSP90, HSC70 and GRP75) compared with fish living in normal river water temperature [82].

HSP90 is expressed as a 90 kDa protein and its functional molecule is a homodimer (α/α or β/β) and each monomer consists of three domains. They are NH2- terminal nucleotide binding domain (a binding site for ATP/ADP), the middle domain (the binding site for nuclear localization signal and client proteins) and the Cterminal domain (the site of dimerization and cochaperone binding) [83-84]. The HSP90 family in mammalian cells consists of four major homologs including two cytoplasmic isoforms HSP90α (inducible form) and HSP90β (constitutive form) [85], HSP90B located in endoplasmic reticulum and tumor necrosis factor receptor-associated protein (TRAP) found in mitochondria and the inner membrane space [86] (Table 3). It is interesting to note that HSP90α and HSP90β share 86% amino acid identity and are expressed in all nucleated cells.

 

Table 3. Isoforms of HSP90 (Adapted from [86])

 

HSP90 is a highly efficient, ATP-dependent molecular chaperone involved in the maturation and stabilisation of a wide-range of proteins in both physiological and stress conditions being an important hub in the protein network that maintains cellular homeostasis and function [87]. HSP90 belongs to a family of proteins known as "chaperones," which are solely dedicated to helping other proteins (client proteins) correct folding, function and stability. Indeed, cellular stress causes protein denaturation, and they cannot function properly and must be repaired or eliminated with the help of chaperones [88].

HSP90 deals with more than 200 important clients which are involved in signal transduction, including many steroid hormone receptors, receptor tyrosine kinases, Src family members, serine-threonine kinases, cell cycle regulators, telomerase and many other proteins (Figure 3) [84, 89-90]. It is difficult to overestimate chaperoning functions of HSP90 related to various nuclear proteins regulating DNA replication, DNA repair, DNA metabolism, RNA transcription and RNA processing [84] and the protective action of HSP90 is related to posttranslational modifications of soluble nuclear factors as well as histones [76]

 

Figure 3. HSP90 interactions with client proteins (Adapted from [76])

 

It was suggested that HSP90 clients are associated with major physiological events including signal transduction, cell cycle progression, transcriptional regulation, natural and acquired immunity and intracellular movement of proteins [84, 91]. In fact, HSP90 participates in many cellular processes including cell cycle control, cell survival, hormone and other signaling transduction pathways, often acting as hormone receptors and is considered to be key player in maintaining cellular homeostasis and adaptive response to stress [87]. In many cases, HSP90-assocoated stress response is orchestrated via HSF1, which under stress conditions upregulates several hundred genes including HSP90. It is known that under physiological condition, as a client protein, HSF1 is kept in an inactive monomeric form through the transient interaction with Hsp90 [84]. During stress, HSF1 dissociates from HSP90, homotrimerizes, undergoes phosphorylation and translocates to the nucleus to perform its gene-expression regulatory functions [84]. As a matter of fact, HSP90 is regulated transcriptionally through direct interactions with the transcription factor HSF [92]. Generally, HSP90 is present in cells in equilibrium between a low chaperoning activity ‘latent state’ in physiological conditions and an ‘activated state’, with increased chaperoning efficiency in stress conditions [93]. HSP90 usually works as a complex with other chaperones and over 20 co-chaperones [94] and increased expression of HSP90 have been shown to be associated with the tolerance of hypothermia, cell proliferation, and cell cycle control [95]. In fact, cochaperones assist HSP90 in its conformational cycling, act as substrate recognition proteins and provide additional enzymatic activity [96] . It seems likely that under heat stress conditions, co-chaperones allow HSP90 to prevent aggregation of unfolded proteins [12]. Indeed, HSP90 involves in the folding, stabilization, activation and assembly of its client proteins through the formation of complexes with co-chaperones such as HSP70, HSP40, Hop, Hip and p23 [97].

The molecular chaperones HSP90 and HSP70 form a multichaperone complex, in which both are connected by a third protein called Hop. Indeed, Hop (HSP70/HSP90 organizing protein) facilitates interaction between HSP90 and HSP70 helping substrate to be efficiently transferred from HSP70 to HSP90 [98]. It seems likely that the interplay between the two chaperone machineries affecting the trafficking and turnover of several hundred signaling proteins as well as removal of damaged and aberrant proteins via the ubiquitinproteasome pathway is of great importance for cell viability and adaptability. HSP90 is shown to possess an ATPase activity, which is known to be essential to modulate the conformational dynamics of the protein. In fact, ATP hydrolysis is associated with the HSP90 dimer transitioning into its ‘‘open’’ conformation and releasing the client protein [91]. The system is regulated by posttranslational modifications including phosphorylation, acetylation, nitrosylation and methylation and uses a range of co-chaperones mediating interactions with HSP90 client proteins [84, 87]. Therefore, HSP90 has been considered to be a key factor at the crossroads of genetics and epigenetics [76]

A cDNA clone for the 90kDa heat-shock protein was isolated by direct immunological screening of a chicken smooth muscle cDNA expression library [99]. It was shown that HSP90 is increased in heat-shocked chick embryo fibroblasts [100]. Furthermore, HSP90 from chicken liver has been purified and physically characterised [101]. The protein was shown to be an elongated dimer with a molecular weight of 160,000 and a frictional ratio of 1.6, extensively phosphorylated and partitioned totally into the aqueous phase. A comparison of the amino acid sequence of the chick HSP90 to that of the homologous HSP90 from yeast to man, reveals 64- 96% identity respectively [102]. The authors suggested that two hydrophilic regions A and B may play a role in the interaction of HSP90 with other proteins such as steroid hormone receptors. In fact, the dimeric form of the HSP90 was confirmed and its structure was shown to be stabilized by hydrogen bonds [103]. Furthermore, the cDNA-derived amino acid sequence of chick HSP90 revealed a "DNA like" structure: potential site of interaction with steroid receptors [102]. The nucleotide sequence of a 2652 bp derived from a chicken HSP90 genomic clone was reported and two introns have been identified [104]. It was proven that HSP90 gene expression is constitutive and heat inducible. In the chick oviduct cells, HSP90 was located in the cytoplasm as aggregates, often inside small vesicles, while in the apical part of the cell, HSP90 was located at the Golgi complex [105]. The epithelium also exhibited some cells with high levels of HSP90. It is interesting to note that HSP90 is associated with both microtubules and microfilaments [106]. In fact, C-terminal half of HSP90 contains a sequence which is responsible for the cytoplasmic localization of the protein and the cytoplasmic anchoring signal is located between amino acids 333 and 664 [107].

It was shown that in contrast to HSP70, the 35S metabolically-labelled HSP90, which accumulates in the cytosoluble fraction 6 - 8 h after serum treatment, is not preferentially translocated to the nuclear compartment, although a small fraction is always present in the nucleus [108]. It was also demonstrated that serum- or insulininduced accumulation of HSP90α mRNA results from an activation of gene transcription and that hsp90α promotor activity is induced approximately fivefold after serum stimulation. Therefore, chicken HSP90 constitutively expressed in most cells, is up-regulated by thermal stress and by developmental and mitogenic stimuli. Indeed, a transient induced expression of the HSP90α gene takes place at both the messenger RNA and the protein synthesis level. This response is protein synthesis dependent and DNA synthesis independent. A possible link between cell cycle and HSP90α regulation was suggested [109]. It seems likely that the HSP90 alpha and beta genes are the result of a gene duplication event that occurred at the time of the emergence of vertebrates [110]. Furthermore, avian HSP90β mRNA is not inducible by thermal stress or mitogenic stimuli, contrary to the mouse and human HSP90 alpha and beta mRNAs. Indeed, chicken HSP90β is the only vertebrate HSP90 insensitive to heat shock and there are some specific features of HSP90 beta gene structure and location explaining why chicken HSP90 beta mRNA is generally less abundant than alpha and is not inducible by heat shock or serum/growth factor stimulation [111].

The importance of ATP binding and hydrolysis by HSP90 in formation and function of protein heterocomplexes was shown [112]. Chicken HSP90 hydrolysing ATP activity was found to be 10-100-fold lower than that in yeast HSP90 and TRAP1, an HSP90 homologue found in mitochondria [113]. The authors showed that sequences within the last one-fourth of HSP90 regulate ATP hydrolysis. The N-terminal ATP binding domain of HSP90 is necessary and sufficient for interaction with estrogen receptor [114].There are two sites in HSP90 binding ATP. In fact, HSP90 N-terminal domain has a nonconventional nucleotide binding site and HSP90 possesses a second ATP-binding site located on the C-terminal part of the protein [115]. HSP90 chaperone activity was shown to require the full-length protein and interaction among its multiple domains, indicating that the cooperation of multiple functional domains is essential for active, chaperone-mediated folding [116].

The expression of HSP90 increased in the heart, liver and kidney of broilers after exposure to increased temperature for 2 h [117]. In the heart and kidney, HSP90 mRNA transcription levels exhibited the same trend as the protein expression of HSP90. Induction of HSP90 mRNA and HSP90 protein at an early stressing stage indicated that heat stress can directly stimulate and quickly initiate the transcription of HSP90 mRNA and translation of HSP90 protein to protect cells. The HSP90α gene is shown to play an evolutionarily conserved role during somitogenesis in vertebrates in addition to providing protection to all cells of the embryo following stress [118].

It was suggested that HSP90 can participate directly in the function of a broad range of cellular signal transduction components, including retinoid receptor signal transduction [119]. In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity [120]. In fact, chicken HSP90 participates in folding and stabilization of signaltransducing molecules including steroid hormone receptors and protein kinases and both amino- and carboxyl-terminal domains of HSP90 interact to modulate chaperone activity [121]. Depletion of HSP90β induces multiple defects in B cell receptor signaling [122]. Indeed, inhibition of HSP90 with geldanamycin resulted in the inactivation of MAPK/ERK and PI3K/AKT pathways leading to significantly reduced levels of IFN-γ, IL-6 and NO mRNAs in avian macrophages [123].

Therefore, in contrast to mammals, HSP90α but not HSP90β may play a major role in CpG ODN(2007) induced immunoactivation in avian macrophage cells. Collectively, these observations strongly suggest that signaling roles of HSP90 in avian species need further investigation. Recently, four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica have been described [124].

The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. Furthermore, CjHSP90AA1 exhibited a robust response to heat shock treatment.

HO-1 is the stress-inducible isoform of the three HO isoforms described to date, serving as a critical protective mechanism in vertebrate systems responsible for adaptation to oxidative, inflammatory, and cytotoxic stress [125-126]. In fact, HO-1 (32 kDa), also known as heat shock protein-32 (HSP32), is shown to be expressed at a relatively low level in most tissues. It is proven that HO-1 is endoplasmic reticulum phase II enzyme catalysing the rate-limiting step in heme degradation, producing free iron (Fe²⁺), carbon monoxide (CO) and biliverdin [127]. Biliverdin is subsequently reduced to bilirubin by biliverdin reductase. It is interesting to mention that the products of the aforementioned reaction can trigger signalling cascades leading to improvement of antioxidant defences and protection against oxidative stress. In particular, CO can modulate the production of proinflammatory or anti-inflammatory cytokines and mediators having immunomodulatory effects with respect to regulating the functions of antigen-presenting cells, dendritic cells, and regulatory T cells [128]. It seems likely that products of the HO-1 reaction namely CO and biliverdin have also cytoprotective, anti-inflammatory and anti-apoptotic properties in stress conditions [129-131]. Cells exposed to low concentrations of CO were shown to respond by an increase in ROS formation (e.g. oxidative conditioning) with important consequences for inflammation, proliferation, mitochondria biogenesis, and apoptosis [132]. Actually, the degradation of heme by HO-1, the signaling actions of CO, the antioxidant protective action of biliverdin/bilirubin, and the sequestration of Fe²⁺ by ferritin are suggested to contribute to the anti-inflammatory effects of HO-1 [133] and increase stress resistance. Furthermore, recent studies have demonstrated that HO-1 inhibits stress-induced extrinsic and intrinsic apoptotic pathways in vitro [134]. The vital importance of HO-1 in stress adaptation have been confirmed in HO-1-deficient mice models showing atypical proinflammatory immune response [135] with increased vulnerability to endotoxin sepsis [136], defective expression of interferon-β [137] and increased susceptibility to apoptosis [138-139]. Morover, HO-1 knockout mice were characterised by very low survival (~1%–5% of litters) and high levels of oxidative stress with a shortened life span [140]. In fact, HO-1 knockout mice were shown to be extremely sensitive to oxidative stress caused by ischemia and reperfusion [141-142] and to develop anemia associated with hepatic and renal iron overload leading to oxidative tissue injury and chronic inflammation [136]. The aforementioned observations provide substantial evidence to support the implication of HO-1 in stress response.

The half-lives of HO-1 mRNA and protein are shown to be approximately 3 hours and 15–21 hours, respectively [143]. In humans, the HO-1 gene (Hmox1) is located on chromosome 22q12 and consists of four introns and five exons. The regulatory region of the mammalian HO-1 gene has a promoter, a proximal enhancer, and two or more distal enhancers (for review see [144]). The Hmox1 promoter is shown to exhibit a range of binding sites (for AP-1, AP-2, NF-κB, and HIF- 1), as well as HSE sequences, metal response elements and stress-response elements. Therefore, the complex gene structure explains its high sensitivity to induction by diverse pro-oxidant and inflammatory stimuli including heme, dopamine, TNF-α, IL-1β, cysteamine, β-amyloid, H₂O₂, hyperoxia, UV light, heavy metals, lipopolysaccharide, etc. [144]. In vertebrates HO-1 is shown to be upregulated by its substrate heme as well as by a wide variety of stressors including heavy metals, heat shock, ischemia, ROS, RNS, bacterial endotoxins, radiation, hypoxia, H₂O₂, nitric oxide, etc. [145-146]. Furthermore, inflammatory mediators such IL-1, TNF-α, LPS are also shown to upregulate HO-1 in vitro [147- 148]. At the cellular level, HO-1 is highly expressed in the organs participating in degrading senescent red blood cells, including spleen, reticuloendothelial cells of the liver and bone marrow [149] as well as in macrophages [150] and dendritic cells [151]. In fact, HO-1 upregulation in various cells is shown to attenuate the expression of various proinflammatory genes [152-153]. Furthermore, HO-1 is of great importance for building immunocompetence. Indeed, induction of HO-1 in dendritic cells alters their maturation state and interaction with other cells [151, 154], including T lymphocytes [155-156] and macrophages [140, 146, 157-158].

Regulation of HO-1 activity as an adaptive response to stress is mediated via several key initiator and feedback control processes. In particular, the transcriptional regulation of the HO-1 gene is shown to be attributed to several transcription factors including Nrf2, Bach1[159-160], HIF-1 [161] and PPARs [162]. It seems likely that MAPK signaling is involved in HO-1 induction [163]. In particular, the anti-inflammatory cytokine IL-10 was shown to induce HO-1 expression via a p38 MAPKdependent pathway [152]. Indeed, the antiapoptotic effect of CO was shown to be mediated by the activation of the p38 MAPK signal transduction pathway and required the activation of the transcription factor NF-κB [164]. Furthermore, the phosphatidylinositol-3 kinase (PI3K)/Akt signaling also modulates HO-1 activity [165]. In addition, HO-1 is involved in suppression of the expression of the pro-inflammatory cytokine TNF-α, while an HO-1 inhibitor (zinc protoporphyrin) attenuated this effect [152]. Furthermore, HO-1 is an important regulator of cellular metabolism, and its activity may affect NADPH- and oxygen-consuming pathways, including fatty acid synthesis, oxidative metabolism of cytochrome p450, or modulation of ROS generation in phagocytes [140].

Data on HO-1 expression and its protective actions in poultry production are very limited. In early 1990th , HO-1 was purified from liver microsomes of chicks pretreated with cadmium chloride [166]. The molecular weight of the enzyme was shown to be 33,000 Da and the pH optimum of the reaction was 7.4. It was also shown that Hg²⁺ inhibited HO-1 activity by 67% at 10 μM and totally at 15 µM. Comparison of sequences to those derived from cDNA sequences for the major inducible rat and human HO-1 showed 69% and 76% similarities, respectively [166]. Next year, a cDNA from a chick liver library that encodes for HO-1 has been cloned and sequenced [167]. The protein corresponding to this fragment of DNA was found to compose of 296 amino acid residues and has a molecular mass of 33,509 Da. The similarity of chick HO-1 to rat and human HO-1 (nucleotides 66% and amino acids 62%) was confirmed to be moderately high. It was also shown that Cd-dependent induction of HO-1 was due to increased transcription of the gene or stabilization of its message [167]. Similar to mammalian HO-1, chicken HO-1 has five exons and four introns [168]. In the DNA sequence there are consensus sequences corresponding to numerous transcription factor recognition elements, including AP-1, AP-2, NF-kB, C/EBP, c-Myc and a metal-responding element identified in the promoter region [168]. Furthermore, chick HO-1 promoter region responded to sodium arsenite, H2O2 and transition metals, but not to heme. The chick HO-1 promoter region also contains a unique sequence that localized at -3.7 kb upstream of the transcription start site of the chick HO-1 gene and subserves up-regulation of the gene by metalloporphyrins [169-170]. Furthermore, the chick HO-1 promoter region was shown to contain "expanded" by three base pairs AP-1 sites that are important for up-regulation of the gene by heme and cobalt protoporphyrin, but not other metalloporphyrins [170].

HO-1 could be detected in microsomes from all chick or rat organs studied, including spleen, testis and brain [171]. The effects of heme on the induction of mRNA and protein synthesis for HO-1 have been studied in primary cultures of chick embryo liver cells [172]. It was shown that heme increased (up to 20-fold) the amount of mRNA and the rate of HO-1 gene transcription in a dose-dependent fashion. In fact, 7-15 h after heme addition, the half-life of HO-1 mRNA was 3.5 h in the presence or absence of actinomycin D, while the half-life of heme-induced HO-1 protein was 15 h [172]. Similarities were observed with respect to regulation of HO-1 expression in primary chick embryo hepatocytes and chicken hepatoma cells [173]. It seems likely that HO-1 synthesis is under hormonal control. For example, the effects of various hormones on the induction of HO-1 in monolayer cultures in chick embryo hepatocytes were examined [174]. Indeed, insulin is shown to suppress the activity of basal as well as Co²⁺ -induced HO-1, while hydrocortisone suppressed the basal enzyme activity and slightly enhanced Co²⁺ -induced enzyme activity. In contrast, triiodothyronine caused a slight increase of both uninduced and induced levels of the enzyme [174].

There is a range of in vitro studies, mainly with embryonic chick cells, to address possible mechanisms of HO-1 induction by various metals. For example, in primary cultures of embryonic chick liver cells HO-1 activity was shown to be upregulated by inorganic cobalt [175]. Treatment of isolated chick embryo liver cells in vitro with sodium arsenite or melarsoprol also showed a potent induction of HO-1 [176]. In monolayer cultures of chick embryo liver cells the most potent HO-1 inducing action was exhibited by Co²⁺, Cd²⁺, Sb³⁺, As³⁺, and Au¹⁺ followed by lower induction observed with Cu²⁺, Fe²⁺, and Fe³⁺ [177]. In contrast, adding Zn²⁺ (20 μM), Mn²⁺ (50 μM) or cysteine (400 μM) to Co²⁺ -treated cells blocked/inhibited the HO-1 induction. It seems likely that increased HO-1 activity by metal treatment is dependent on fresh RNA and protein synthesis since cycloheximide and actinomycin D blocked the induction of HO-1 [177]. The activity of HO-1in chick embryo is shown to be enhanced by cadmium chloride treatment [178]. It has been suggested that induction of HO-1 by drugs and metals occurs by different mechanisms. For example, a drug phenobarbitone induced HO-1 by increasing hepatic haem formation, while increases in HO activity by metals (cobalt, cadmium or iron) were not dependent on increased haem synthesis and were not inhibited by 4,6- dioxoheptanoic acid [179]. In cultured chick embryo liver cells, synergistic induction of HO-1 by iron, added with the phenobarbital-like drug, glutethimide was hemedependent [180]. Addition of an inhibitor of heme biosynthesis abolished the synergistic induction of heme oxygenase providing evidence for the heme-dependent mechanism of induction. Both HO-1 mRNA and protein levels were shown to correlate with changes in HO-1 activity indicating that glutethimide and iron induce HO-1 at the transcriptional level. Induction of the HO-1 gene by heme is shown to be fundamentally different from that produced by transition metals or sodium arsenite and expression of the HO-1 gene is highly conserved across species [181]. Notably, in chick embryo liver cell cultures, HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. Although 2.5 μM arsenite is shown to induce HO-1 four- to six-fold, this had no effect on degradation of exogenous heme [182].

It seems likely that similar to mammals, in birds HO-1 induction in stress conditions is mediated by various signaling pathways. For example, in chicken hepatoma cells, MAP kinases ERK and p38 are shown to be involved in the induction of HO-1, and at least one AP-1 element is involved in this response [183]. In particular, it was shown that the phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases, upregulated HO-1 gene activity in dose- and time-dependent fashion and both an AP-1 element and a metal responsive element were involved in the PAO-mediated induction of the HO- 1 activity [184]. Indeed, a short (1-15 min) exposure of normal hepatocytes to low concentrations (0.5-3 μM) of PAO is shown to produce a marked increase in mRNA and protein of HO-1, which occurs without producing changes in cellular glutathione levels or stabilization of HO-1 message [185]. Furthermore, preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO-1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO-1 in a dose-dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme [185]. It was shown that induction of the chicken HO-1 gene by sodium arsenite or cobalt chloride is mediated through oxidative stress pathway(s) by activation of AP-1 proteins [186]. It seems likely that vascular endothelial growth factor upregulates HO-1 protein expression in vivo in chicken embryo chorioallantoic membranes by a mechanism dependent on an increase in cytosolic calcium levels and activation of protein kinase C [187].

In chick embryo hepatocytes heme breakdown occurred predominantly, if not solely, by heme oxygenase [188]. It seems likely, that increased HO-1 expression in chicken embryos between internal (day 19) and external pipping (day 20) [189] is an adaptive mechanism responsible for increased protection of tissues during this stressful period of the ontogenesis. Similarly, increased concentrations of vitamin E and carotenoids were observed in chicken embryonic tissues at the same period of time [63], providing an effective protection at hatching. It is well known, that various phytochemicals can affect HO-1 activity [190-191], however, more research is needed to understand molecular mechanisms of their interactions. For example, sulpharaphane containing broccoli extract and four different essential oils were tested in the 2 week old broilers as feed additives for 3 weeks. The phytogenic feed additives increased HO-1 activity in the jejunum, but decreased it in the liver [192]. It is interesting to note that relative mRNA expression of HIF-1 (heart) was increased and HO-1 (heart and liver) was decreased at week 4 in broilers fed with high ME and protein diet [193]. From the aforementioned data it is clear that HO-1 is well investigated in avian species, however, its response to different stresses in commercial and wild birds are still not fully characterised.

Thus, an analysis of the published data leads to the conclusion that HSPs play a significant role in cell/organism protection against various stresses being an integral part of the vitagene network responsible for proteostasis maintenance.

 

The university of the HSR and conservation of proteins induced by heat stress were shown in experiments with various species. As mentioned above, studies on effects of heat stress on the expression of HSPs in avian species started in early 1980th [49-51]. Similarly, exposure of chick myotube cultures to an increased temperature (45°C) caused extensive synthesis of three major HSPs (25 kDa, 65 kDa and 81 kDa). When experimental cells were allowed to recover from heatshock treatment at 37°C for 6-8 h, HSP synthesis declined to levels comparable to those in control cultures maintained at 37°C [194-195]. Therefore, four major chicken stress mRNAs coding HSPs with apparent molecular weights of 88 kDa, 71 kDa, 35 kDa and 23 kDa were separated and their properties were studied [196]. Exposure of the 11-day embryonic chicken lens to elevated temperature (45°C) dramatically increased the synthesis of three HSPs with subunit molecular weights of 89,000, 70,000 and 24,000 Da. Furthermore, the functional half-lives at 37°C of the mRNAs encoding the lens HSPs were about 3-5 hr [197].

The intracellular distributions of the major heat shock proteins, HSP89, HSP70, and HSP24 were studied in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed [198]. It was shown that HSP89 was localized primarily to the cytoplasm and during the restress a portion of this protein was associated with the nuclear region. In contrast, significant amount of HSP70 was shown to move to the nucleus during stress. In general, the nuclear HSPs reappeared in the cytoplasm in cells allowed to recover at normal temperatures. It is interesting to note that sodium arsenite also induces HSPs and their distributions were similar to that observed after heat shock, except for HSP89, which remained cytoplasmic [198]. Reticulocytes, purified from the blood of quail and chickens, responded to heat shock by the synthesis of HSP90, HSP70 and HSP26 (quail) or HSP24 (chicken) and the depressed synthesis of many other proteins normally produced at a physiological temperature [199]. It was shown that the expression of each protein depended upon the particular temperature and duration of heat exposure. It was noted that HSP70 was constitutively synthesized and selectively partitioned between cellular compartments. Furthermore, heat shock induced synthesis of the HSP90, HSP70 and HSP26 in quail was prevented by actinomycin D [199].

Heat shock response is a universal biological protective mechanism in stress conditions. Indeed, cultured bovine, equine, ovine and chicken lymphocytes responded to heat stress by the increased synthesis of HSPs. In particular, HSP70 and HSP90 were synthesized in all species and induction time of the HSPs synthesis comprised 30-60 minutes [200]. Heat shock response is an important mechanism of immune cells protection. Actually, heat-induced chicken macrophages synthesized HSP23, HSP70 and HSP90. The optimal temperature and time for induction of these HSPs was 45-46°C for 1 h, with a variable recovery period for each HSP [201]. A comparison of HSP synthesis among peritoneal macrophages (PM) from chickens, turkeys, quail, and ducks shows the highly conserved nature of heat-shock response within birds. In fact, macrophage cultures from each avian species expressed the three major HSPs (HSP23, HSP70 and HSP90) following heat-shock exposure (1-h heat shock at 45°C) [202]. There was also increased expression of a new HSP called P32, which probably was HSP32 (known as HO-1) in all 4 species. The authors also showed that the duck P32 and HSP23 were lower in molecular mass than their respective homologues expressed in chickens, turkeys, and quail macrophage cultures indicating some species-specific differences between HSPs in avian species [202]. Chicken macrophages (mononuclear phagocytic cell line MQNCSU) exposed to LPS under control (41°C) temperatures expressed enhanced synthesis of classical HSP23, HSP70, and HSP90, as well as heat-inducible 32- kDa protein (P32), and a novel LPS-induced 120-kDa protein (P120). In comparison to LPS treatment, MQNCSU cells exposed to 45°C (HS) expressed HSP23, HSP70, HSP90, and P32 but not P120 [203]. It is interesting to note that lead acetate caused a similar upregulation of the same four HSPs (HSP23, HSP70, HSP90 and P32) previously expressed by macrophages after in vitro and in vivo heat treatment [204]. It seems likely that various nutritional deficiencies could affect HSP response. For example, during acute in vivo heat stress, a HSP response was not inducible in chickens deficient in inorganic phosphorus [205] and they were more susceptible heat stress.

Increased HSPs expression in response to various stresses, including heat stress, is shown to be a universal mechanism in various chicken tissues. For example, both the amount and polyadenylation of HSP70 and ubiquitin transcripts increased when male germ cells from adult chicken testes were exposed to elevated (46°C) temperatures [206]. Similarly, there was a marked increase in HSP70 expression in the brains of female broiler chickens after 4 days (from d35 to d38) of heat treatment (38+/-1 degrees C for 2 h/d) [207]. In addition, in chicken pineal cells several heat shock proteins (HSPs 25, 70, and 90) are shown to be synthesized under temperature conditions [208]. Thermal stress (41°C) caused induction of HSP90α and HSP90β in chicken heart, liver and spleen, but HSP90α and HSP90? mRNA levels were stable in brain. Transcription of HSP70 also increased in all organs from chickens in heat stress groups when compared to chickens in control groups [209]. The elevation of the three HSPs in heart, may act as protective mechanism in adverse environments. For example, three main chicken HSPs (HSP60, HSP70, HSP90), and their corresponding mRNAs in the heart tissue of heat-stressed (37°C for 2-10 hours) broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the level of HSP60 protein in the heart increased and maintained throughout heat exposure [67].

Indeed, there is a great diversity in heat shock response in different tissues. For example, thirty two week old broiler breeders were subjected either to acute (step-wisely increasing temperature from 21 to 35°C within 24 hours) or chronic (32°C for 8 weeks) high temperature exposure. There was a tissue specificity in the response to acute and chronic stress [210]. For example, in the heart, acute heat challenge increased lipid peroxidation and upregulated gene expression of all four HSFs. Furthermore, during chronic heat treatment, the HSP70 mRNA level was increased and HSP90 mRNA was decreased. At the same time, in the liver, protein oxidation was alleviated during acute heat challenge and gene expression of HSF2, 3 and 4 and HSP70 were highly induced. In addition, HSP90 expression was increased by chronic thermal treatment. In the muscle, both types of heat stress increased protein oxidation, but HSF and HSP gene expression remained unaltered and only tendencies to increase were observed in HSP70 and HSP90 gene expression after acute heat stress [210]. The expression of HSP27, HSP70, and HSP90 mRNA in the bursa of Fabricius and spleen of 42-d old chickens were increased due to heat stress (37 ± 2°C for 15 d). However, under the same stress conditions the expression of HSP27 and HSP90 mRNA in the thymus was decreased. In the testes of heat-stressed cockerels (38 °C for 4 hours) the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated [211]. Therefore, acute heat stress impairs the processes of translation, protein folding, and protein degradation resulting in apoptosis and spermatogenesis disturbance. Heat stress in 21-day-old broilers was associated with upregulation of the rectal temperature and the mRNA expression of HSP70 in the liver [212]. Heat stress (40°C for 2 h) in the growing chickens (41 day old) caused significant increases in sera corticosterone, LDH, MDA and SOD, the expression of HSP90 and HSP70 in the pectoralis major. Furthermore, HSP90 was shown to positively correlate with corticosterone and SOD activities [213]. In the chicken hypothalamus the transcripts of HSP90 decreased while HSP40 increased in response to thermal stress (34°C for 24 h) [214].

It seems likely that gene expression changes due to heat stress are of great importance for cell adaptation to stress. For example, heat stress (38°C for 4 hours) was associated with upregulation of 169 genes and downregulation of 140 genes in rooster testes [215]. Differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. Indeed, HSP genes (HSP25, HSP70 and HSP90AA1) and related chaperones were the major upregulated groups in chicken testes after acute heat stress. Heat stress in chickens was associated with 166 differentially expressed genes in the brain, 219 in the leg muscle and 317 in the liver [216]. Six of these genes were differentially expressed in all three tissues and included heat shock protein genes (HSPH1-heat shock 105kDa/110kDa protein 1 and HSP25), apoptosis-related genes (RB1CC1, BAG3), a cell proliferation and differentiation-related gene and the hunger and energy metabolism related gene. Various functional clusters were related to the effects of heat stress, including those for cytoskeleton, extracellular space, ion binding and energy metabolism [216]. Terefore, it is proven that HSP expression in response to increased temperature is a universal cellular mechanism protecting proteins against unfavourable changes, including misfolding and molecular mechanisms of HSR need further research.

It has been noted that exposure of cells to nonlethal elevations in temperature activates cellular stress responses and induces a state of thermotolerance, characterised by increased cell resistance to subsequent lethal insults. Indeed, preconditioning is a phenomenon in which a prior stress provides protection against a subsequent and more severe stressful exposure/dose [217- 218], probably, via hermetic mechanisms. In fact, in early 1980th the idea of thermotolerance received substantial attention. In particular, in studies with Chinese hamster ovary cells thermal tolerance was shown to be developed during chronic or acute heating. For example, cells that expressed thermal tolerance as a result of a chronic heat treatment at 42°C also expressed thermal tolerance to a subsequent acute treatment at 45.5°C [219]. Also, cells heated acutely showed tolerance to chronic hyperthermia. Therefore, when cells are tolerant to chronic hyperthermia they are also tolerant to acute hyperthermia and the reverse is also true.

Physiological studies with wild birds and observations on the natural incubation of eggs by domestic hens indicate that egg incubation temperature has a great fluctuation [220] and it was hypothesized that this could have a positive effect on adaptive ability of chickens [221-222]. Therefore, exposing embryos to periods of high or low temperature during incubation could potentially affect their thermotolerance reflecting the programming effect of early development on the subsequent performance of chicken. Indeed, increasing the incubation temperature during important stages of the embryo development, associated with thermoregulation and stress, was shown to be an effective and long-lasting means of acquiring thermotolerance in growing chickens reared in cages [223-224]. Similarly, exposure of chicken embryo to a high incubation temperature (39.5 °C for 12 h/d between E7 to E16) reduced abdominal fat pad by 8% and increased breast muscle yield [225], and improved FCR [226]. Recently it has been shown that an increasing incubation temperature during early embryogenesis positively influences the growth and carcass traits of the broilers, accompanied with a partly negative impact on meat quality (drip loss, shear force, lightness) [227]. It is interesting to note that the growth effects were sexdependent, as significant weight differences could be only found in female broilers. Furthermore, it was concluded that thermal manipulation (TM) during turkey embryogenesis might have altered the thermoregulatory set point, and thus lowered the embryo metabolic rate with a long-lasting posthatch effects [228]. In fact, during the first week posthatch, myoblast proliferation activity was significantly higher in TM groups compared to controls; however, at 2 wk of age it was significantly lower [229]. Therefore, TM of the chick embryo has been suggested to improve the ability of the chicks to reduce their heat production during thermal stress later in their life. Furthermore, TM was shown to affect embryo physiology, growth, meat yield and processing quality [ 230-234] as well as heat tolerance, associated with lower body temperature until slaughter age [223-224].

However, the data on the effect of TM on thermotolerance published to date are not consistent and several studies reported no effect. Indeed, short periods 1 or 2 °C changes in incubation temperature did not influence hatching efficiency of broilers [231-232, 234- 237] or laying chicks [238]. For example, a higher egg shell temperature within the first embryonic week increases the weight of 21.5 d old broiler embryos but not of 7 d old chicks [239]. Similarly, incubation at higher temperatures between ED 7 and 10 resulted in comparable weights of broilers at day 36 post-hatch [240]. Lower carcass yields of 33 d old broilers and lower live weights and leg weights of 69 d old birds was reported after increasing incubation temperature (+1?C) between ED 3 and 6 compared to embryos at the normal incubation temperature [241]. In general, there was no negative effect of higher or lower temperatures from day 10 to 18 of incubation on hatching performance and hatch weight of laying chicks and prenatal temperature conditioning of laying hen embryos had no advantage on laying performance of hens under temperature stress conditions [242]. Principally, a critical time window of thermal manipulation of the chicken embryo is quite narrow since eggshell temperature manipulations (38.4- to 39.0?C for three hours daily) applied during hatching term (days 19 to 21) negatively affected incubation results and broiler performance, especially mortality due to ascites [243].

It seem likely that inconsistency of the results of TM during avian embryonic development reflects lack of understanding molecular mechanisms of the acquisition of TM. Therefore, even small variations in experimental set up (breed, sex, temperature as well as time and duration of its application, etc.) could substantially affect experimental results. There is no doubt that TM during embryogenesis has long-lasting effects on physiology, which may potentially modulate gene expression and metabolism in peripheral tissues being a background for an adaptive mechanism such as heat tolerance [225]. Without affecting hatchability, TM resulted in lower body temperature at hatching and until d 28 post-hatch and significant changes in plasma thyroid hormone concentrations. Notably, fine tuning of incubation conditions, taking into account the level and duration of increases in temperature and relative humidity during a critical period of embryogenesis is considered to be a powerful tool for improvement thermotolerance (resistance to environmental changes) and growth and development of the posthatch chicks [244]. It seems likely that the induction of epigenetic mechanisms related to control body temperature provides long lasting effect in postnatal development of birds. In fact, DNA methylation and histone modification patterns during the late embryonic and early postnatal development of chickens have been recently described [245] and it would be important to study effects of TM on these epigeneticrelated processes.

Recently the effects of increasing the incubation temperature by 1 °C from day 11 to 20 on the embryonic and posthatch skeletal muscle development of the Peking duck were investigated and gene expression profile of leg muscle tissues from thermally manipulated ducks was assessed [246]. Indeed, altering the incubation temperature had immediate and long-lasting effects on phenotypic changes in the embryonic and post-hatching muscle development. In particular, expression levels of total 1370 genes were altered in muscle tissues by the thermal treatments. In fact, cellular processes including metabolism, cell cycle, catalytic activity, and enzyme regulatory activity may have involved in the muscle mass impacted by thermal manipulation [246]. The transcriptome studies confirmed the complexity of the heat stress response. In fact, heat stress responsive genes in the chicken male white leghorn hepatocellular cell line have been identified [247]. The transcripts of 812 genes were shown to respond to heat stress with 235 genes upregulated and 577 downregulated following 2.5 h of heat stress. Genes whose products function as chaperones, as well as genes affecting collagen synthesis and deposition, transcription factors, chromatin remodelers, and genes modulating the WNT and TGF-β pathways were upregulated. At the same time, genes affecting DNA replication and repair along with chromosomal segregation were found to be predominant among the downregulated genes [247].

As mentioned above, molecular mechanisms of acquired thermotolerance are not fully understood but HSP expression and synthesis are thought to play an essential part in this process. Initial work on effect of early chick exposure to heat stress on the HSP expression and thermotolerance in later life started at the end of 1990th. In fact, when chicks at early age were exposed to heat (conditioning; 36°C for 24h at age of 5 days) to improve thermotolerance, lower levels of HSP induction in the heart and lung tissues was observed in the treated chickens [248]. It was shown that the induction of HSP in the heart and lung tissues of the whole animal correlates with the body temperature. Indeed, at the age of 42 days, challenge with acute heat stress (35°C) resulted in a large increase in cloacal temperature of the control chickens and by a more moderate increase in the conditioned chickens. Mortality during the thermal challenge was significantly higher in the control chickens than in the conditioned ones. However, the synthesis rate of HSP70 and HSP90 during the first hour of heat challenge, accelerated gradually in control chickens, whereas in the conditioned chickens it accelerated only after 3 hours and in a more moderate response [249]. The authors suggested that HSP response does not represent a part of the longterm mechanism that is evoked by the early age conditioning. However, next year that conclusion was challenged. In fact, heat conditioning (41°C for 1 h, daily) in both broiler chickens and turkey poults enhanced expression of HSP90, HSP70, and HSP23 in peripheral leukocytes when these cells were heat stressed in vitro [250]. In chickens, 1wk conditioning was sufficient to enhance the HS response when the leukocytes were heat stressed in vitro in the following week. However, in turkey a 3-wk heat conditioning period, followed by a 2- wk rest period, was associated with maximal induction of the three HSP studied in these experiments. It is interesting to note that enhancement in HSP expression was evident for periods up to 4 wk after termination of the daily heat conditioning episodes [250]. This was further developed in a study where resistance to acute heat stress (41°C, days 39-42) and concentration of HSP70 were increased in chickens subjected to early heat stress during rearing (3 heat stress episodes at 35°C for 4 h per week) [251]. Furthermore, a positive correlation was observed between HSP70 concentration and the time taken for a 3°C increase in rectal temperature. It was shown that a combination of early feed restriction (days 4-6) and heat stress (36°C for 1 hour from day 1 to day 21, FRHT) was associated with better HSP70 response after heat stress (d 41). Furthermore, early stress improved weight gain and resistance to IBD in male broiler chickens under heat stress conditions [252]. The authors attributed the improved heat tolerance and disease resistance in FRHT birds to better HSP 70 response.

Ten years later the interest in role of HSPs in chicken thermotolerance was renewed. TM during embryogenesis (39°C for 9-18h) resulted in a significant increase in mRNA levels of HSP90, HSP60 and HSF1 in muscle, heart and brain tissues during embryogenesis and during thermal challenge (43°C) at days 10 and 28 posthatching [253]. The authors suggested that the changes in HSP90, HSP60 and HSF1 gene expressions could be associated with improved thermotolerance acquisition in TM chicks. However, a recent research from Rajkumar et al. [254] showed that exposure to increased temperature (by 2°C) during incubation (days 16-18) resulted in reduced expressions of HSP70 mRNA in various tissues indicating better thermotolerance of the heat-exposed birds. It seems likely that temperature, duration of heat treatment, stages of embryo development and other differences in expe-rimental set up could be responsible for a variability in HSP response.

It seems likely that long-term results of TM of the avian embryo and thermotolerance acquisition are mediated not only by HSPs, but other molecular mechanisms are also involved. For example, the role of HSP in acquisition of thermotolerance was questioned, since a major inducible HSP, HSP68, was not required for the development of thermotolerance in rat fibroblasts [255] or mouse plasma cytoma cells [256]. Furthermore, it was demonstrated that P388D1, a mouse macrophage tumour cell line, failed to induce HSPs in response to either heat stress (42°C, 1 h) or to heavy metal stress induced by arsenic trioxide (5–20 µM), however, cells exhibited thermotolerance in the absence of induced HSPs. The tolerance was shown to be abrogated in cells treated with cycloheximide (250 ng/ml) suggested that thermotolerance was dependent on de novo protein synthesis [257]. Furthermore, posttranslational histone modifications in the promoters of HSP80 and HSP90 are suggested to be involved in formation of heatacclimation-mediated cytoprotective memory [258]. Indeed, there is a need for more research in this area to elucidate molecular mechanisms of thermotolerance with a specific emphasis to HSPs and their interactions with various elements of the antioxidant defence system, including transcription factors.

Heat shock proteins are important cellular tools in protection against heavy metal toxicity. For example, splenocytes harvested in presence of sodium arsenite were characterised by increased expression of HSP70 and serum levels of HSP70 in broiler chicken also increased after continuous supplying sodium arsenite in drinking water [259]. Similarly, the levels of HSP mRNA (HSP90, HSP70, HSP60, HSP40 and HSP27) and protein (HSP70 and HSPp60) expression in immune organs of chickens were significantly increased in the As₂O₃ treatment groups compared with the corresponding control groups [260]. However, HSP response depends on the toxicant dose used. For example, addition of lead to the chicken diet (200 mg lead acetate/kg diet) significantly decreased feed intake, body weight gain, and feed efficiency, upregulated the antioxidant enzymes gene expression together with the downregulation of glutathione Stransferase and HSP70 in the jejunum [261]. In an in vitro study, Mn had a dosage-dependent effect on HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA expression in chicken spleen lymphocytes: the mRNA expression of the heat shock proteins was induced at lower concentrations of manganese and was inhibited at higher concentrations [262]. Therefore, as manganese concentration increased, the mRNA expression of the heat shock proteins first increased and then decreased.

Since all antioxidants in the body are working together to build the effective antioxidant defence network, the increased concentration of one antioxidant can be associated with down-regulation of another antioxidant element in stress conditions.

Vitamin E is considered to be a main chainbreaking antioxidant in biological systems and its roles in poultry production are greately appreciated [3, 63, 263- 264Ç. It was shown that vitamin E, added to the Vero cell culture prior mycotoxins (citrinin, zearalenone and T2 toxin) was able to prevent an induction of HSP70 expression due to mycotoxins [265]. In isolation-stressed quail, vitamin E or vitamin C were shown to prevent an increase in HSP70 expression in the brain and heart [266]. In crossbred cows, treatment with α-tocopherol acetate during dry period resulted in reduced oxidative stress and HSP70 [267]. In cultured rat hepatocytes vitamin E significantly counteracted the effect of cyclosporine Ainduced increase in HSP70 [268]. Furthermore, in young men, α-tocopherol was shown to prevent the exercise induced increase of HSP72 in skeletal muscle as well as in the circulation [269]. However, in most of cases effect of vitamin E on HO-1 expression is different from the aforementioned effects on HSP70. Indeed, recently it has been shown that vitamin E activated the HO-1 promoter via the cAMP-response element, but not the ARE enhancer, through the extracellular signal-regulated kinase and protein kinase A [270]. It was shown that α-Tocopheryl succinate (α-TOS) increases nuclear translocation and electrophile-responsive/antioxidantresponsive elements binding activity of Nrf2, resulting in up-regulation of downstream genes cystine-glutamic acid exchange transporter and HO-1, while decreasing NF-κB nuclear translocation [271]. It seems likely that α- tocopherol protects human retinal pigment epithelial cells from acrolein-induced cellular toxicity, not only as a chain-breaking antioxidant, but also as a Phase II enzyme inducer, including Nrf-2, SOD and HO-1 induction [272]. Similarly, in a murine prostate cancer model γ-tocopherol-enriched mixed tocopherols significantly upregulated the expression of Nrf2 and its related detoxifying and antioxidant enzymes, including SOD and HO-1 [273]. In rats, protective effect of vitamin E against focal brain ischemia and neuronal death was shown. In fact, vitamin E induced the expression of the alpha subunit of hypoxia-inducible factor-1 (HIF-1) and its target genes, including vascular endothelial growth factor (VEGF) and heme oxygenase-1 [274].

Ascorbic acid is the most important water-soluble antioxidant provided with feed and synthesised within the animal/chicken body [275]. It has been shown that chickens experience a less severe stress response after exposure to high temperatures when they are provided dietary ascorbic acid. In fact, heart HSP70 expression decreased in ascorbic acid-fed chickens and the HSP70 increase after heat was two-fold lower in ascorbic acidfed birds in comparison with the control chickens. Furthermore, plasma corticosterone and heart HSP70 were positively correlated, while plasma ascorbic acid and heart HSP70 were negatively correlated [276]. In the ascorbic acid-fed chickens, neither the lower constitutive HSC70 nor the decreased HSP70 response to heat stress (42°C) in the heart and liver were sex-dependent [277]. A lower expression of HSP70 associated with lower body temperature in heat-stress conditions reflected a lower stress response in the ascorbic acid-fed birds. Indeed, ovary and brain HSP70 expression linearly decreased as dietary vitamin C or vitamin E supplementation increased in heat-stressed quail. However, HSP70 expression of ovary and brain was not affected by vitamin C or E supplementation under thermo-neutral conditions [278].

Effects of ascorbic acid on HSP70 expression were also evaluated in experiments with laboratory animals or in human trials. For example, lymphocytes from nonsupplemented subjects responded to hydrogen peroxide with increased HSP60 and HSP70 content over 48 h. In fact, in vitamin C supplemented subjects, baseline HSP60 (lymphocytes) and HSP70 (muscles) content were elevated, but they did not respond to hydrogen peroxide or exercise [279]. In elderly, increased concentration of vitamins C and E was associated with a reduction in oxidative stress and leukocytes HSP72 [280]. Ascorbic acid was shown to attenuate increase in HSP expression due to various toxic agents or heat stress. For example, human brain astrocyte cells enriched with ascorbic acid before being exposed to ethanol, were reported to be better protected against the alcohol-mediated toxicity than non-supplemented cells, and showed significantly lower concentrations of HSP70 [281]. Ascorbic acid significantly attenuated Cd-induced upregulation of GRP78 in mouse testes [282]. ?yclic heat stress (23 to 38 to 23°C, for 2 h on each of seven consecutive days) activated hepatic HSP70, TNF-α, iNOS, and GSH-Px genes, whereas vitamin C (0.5% in water) during heat stress ameliorated heat stress-induced cellular responses in rats [283]. It is interesting to note that there was a specific disappearance of HSP70 in the testes of 20-dayold ascorbic acid-deficient mice [284]. It seems likely that effects of ascorbic acid on HSPs is not universal and for HO-1 is different from HSP70. Indeed, the HO-1 mRNA and protein level in rat kidney, liver, and lung were highly induced by ascorbate treatment (100 mg/kg b.w.) under normal and HS conditions. In particular, in HS the HO-1 activity in tissues was enhanced by both ascorbate preand post-treatment [285].

Vitamin D is known for its classical functions in calcium uptake and bone metabolism. However, recently, vitamin D has been recognized for its non-classical actions including modulation of antioxidant defences [286-287] through regulating oxidant and antioxidant enzyme genes. It was shown that HO-1 was downregulated in the livers of mice fed the vitamin D deficient diet [288]. At the same time, vitamin D deficiency increases the expression of the hepatic mRNA levels of HO-1 in obese rats [289]. In a model of reperfusion of bilateral femoral vessels pre-treatment of rats with vitamin D3 results in a significant increase in leukocyte HO-1 expression in rat model of reperfusion [290]. By employing microarray technology, the effect of a single dose of 1,25-(OH)2D3 on gene expression in the intestine of vitamin D-deficient rats was evaluated. Indeed, at 3 h, there was a 1.9-fold increased expression of HO-1 [291]. The effects of 1,25-D3 treatment on HO-1 expression following focal cortical ischemia elicited by photothrombosis in glial cells were studied. Postlesional treatment with 1,25-D3 (4 μg/kg body weight) resulted in a transient, but significant upregulation of glial HO-1 immunoreactivity [292].

Carnitine is considered as a novel mitochondriatargeted antioxidant with a range of antioxidant actions [1], while betaine is reported to have antioxidant properties in various oxidative stress-generating model systems [293]. In human endothelial cells in culture carnitine was shown to increase gene and protein expression of HO-1 [294]. Furthermore, in humans and in an animal model it was shown that carnitine-mediated improved response to erythropoietin involves induction of HO-1 [295]. Indeed, L-carnitine treatment was associated with an increased level of HO-1 in the retinal ganglion cells [296]. L-Carnitine prevented increase in HSP70 in the testes of cadmium-exposed rats [297]. It was shown that Acetyl-L-carnitine-induced up-regulation of heat shock proteins protects cortical neurons against amyloidbeta peptide 1-42-mediated oxidative stress and neurotoxicity [298]. Acetylcarnitine induces heme oxygenase (increased the amount and activity of HO) in rat astrocytes and protects against oxidative stress [299]. From the aforementioned data it is clear that carnitine can be considered as an important regulator of the vitagene network.

The influence of hyperosmotic shrinkage and the osmolyte betaine on heme oxygenase expression was studied in cultured rat hepatocytes. Hyperosmolarity transiently suppressed HO-1 induction in response to hemin or medium addition at the levels of mRNA and protein expression. Pretreatment of the cells with betaine largely restored induction of both HO-1 mRNA and protein under hyperosmotic conditions [300].

Selenium is a central part of the antioxidant defence network via at least 25 selenoproteins [32]. The protective effect of selenium against cadmium-induced cytotoxicity in chicken splenic lymphocytes was shown to be mediated via the HSP pathway [301]. Indeed, the mRNA expression of HSPs (HSP27, HSP40, HSP60, HSP70 and HSP90) exposed to 10^-6 mol/L Cd showed a sustained decrease at 12-48 h exposure. Notably, adding to the medium Se (10^-7 mol/L) was associated with a significant increase in the mRNA expression of HSPs, as compared to the control group of chicken splenic lymphocytes. Concomitantly, treatment of chicken splenic lymphocytes with Se in combination with Cd prevented a decrease in the mRNA expression of HSPs due to Cd treatment. A different HSP response to arsenic was observed. The expression of HSPs mRNA and protein (HSP70 and HO-1) in rat liver were increased by 5- and 3-folds in the arsenic-fed animals compared with the control group, and selenium prevented the occurrence of oxidative damage from arsenic and significantly reduced expression of HSPs mRNA and protein [302].

The HSP70 response was shown to be significantly lower in the chickens fed selenium and challenged with either enteropathogenic Escherichia coli or heat stress than in those chickens given no supplemental selenium [303-304]. An acute heat stress induced HSP70 in 22d turkey embryos and the embryos from selenium-fed dams were shown to have less HSP70 after the 3 h post-heat stress recovery period [305] demonstrating that selenium had the ability to reduce the impact of heat stress. In fact, heat stress enhanced HSP70 and HSP27 expression and concentration in chicken spleen and dietary Se prevented the aforementioned increase in HSPs [306]. Similarly, in piglets under heat stress conditions selenium can downregulate the mRNA levels of HSPs in various tissues [307]. The relative messenger RNA (mRNA) and protein expression of HSP60, HSP70, and HSP90 in PBMC was observed highest in heat-stressed goats and Se + vitamin E supplementation decreased the HSP expression [308].

In contrast, Se deficiency increased the mRNA levels of HSPs (HSP90, 70, 60, 40, and 27) in chicken neutrophils [309]. Indeed, HSPs played an important role in the protection of the chicken liver after oxidative stress due to Se deficiency. For example, the mRNA levels of HSPs and the protein expression of HSPs (HSP60, 70, and 90) increased significantly in the Se-deficient group compare to the corresponding control group [310]. In exudative diathesis (ED) broiler chicken model caused by Se deficiency, the antioxidant function was shown to decline remarkably, and most of the HSP expression levels increased significantly in the spleen, thymus, and bursa of Fabricius of the broiler chicks with ED [311]. Indeed, Se deficiency causes defects in the chicken bursa of Fabricius associated with decreased selenoprotein expression [312]. As a compensatory response to changes due to Se deficiency, the mRNA and protein expression levels of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) were significantly increased. Similar observations with Se deficient mouse were recorded. For example, Se deficiency was shown to increase HSP70 levels in mouse testes [313]. A significant increase in the stress-inducible HSP70 gene and protein expression was observed in the mice fed Se-deficient or Se-excess supplemented diet as compared with Se adequate fed group [314]. It is interesting to note that the testis-specific HSP70-2 expression significantly decreased as result of Se deficiency. It is clear that increased expression of HSPs in response to toxic metals is an adaptive mechanism to deal with oxidative stress imposed by such toxicants. Similarly, in the case of Se deficiency increased HSP expression is also an adaptive mechanism to compensate for lack of synthesis of selenoproteins and their antioxidant protective functions. As mentioned above, HSP response to various stressors and to nutritional supplements would depend on many factors, including the model used, stressor nature and strength, etc. For example, in human lens epithelial cells sodium selenite gradually increased the expression of HSP70 in a timedependent manner [315]. In rat hippocampus with ischemia-induced neuronal damage, selenium pretreatment was shown to significantly increase the level of HSP70 when compared with ischemic group [316]. In fact, a significant increase in hippocampal HSP70 expression in the ischemic group was observed but the expression was even higher in the selenium-pretreated group than ischemic group.

Regulatory and health promoting properties of various phytochemicals and their effects on HSPs have received substantial attention and there is a range of comprehensive reviews covering the subject [317-320]. They are beyond the scope of the present review. Therefore, only effects of silymarin, possessing a range of antioxidant-related activities [2], are reviewed below.

It seems likely that SM, similar to other flavonoids, can affect the vitagene network. In fact, SM/silybin affects HSP32 (HO-1) activity in different model systems. For example, As-intoxicated rats showed a significant upregulation of myocardial NADPH (NOX) oxidase subunits such as NOX2 and NOX4 as well as Keap1 and down-regulation of Nrf2 and vitagene HO-1 protein expressions. Pre-administration of silibinin (75 mg/kg/BW) recovered all these altered parameters to near normalcy in As-induced cardiotoxic rat [321]. Similarly, in a model of liver injury caused by alcohol plus pyrazole, SM administration (50 mg/kg/BW) had a protective effect with a trend in restoring the decreased activity of HO-1 and Nrf2 [322].

SM (250 mg/kg/BW) possesses substantial protective effect against B(a)P-induced damages by increasing (restoring) HO-1 (vitagene) activity [323]. Similarly, in vitro SM (500 μM) reduced tBH-induced hepatocyte toxicity by activating HO-1 gene expression [324]. Indeed, the enzyme HO-1 is an important regulatory molecule present in most mammalian cells. In fact, the main function of HO-1 is to break down the prooxidant molecule heme into three products; carbon monoxide (CO), biliverdin and free iron and actively participate in the antioxidant defence in the human/animal body [325]. Indeed, HO-1 is a stress-inducible protein and can be induced by various oxidative and inflammatory signals. From the data presented above it is clear that SM/silibinin can upregulate HO-1 and improve antioxidant defences. It is likely that SM/silibinin can affect other HSPs including HSP70. Indeed, in an in vitro system based on CHO-K1 cells treated with As, SM (5 μM) significantly decreased HSP70 expression previously elevated by As [326]. In another in vitro system based on heat-induced chicken hepatocytes, SM (259 μM) affected HSP70 expression significantly, preventing its alleviation by heat stress [327]. A similar protective effect of SM (100 mg/kg/BW) on HSP70 was seen in rats given SM for 7 days prior to mesenteric ischemia-reperfusion (I-R) compared to I-R group [328]. It is interesting to note that silybin was identified as a novel HSP90 inhibitor [329]. Therefore, silibinin can decrease HSP70 expression in stressed cells indicating improved AO defences and decrease stress by other means (e.g., Nrf2-related increased AO synthesis). Indeed, effects of silymarin on HSPs in avian species awaits investigation, while other phytochemicals are shown to be effective. For example, resveratrol, a plant phytochemical possessing antioxidant activities, attenuated the heat stress-induced overexpression of HSP27, HSP70, and HSP90 mRNA in the bursa of Fabricius and spleen and increased the low expression of HSP27 and HSP90 mRNA in thymus in 42 d old chickens upon heat stress [330]. Indeed, there is a need for more detailed investigation of the relationship between nutritional antioxidants and HSP expressions in physiological and stress conditions.

The aforementioned data clearly indicate that vitagenes can be modulated by nutritional means. Indeed, Vitamins E, D, C, carnitine, betaine, selenium and some phytochemicals can affect HSP expression and concentration in various stress conditions. It is interesting that the same compounds can affect other vitagenes, namely thioredoxins, sirtuins and SOD [331]. Therefore, it would be of considerable interest to develop an antioxidant-based composition/supplement for decreasing negative consequences of various stresses in poultry and pig production. Such a composition should meet at least five important requirements [1]:

Inclusion of various protective compounds into the diet of farm animals and poultry to decrease negative consequences of stress conditions is complicated, firstly, by a decreased feed consumption at time of stress. Secondly, such an approach has a low flexibility, since the existing feeding systems do not allow to include anything into the feed loaded into the feed storage bins located near the poultry/pig house (usually several tons of feed for several days feeding). Therefore, before the previous feed is consumed, nothing can be added to the feed. However, sometimes it is necessary to supplement animals/poultry with specific additives very quickly to deal with consequences of unexpected stresses (e.g. mycotoxins in the feed, immunosuppression, high temperature, etc.). In such a case, additive supplementation via drinking system is a valuable option [331]. In fact, modern commercial poultry and pig houses have water medication equipment installed, which can be perfectly used for the aforementioned supplementations. For example, an attempt to address the aforementioned option was implemented in a commercial product PerforMax, containing a vitagene-regulating mixture of 28 compounds, including antioxidants (vitamins E and C), carnitine, betaine, minerals (Zn and Mn) and essential amino acids, and supplied via drinking water. Its efficacy in fighting stresses in commercial poultry production has been recently reviewed [4] and prospects of its use to maintain gut health in weaned piglets and newly hatched chicks was considered [5].

Indeed, it is well known that commercial animal/poultry production is associated with a range of stress conditions including environmental (high temperature), nutritional (mycotoxins and oxidized fat) or internal (vaccinations, disease challenges, etc.) stresses [4, 32, 63]. In such conditions, supplying the PerforMax with drinking water was shown to have protective effects in growing birds [332-333] as well as in adult birds [4] helping maintain their health, productive and reproductive performance. Therefore, the aforementioned results are the first step to go from the development of the vitagene concept to designing a commercial product and testing it in the commercial conditions of poultry and pig production. We can suggest that this idea could be realized in human nutrition as well. Clearly more research is needed to understand a fundamental role of vitagenes in adaptation to various stresses.

 

From the aforementioned analysis of the data related to HSPs in poultry physiology and adaptation to stresses it is possible to conclude:

 

ACKNOWLEDGEMENT: None

CONFLICT OF INTEREST: The authors declare that they have no conflict of interest.

 

 

 

 

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