Luisa Cigliano

Carlo Nebbia

Guido Rychen

Cyril Feidt

Flavia Girolami

Cristina Rossetti

Maria Stefania Spagnuolo

Evaluation of serum markers of blood redox homeostasis and inflammation in PCB naturally contaminated heifers undergoing decontamination

Keywords: Biomarkers, Environmental pollution, PCB decontamination, Redox homeostasis, Haptoglobin, Oxidativestress

Dioxins and polychlorinated biphenyls (PCBs) are widely spread and long persistent contaminants. The aim of this study was to evaluate physiological changes associated with the decontamination of animals previously exposed to environmental pollutants. Eight Limousine heifers were removed from a polluted area and fed a standard ration for six months. The extent of contamination was defined by measuring total toxic equivalents (TEQ) values of dioxin like-PCBs (DL-PCBs), polychlorinated dibenzop-dioxins (PCDDs), and polychlorinated di-benzofurans (PCDFs), and NDL-PCBs amount in pericaudal fat two weeks after the removal from the contaminated area (day 0) and then bimonthly for six months during the decontamination (days 59,125, and 188). The concentrations of both DL-PCBs + PCDD/Fs and NDL-PCBs at the start of decontamination (day 0) were higher than those legally admitted, and they were strongly decreased at the end of the experimental period. Specific indices of blood redox homeostasis and inflammation were also measured at each time. Serum concentrations of Retinol, Tocopherol and Ascorbate, the total antioxidant capacity (TAC) and the activities of superoxide dismutase and glutathione peroxidase were lower at day 0 than after 59,125 or 188 days of decontamination. Protein-bound carbonyls (PC), nitro-tyrosine (N-Tyr), and lipid hydroperoxides concentrations were higher at day 0 than during decontamination. Inaddition, TAC, PC and N-Tyr levels correlated with both DL-PCB and NDL-PCB concentrations only at day 0. Serum concentrations of TNF-alpha and Haptoglobin were higher in samples collected at day 0 than in those obtained during decontamination. As Haptoglobin and TNF-alpha levels correlated with both DL-PCB and NDL-PCB concentrations at day 0 and at day 59 (when these concentrations are still over legal limit), they might represent easily measurable parameters for assessing acute exposure to pollutants. Further both N-Tyr and TNF-alpha concentrations could be used asbio-monitoring markers of the decontamination procedure.

Introduction __________________________________________________________________

Persistent organic pollutants (POPs) are toxic to human health and ecosystems, and are largely transferred in the food chain [START_REF] Rychen | Terms and risk of transfer of persistent organic pollutants into milk[END_REF][START_REF] Antignac | Studying variations in the PCDD/PCDF profile across various food products using multivariate statistical analysis[END_REF]. Several POPs, such as dioxin-like and non-dioxin-like polychlorinated biphenyls (DL-PCBs and NDL-PCBs respectively) are voluntary produced, for commercial purposes, as a result of industrial activities, while other pollutants, such as polychlorinated dibenzo-para-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), designed as dioxins, are unintentional byproducts of industrial processes. Dioxins and PCBs are toxic chemical compounds of great public health concern, and represent the major sources of environmental contamination. Due to their structural stability and volatility are widely spread, thus affecting agricultural areas near or far away from emitting sources [START_REF] Beyer | Assessing longrange transport potential of persistent organic pollutants[END_REF][START_REF] Lohman | Atmospheric fate and transport of dioxins: local impacts[END_REF]. They are also resistant towards chemical and biological degradation processes, thus showing a long persistence in the environment. Although the Stockholm Convention on Persistent Organic Pollutants in 2001 banned PCB production internationally (UNEP, 2001), PCBs are still found in soil, fresh water, aquatic wildlife, and mammals, essentially because of the seepage into the environment, accidental spills, and improper disposal. Food chain contamination occurs as a consequence of the animal ingestion of contaminated water, soil and forage [START_REF] Mclachlan | Mass balance of polychlorinated biphenyls and other organochlorine compounds in a lactating cow[END_REF]Thomas et al., 1999a[START_REF] Thomas | Polychlorinated dibenzo-pdioxin and furan (PCDD/F) uptake by pasture[END_REF][START_REF] Brambilla | Review of aspects pertaining to food contamination by polychlorinated dibenzodioxins, diben-zofurans, and biphenyls at the farm level[END_REF][START_REF] Rychen | Exposure of ruminants to persistent organic pollutants and potential of decontamination[END_REF]. Indeed, dioxins and PCBs are lipophilic and accumulate in animal body, mainly in the liver and in the adipose fraction of organs and tissues [START_REF] Larsen | Risk assessments of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and dioxin-like polychlorinated biphenyls in food[END_REF], being also transferred into milk and eggs. Therefore, the consumption of animal products rich in fat represents by far the major source of exposure for humans (Thomas et al., 1999b;[START_REF] Schecter | Dioxins: an overview[END_REF]. However, ruminants were reported to be decontaminated [START_REF] Mclachlan | Model of the fate of hydrophobic contaminants in cows[END_REF]Thomas et al., 1999b;[START_REF] Huwe | Laboratory and on-farm studies on the bioaccumulation and elimination of dioxins from a contaminated mineral supplement fed to dairy cows[END_REF][START_REF] Rossi | Monitoring of the declining trend of polychlorobifenyls concentration in milk of contaminated dairy cows[END_REF][START_REF] Rychen | Exposure of ruminants to persistent organic pollutants and potential of decontamination[END_REF], essentially via milk excretion (Thomas et al., 1999b;[START_REF] Glynn | Declining levels of PCB, HCB and p,p′-DDE in adipose-tissue from food producing bovines and swine in Sweden 1991-2004[END_REF][START_REF] Rossi | Monitoring of the declining trend of polychlorobifenyls concentration in milk of contaminated dairy cows[END_REF], or through the allometric increased volume of adipose tissue during growth [START_REF] Chamberland | Clinical chemistry, growth and PCB levels in beef cattle exposed to a PCB fire[END_REF][START_REF] Glynn | Declining levels of PCB, HCB and p,p′-DDE in adipose-tissue from food producing bovines and swine in Sweden 1991-2004[END_REF][START_REF] Rychen | Exposure of ruminants to persistent organic pollutants and potential of decontamination[END_REF].

Dioxins and DL-PCBs share a common toxicity mechanism, that is mediated via binding to a specific intracellular receptor, the aryl hydrocarbon receptor (AhR) [START_REF] Alsharif | Oxidative stress induced by 2,3,7,8tetrachlorodibenzo-p-dioxin is mediated by the aryl hydrocarbon (Ah) receptor complex[END_REF][START_REF] Mandal | Dioxin: a review of its environmental effects and its aryl hydrocarbon receptor biology[END_REF], whose activation is responsible for the enhanced expression of genes coding for cytochrome P450 1 family enzymes in liver of several species, including cattle [START_REF] Safe | Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans[END_REF][START_REF] Whitlock | Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzop-dioxin action[END_REF][START_REF] Machala | Effects of chronic exposure to PCBs on cytochrome P450 systems and steroidogenesis in liver and testis of bulls (Bos taurus)[END_REF][START_REF] Matsumura | On the significance of the role of cellular stress response reactions in the toxic actions of dioxin[END_REF][START_REF] Guruge | Differential induction of cytochrome P450 1A1 and 1B1 mRNA in primary cultured bovine hepatocytes treated with TCDD, PBD D/Fs and feed ingredients[END_REF].

In particular, PCB 126, the most potent AhR agonist among DL-PCBs [START_REF] Bandiera | Binding of polychlorinated biphenyls classified as either phenobarbitone-, 3-methylcholanthrene-or mixed-type inducers to cytosolic ah receptor[END_REF], not only alters the expression of genes coding for CYP1A1, but also impairs the expression of genes coding for antioxidant enzymes, resulting in oxidative stress in the liver [START_REF] Hassoun | Induction of oxidative stress in the tissues of rats after chronic exposure to TCDD, 2,3,4,7,8pentachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl[END_REF][START_REF] Parkinson | Immunochemical quantitation of cytochrome p-450 isozymes and epoxide hydrolase in liver microsomes from polychlorinated or polybrominated biphenyl-treated rats. A study of structure-activity relationships[END_REF]. Dioxin exposure was reported to promote, via AhR activation, highly reactive oxygen species (ROS) production [START_REF] Slezak | Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)[END_REF][START_REF] Nebert | Role of the aromatic hydrocarbon receptor and (Ah) gene battery in the oxidative stress response, cell cycle control, and apoptosis[END_REF][START_REF] Dalton | Induction of cellular oxidative stress by aryl hydrocarbon receptor activation[END_REF], and to depress several ROS quenching systems [START_REF] Ishida | Attenuation of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity by resveratrol: a comparative study with different routes of administration[END_REF], thus inducing increased DNA fragmentation, as well as production of superoxide anion, thiobarbituric acid reactive substances, and hydroperoxides [START_REF] Shertzer | Enzyme induction by L-buthionine (S,R)-sulfoximine in cultured mouse hepatoma cells[END_REF][START_REF] Zhao | Modulation of hepatocyte gene expression by the carcinogen benzo[a]pyrene[END_REF][START_REF] Slezak | Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)[END_REF][START_REF] Slezak | TCDDmediated oxidative stress in male rat pups following perinatal exposure[END_REF]. This leads to oxidative conditions [START_REF] Shertzer | Dioxin causes a sustained oxidative stress response in the mouse[END_REF][START_REF] Slezak | Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)[END_REF][START_REF] Senft | Dioxin increases reactive oxygen production in mouse liver mitochondria[END_REF] that may induce oxidative stress-related processes [START_REF] Mandal | Dioxin: a review of its environmental effects and its aryl hydrocarbon receptor biology[END_REF][START_REF] Pelclova | Increased oxidative/nitrosative stress markers measured non-invasively in patients with high 2,3,7,8-tetrachloro-dibenzo-p-dioxin plasma level[END_REF], that are associated with modifications of physiological and metabolic functions [START_REF] Halliwell | Free radicals, other reactive species and disease[END_REF]. NDL-PCBs, due to their chemical properties, have a low affinity for AhR, and are mainly involved in alterations of signal transduction systems, neurotoxicity, immune suppression and endocrine disruption [START_REF] Fischer | Symposium overview: toxicity of non-coplanar PCBs[END_REF][START_REF] Selgrade | Immunotoxicity: the risk is real[END_REF]. NDL-PCB bioaccumulate preferentially in adipose tissue causing disruption of lipid metabolism and induction of IL-6 and TNF-alpha production [START_REF] Ferrante | Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes[END_REF]. NDL PCBs were also reported to reduce cell viability and induce oxidative stress [START_REF] Westerink | Modulation of cell viability, oxidative stress, calcium homeostasis, and voltage-and ligand-gated ion channels as common mechanisms of action of (mixtures of) non-dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers[END_REF][START_REF] Abella | Nondioxin-like polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) induce chondrocyte cell death through multiple pathways[END_REF].

In physiological conditions, the antioxidant defence system, provided by enzymes and antioxidants, scavenges ROS, thus limiting or preventing oxidative damage [START_REF] Halliwell | Free radicals, other reactive species and disease[END_REF]. Oxidative stress occurs as consequence of an imbalance between ROS production and neutralizing capacity of antioxidant mechanisms [START_REF] Halliwell | Free radicals, other reactive species and disease[END_REF], and is involved in the aetiology of several diseases and metabolic disorders [START_REF] Lomba | Influence of dietary cation-anion and oxidative-antioxidants balances on diseases occurring around parturition in the dairy cows[END_REF][START_REF] Bernabucci | Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season[END_REF][START_REF] Bernabucci | Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows[END_REF][START_REF] Castillo | Oxidative status during late pregnancy and early lactation in dairy cows[END_REF][START_REF] Wilde | Influence of macro and micro minerals in the periparturient period on fertility in dairy cattle[END_REF], also contributing to the reduction of fertility in dairy cows [START_REF] Wathes | Associations between lipid metabolism fertility in the dairy cow[END_REF]. Therefore, the evaluation of blood redox homeostasis has increasingly contributed to knowledge of the processes involved in reproductive and metabolic disorders [START_REF] Campbell | Effect of supplemental dietary vitamin E and zinc on reproductive performance of dairy cows and heifers fed excess iron[END_REF][START_REF] Kankofer | Placental release/retention in cows and its relation to peroxidative damage of macromolecules[END_REF][START_REF] Sordillo | Impact of oxidative stress on the health and immune function of dairy cattle[END_REF], and it has become important as a complementary tool for the evaluation of health and metabolic status of dairy cows [START_REF] Bernabucci | Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows[END_REF][START_REF] Castillo | Oxidative status during late pregnancy and early lactation in dairy cows[END_REF][START_REF] Castillo | Plasma malonaldehyde (MDA) and total antioxidant status (TAS) during lactation in dairy cows[END_REF].

The main objective of this investigation was to evaluate the effect of a decontamination procedure, based on the removal of Limousine heifers from an agricultural area contaminated by emissions of an industrial plant specialized in PCB treatment, followed by the feeding a standard ration, on specific indices of blood redox homeostasis and inflammation, in order to assess the changes associated with decontamination, and to obtain tools for monitoring such a process. We focused on the analysis of oxidative and inflammatory status essentially because both DL-PCBs and NDL-PCBs, although through different pathways, are able to disrupt redox homeostasis and to induce inflammatory response.

Material and Methods __________________________________________________________

Materials

Bovine serum albumin fraction V (BSA), chemicals of the highest purity, Rabbit anti-human Hpt IgG, Goat anti Rabbit IgG-horseradish peroxidase linked (GAR-HRP), Rabbit antidinitrophenylhydrazine (anti-DNP) IgGs, and standards for high performance liquid chromatography (HPLC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The Nucleosil 100-NH2 column (5 μm particle size, 250 × 4.6 mm i.d.) and the Nova-PAK C18 column (4 μm particle size, 125 × 2 mm i.d.) were obtained from Macherey-Nagel (Duren, Germany). Organic solvents were purchased from Romil (Cambridge, UK). Polystyrene 96-wells plates were purchased from Nunc (Roskilde, Denmark). Nitrated BSA and the kit for titration of lipoperoxide of Cayman Chemical, as well as rabbit anti-nitrotyrosine IgG of Covalab were purchased by Vincibiochem (Vinci, Italy). The dye reagent for protein titration, recombinant bovine TNF-alpha, mouse anti-bovine TNF-alpha IgG, biotinylated anti-TNF-alpha IgG, and Streptavidin conjugated to HRP were from AbD Serotec (Bio-Rad Laboratories, Hercules, CA).

Farm selection and animals

The study was carried out on eight one year old contaminated Limousine heifers reared in a farm close to an industrial plant (Grez en Bouère, 53, Mayenne, France). During the first year of their life, the animals were exposed to emissions of organic pollutants, particularly DL-PCBs and NDL-PCBs. In detail, the heifers here studied were reared in an agricultural area near to Aprochim, an industrial plant specialized in treatment and decontamination of equipments, transformers, and oils contaminated by PCBs. The accidentally exposure to pollutants was essentially due to the ingestion of forages contaminated by emissions of the factory Aprochim. This pollution was identified as "PCB contamination" because a control performed by the local prefecture revealed that samples of milk, meat, forages and dairy products, collected in 8 farms located within 3 km from the industrial plant, contained a PCB concentration higher than that legally admitted. The commercialization of meat and dairy products from these farms was then forbidden, and in several farms animals were slaughtered. Indications on the PCB contamination around Aprochim are available online (www.leparisien. fr/.../pcb-troiselevages-bovins-contamines-abattus-en-m, http://www. vedura.fr/actualite/6945-pollution-pcb-mayenne-troupeauxabattus-usine-aprochim-mise-demeure; http://www.parismatch.com/Actu/ Environnement/Usines-Aprochim-pollueur-vainqueur-595816).

The extent of contamination was evaluated by measuring total toxic equivalent (TEQ) values of DL-PCB + PCDD/F, and NDL-PCB amount in pericaudal fat of each animal. The heifers were removed from the contaminated area (Mayenne, 53) and housed in an experimental facility located far away this area (Meurthe et Moselle, 54, at the "Domaine experimental de la Bouzule", Lorraine University-Ecole Nationale Supérieure d'Agronomie et des Industries Alimentaires; Vandoeuvre, France) for six months. The animals were reared under controlled conditions, and fed a mixed ration based on grass silage, hay, straw, soybean and corn, with the objective of a weight gain of 1000 g/d. The animals were weighed, blood sampled and submitted to a pericaudal biopsy to get 2 g fat samples two weeks after their arrival in the experimental facility (day 0, sampling A) and bimonthly during the decontamination period (59, 125 and 188 days after starting the decontamination; samplings B, C, and D respectively). The fat samples were stored at -20 °C before being analysed.

TEQ values of DL-PCBs + PCDD/Fs in pericaudal fat were measured, in order to monitor and evaluate the efficiency of the decontamination procedure. The analyses were carried out on pericaudal fat because pericaudal biopsies are minimally invasive, and the animals rapidly recover. In addition, a correlation between the dioxins level in caudal and internal (perirenal) fat was demonstrated (EU-RL, 2009), and it was proposed that the easily accessible subcutaneous fat represents a reliable estimate of adipose tissue contamination by pollutants [START_REF] Kim | Fate and complex pathogenic effects of dioxins and polychlorinated biphenyls in obese subjects before and after drastic weight loss[END_REF].

Eight one year old Charolaise heifers reared at the "Domaine experimental de la Bouzule (Meurthe et Moselle, 54)", i.e. far away from any known PCBs source, were also included in the study as control group and sampled on December 2011 (sampling K1) and March 2012 (sampling K2). Charolaise heifers can differ from the Limousine heifers for fat body mass, and for other general characteristics, so they were used as "control" only because represent heifers never exposed to PCB pollution and reared in the experimental farm where the decontamination took place.

Blood samples were centrifuged at 500 g (20 min; 4 °C), and sera were used for measuring the concentration of nonenzymatic antioxidants (Retinol, alpha-Tocopherol, and Ascorbate), and the activities of enzymatic antioxidants, superoxide dismutase (SOD) and glutathione peroxidase (GPx), here used as indices of the antioxidant defence system. The total antioxidant capacity (TAC) was also assessed, as it well reflects the overall antioxidative potential of the whole organism [START_REF] Kankofer | Comparison of antioxidative/oxidative profiles in blood plasma of cows with and without retained fetal placental membranes[END_REF], and effectively describes the equilibrium between pro-oxidants and antioxidants in blood [START_REF] Ghiselli | Total antioxidant capacity as a tool to assess redox status: critical view and experimental data[END_REF]. Oxidative modifications were monitored by measuring serum concentrations of nitro-tyrosine (N-Tyr) and protein-bound carbonyls (PC), for evaluating the extent of oxidative damage to protein, and level of lipid hydroperoxides (LPOs) for assessing the extent of lipid peroxidation, induced by the interaction of free radicals with polyunsaturated fatty acids. Haptoglobin and TNF-alpha concentrations were measured as inflammation markers.

Protein concentration in each serum sample (previously diluted 1:80 in 130 mM NaCl, 20 mM Tris-HCl, pH 7.4) was measured by Bradford assay [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

DL-DL-PCBs and NDL-PCB analysis PCBs and NDL-PCB analysis

Chemical analyses of PCBs were performed at the French National Reference Laboratory (LABERCA, ONIRIS, Nantes) according to the requirements of the quality assurance parameters of the Commission Directive 2002/69/EC and 2002/70/EC of July 2002 laying down the sampling methods and the methods of analysis for the determination of PCBs in foodstuffs and feeding stuffs, respectively. Moreover, analyses were performed upon an accredited system ISO 17025. All the methods used have been validated and are accredited ISO 17025. Furthermore, this research project was conducted under a certified system ISO 9001 v. 2000 standard. Briefly, fat samples and feed (~2-5 g) were weighed, and 18 13Clabelled PCBs from Cambridge Isotope Laboratories and Wellington Laboratories were added as internal standards to each sample. The solid samples were extracted by pressurized liquid extraction (ASE 300, Dionex, Sunnyvale, CA, USA), using mixed toluene (Picograde -1350 Promochem) and acetone (Picograde -1142 Promochem) at 70/30 (v/v). Final measurement of PCBs was performed by GC-HR-MS (gas chromatograph-HP-5ß90) from Hewlett Packard (Palo Alto, CA, USA). The mass spectrometer (JMS 700 D, Jeol, Tokyo, Japan) was set at a resolution of 10,000, in electron ionization mode.

Single Ion Monitoring (SIM) was used to record the two most abundant signals of the molecular ion (35Cl and 37Cl isotopic contribution). A DB-5MS capillary column (30 m × 0.25 mm i.d.,•0.25 Eim film thickness) from J&W was used in splitless mode.

The GC temperature programme was 120 °C (3 min), 20 °C min-1 to 170 °C (0 min), 3 °C min-1 to 245 °C (0 min) and finally 20 °C min-1 to 275 °C (7 min). The GC programme for Signals was Diok V2 software. All these values were automatically corrected by taking into account the recovery rate of the 13C labelled molecules. The PCBs detection limits in the different tissues and serum analysed including all congeners were better than 30 fg g-1 of the matrix.

Determination of antioxidants and lipid hydroperoxides (LPOs)

Ascorbate (Asc) concentration in serum samples was measured by HPLC, using the anion exchange column Nucleosil 100-NH2, as previously described [START_REF] Spagnuolo | Effect of dioxin exposure on several indices of blood redox status in lactating buffalo cows[END_REF]. Retinol (Ret) and alpha-Tocopherol (Toc) levels were titrated by HPLC, using the reverse phase C18 column Nova-PAK C18, according to a published procedure [START_REF] Spagnuolo | Effect of dioxin exposure on several indices of blood redox status in lactating buffalo cows[END_REF]. The total antioxidant capacity (TAC) was measured by the Trolox Equivalent Antioxidant Capacity Assay, according to [START_REF] Miller | A novel method for measuring antioxidant capacity and its application to monitoring antioxidant status in premature neonates[END_REF]. Samples were reacted with the radical 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) [ABTS], and the antioxidant capacity was measured as change of the absorbance at 734 nm, and expressed as EiM concentration of Trolox equivalents [START_REF] Spagnuolo | Synthesis of ascorbate and urate in the ovary of water buffalo[END_REF].

LPO concentration was measured by a colorimetric quantitative assay, using the Lipid Hydroperoxide Assay Kit of Cayman Chemical, according to the manufacturer's instructions

Determination of nitro-tyrosine (N-Tyr)

Nitrated protein levels in serum samples were measured by ELISA, essentially according to a published procedure [START_REF] Cigliano | Characterization of blood redox status of early and mid-late lactating dairy cows[END_REF]. Briefly, samples were diluted (1:500, 1:1500, 1:3000, and 1:9000) with coating buffer (7 mM Na2CO3, 17 mM NaHCO3, 1.5 mM NaN3, pH 9.6), and incubated in the wells of a microtitre plate overnight at 4 °C. Standard curves were obtained with serial dilutions of nitrated BSA. N-Tyr was detected by incubation with Rabbit anti-N-Tyr antibody (1:1500 dilution in 130 mM NaCl, 20 mM Tris-HCl, 0.05% Tween 20, pH 7.3, supplemented with 0.25% BSA; 1 h, 37 °C), followed by GAR-HRP (1:3000 dilution). Colour development was monitored at 492 nm. Data were reported as nmol of N-Tyr per mg of proteins.

Determination of protein-bound carbonyls

PC concentration in serum was titrated by ELISA. Protein derivatiza-tion, sample dilution, and immunodetection with Rabbit anti-DNP antibody were carried out according to [START_REF] Cigliano | Characterization of blood redox status of early and mid-late lactating dairy cows[END_REF]. Data were reported as nmol of carbonyls per mg of proteins.

Evaluation of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities

GPx activity was measured indirectly by a coupled reaction with glutathione reductase (GR), using the glutathione peroxidase assay kit of Cayman Chemical, according to the manufacturer's instructions. GPx activity was expressed as nmol of NADPH oxidized per minute per ml of sample. SOD activity was measured with the superoxide dismutase assay kit of Cayman Chemical, according to the manufacturer's instructions. SOD activity was expressed Unit/ml. One unit of SOD is defined as the amount of the enzyme needed to exhibit 50% dismutation of the superoxide radical.

. Haptoglobin (Hpt) and TNF-alpha titration

Hpt concentration in individual serum samples was measured by ELISA [START_REF] Spagnuolo | Haptoglobin interacts with apolipoprotein E and beta-amyloid and influences their crosstalk[END_REF]. Samples were diluted (1:4000, 1:8000; 1:16,000) with coating buffer, and incubated in the wells of a microtitre plate (overnight, 4 °C). Hpt was detected by Rabbit anti-Hpt IgG (1:1500 dilution; 1 h, 37 °C), followed by GAR-HRP (1:4500 dilution, 1 h, 37 °C). The calibration curve was obtained by assaying the immunoreactivity of 6, 3,1.5, 1, 0.75, 0.5, 0.25 ng of commercial Hpt standard. TNF-alpha concentration was titrated by sandwich ELISA. Aliquots (50 Eil; 12 Eig/ml) of mouse anti-bovine TNF-alpha IgG were coated into the wells of a microtitre plate (2 h, room temperature). The plate was extensively washed and blocked (1 h, 37 °C) by incubation with 1% BSA in PBS (140 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl). Then aliquots (50 Eil) of serum samples (dilution 1:10, 1:30 in PBS) were incubated into the wells (overnight, 4 °C). TNF-alpha was detected by incubation with biotinylated mouse anti-TNF-alpha IgG (1:100 dilution; 2 h, 37 °C) followed by treatment with Streptavidin-HRP (1:1500 dilution; 1 h, 37 °C). The calibration curve was obtained by assaying the immunoreactivity of 20, 10, 5, 2.5, 1, 0.5 pg of recombinant bovine TNF-alpha.

Statistical analysis

The time effect linked to the PCB levels in fat was analysed using the MIXED procedure of SAS, the individual animal being the experimental unit. The student t test was used to compare the mean values between time points.

The samples for measurement of SOD and GPx activities, PC, N-Tyr, Hpt, TNF-alpha, or LPO concentration were processed in triplicate. The titration of Toc, Ret, and Asc was carried out on duplicates. Values were expressed as mean ± SEM. Significance of statistical differences was evaluated by oneway ANOVA, followed by Dunnett's test, for comparing data from samplings performed at days 59,125 and 188 to data from sampling performed at day 0, or by Tukey's test for multiple comparisons, using the GraphPad Prism 5.01 programme (Graph Pad Software, San Diego, CA, USA). Differences were considered statistically significant when the two-sided P value was less than 0.05. The GraphPad Prism 5.01 programme was also used to calculate correlation coefficient (Pearson's r).

Results ______________________________________________________________________

Animal growing performances and contamination profile in pericaudal fat

During the 6-month experimental period, the average live weight of the animals increased from 337 to 524 kg, i.e. a mean daily weight of about 1000 g/d. This weight gain was in line with expectations for one year old Limousine heifers.

The analysis of PCDD, PCDF, DL-PCBs, and NDL-PCBs was carried out on pericaudal fat samples essentially because the European Union Regulation indicated Maximum Residue Levels of PCB legally admitted in bovine fat. In addition, the amount of PCBs stored in adipose tissue occurs as a result of bioaccumulation of these toxins over a lifetime of exposure, and it was suggested that the measurement of fat-adjusted values accurately describes the total burden of PCBs residing in adipose tissue through-out the body [START_REF] Patterson | Correlation between serum and adipose tissue levels of 2,3,7,8tetrach lorodibenzo-p-dioxin in 50 persons from Missouri[END_REF].

As shown in Table 1, both DL-PCBs and NDL-PCBs significantly contributed to bovine contamination. In particular, at the start of the experimental period (time 0, sampling A; two weeks after the removal of the animals from the contaminated area and their arrival to the uncontaminated experimental farm) TEQ value of DL-PCBs and PCDD/Fs was 25.34 ± 1.405 pg/g fat (Table 1). This value was about 6 fold higher than that legally permitted, fixed at 4 pg/g fat (European Union Regulations No 1881/2006, 1259/2011). DL-PCBs provided by far the highest contribution to the TEQ value (about 96%; Table 2), while PCDD/Fs represented less than 4% of the total TEQ (0.945 ± 0.063 pg/g of fat). The high proportion of DL-PCBs was probably due to the origin of the contamination (ingestion of forages contaminated by the emissions of an industrial plant specialized in treatment and decontamination of PCB).

The analysis of contamination profile (Table 3) demonstrated that the more abundant congener among DL-PCBs was PCB 126 (92% of the DL-PCBs), followed by PCB 169 (5.5%) and PCB 118 (1.6%). In addition, the more represented congeners among PCDD/Fs were 2,3,4,7,8-PeCDF (33%), and 1,2,3,7,8-PeCDD (25%), followed by 2,3,7,8-TCDD (9.6%), 1,2,3,6,7,8-HxCDD (9.2%) and 1,2,3,4,7,8-HxCDF (8.5%).

Depletion curve of DL-PCB + PCDD/Fs (Fig. 1) is described by the equation y = 20.522e-0.011x, and demonstrates that half life of these compounds is 44 days. Further, 148 days were necessary to reach the threshold value for DL-PCB + PCDD/Fs (4 pg WHO-TEQ/g fat), and at the end of the experimental period, all animals were considered decontaminated, as TEQ value in pericaudal fat was lower than that legally admitted (European Union Regulation No 1259/2011). Depletion curves of more abundant congeners among PCDD/Fs are shown in Supplementary Fig. S1. Depletion curves of more abundant congeners among DL-PCBs are shown in Supplementary Fig. S2. Half lives of PCB 126, PCB 169 and PCB 118 were calculated and they were found to be 43, 40, and 59 days respectively. NDL-PCB concentration at the beginning of the decontamination period (165.9 ± 14.42 ng/g of fat) was about 4 fold higher than that legally admitted (Table 1), thus strongly contributing to bovine contamination. As shown in Table 4, the highest contribution to NDL-PCB contamination was from PCB 153 (42%), PCB 138 (29%), and PCB 180 (28%). Depletion curve of NDL-PCB (Fig. 2), described by the equation y = 132.87e-0.01x, demonstrates that half life of these compounds is 47 days, and the threshold value of NDL-PCBs (40 ng/g of fat) was reached within 120 days. The specific contamination profile (Table 4) demonstrated that the more abundant congeners were PCB 153 (42.4% of total; calculated half life 47 days), followed by PCB 138 (29.3%; calculated half life 49 days) and PCB 180 (27.9%; calculated half life 46 days). Depletion curves of each congener are shown in Supplementary Fig. S3.

Analysis of serum antioxidants and total antioxidant capacity

Serum concentrations of Ret, Toc, and Asc, the total antioxidant capacity (TAC), as well as GPx and SOD activities were measured as markers of the antioxidant defence system (Table 5).

Ret concentration was positively correlated with Toc concentration only in control animals (r = 0.805, P = 0.016, sampling K1; r = 0.930, P = 0.007, sampling K2), and in the samplings performed 125 (sampling C) and 188 (sampling D) days after starting the decontamination process (C, r = 0.752, P = 0.027; D, r = 0.769, P = 0.035). As depicted in Table 5, the concentrations of Toc, Ret, and Asc were significantly lower in samples collected at the start of experimental period (day 0; sampling A) with respect to either the control (Toc, 25.88 ± 0.47%; Ret, 47.40 ± 0.21%; Asc, 35.50 ± 3.51%; P = 0.001) or to animals sampled during the decontamination period (Toc, 18.54 ± 0.78%; Ret, 47.82 ± 3.07%; Asc, 30.51 ± 1.27%; P = 0.001). No statistically significant differences between values measured in samples from control cows and those collected from animals undergoing decontamination (sampling B, C, or D) were recorded.

Similarly the TAC, and the activities of SOD and GPx were significantly more elevated (55.36 ± 3.69%, 53.05 ± 0.87%, and 83.48 ± 5.69% respectively; P = 0.001) in serum from control cows than in samples taken at the beginning of decontamination (A), and no differences between control samplings and samplings taken during decontamination (B, C and D) were revealed.

A positive correlation between TAC and Asc concentration was found in control cows and in animals undergoing decontamination (K1, r = 0.853, P = 0.007; K2, r = 0.868, P = 0.025; B sampling, r = 0.793, P = 0.019; C sampling, r = 0.787, P = 021; D, r = 0.912, P = 0.004; data not shown), but not in cows sampled at time 0 (sampling A). Likewise, Asc concentration was positively correlated with SOD activity in unexposed cows and in cows fed the decontamination diet (K1, r = 0.818, P = 0.013; K2, r = 0.823, P = 0.040; B, r = 0.732, P = 0.039; C, r = 0.876, P = 004; D, r = 0.852, P = 0.015; data not shown). Finally TAC was negatively correlated with total TEQ of DL-PCBs + PCDD/Fs (r = -0.8523, P = 0.007; Fig. 3,panel A), and with NDL-PCBs (r = -0.789, P = 0.035; Supplementary Fig. S4,panel A) in samples collected at the beginning of experimental period.

Analysis of serum PC, N-Tyr and LP

As reported in Table 6, serum levels of N-Tyr and PC were found significantly higher insampling performed at time 0 (A) than in those from control animals (40.75 ± 0.38% and 44.78 ± 1.38% respectively; P = 0.001), or in those taken during the decontamination period (33.35 ± 2.73% and 43.80 ± 1.98% respectively; P = 0.001). In addition, the level of N-Tyr was higher (17.43 ± 0.53%; P < 0.05) in samples collected 59 days after the start of decontamination process (sampling B) than in samplings from unexposed animals, and it was also higher (11.66 ± 1.36%; P < 0.05) with respect to the samplings performed after 125 (sampling C) and 188 days (sampling D) of decontamination. Conversely, the level of PC did not differ among these groups. When compared with serum samples collected at time 0 (sampling A), statistically significant lower LPO concentrations were detected in the control cows (51.11 ± 0.35%; P = 0.001), as well as in cows under decontamination (44.09 ± 1.25%; P = 0.001). No differences between control samplings and samplings taken during decontamination (B, C and D) were observed.

Serum titre of N-Tyr was negatively correlated with SOD activity, Asc concentration, and TAC in both samplings from control cows, namely K1 (SOD, r = -0.790, P = 0.020; Asc, r = -0.866, P = 0.006; TAC, r = -0.718, P = 0.045), and K2 (SOD, r = -0.885, P = 0.019; Asc, r = -0.906, P = 0.013; TAC, r = -0.898, P = 0.015). Further, the concentration of N-Tyr was negatively correlated with TAC and SOD activity both in contaminated heifers (sampling A, r = -0.736, P = 0.037; r = -0.765, P = 0.027, respectively) and in sera collected during the decontamination i.e. B (r = -0.0718, P = 0.041; r = -0.853, P = 0.007, respectively), C (r = -0.899, P = 0.002; r = -0.784, P = 0.042, respectively), and D (r = -0.950 P = 0.001; r = 0.756, P = 0.043).

Finally, both PC and N-Tyr concentrations were correlated with total TEQ of DL-PCBs + PCDD/Fs (sampling A, r = 0.942, P = 0.0005; r = 0.880, P = 0.004 respectively; Fig. 3, panels B and C, respectively) and with NDL-PCBs (r = 0.806, P = 0.029; r = 0.842, P = 0.018 respectively; Supplementary Fig. S4, panels B and C, respectively) in samples obtained at the start of decontamination.

Analysis of Hpt and TNF-alpha

As shown in Fig. 4, serum Hpt concentration was found lower in samples collected at the start of decontamination (A) compared with samplings from control cows (111.9 ± 1.66%; P < 0.001), and with samplings taken during the decontamination phase (80.88 ± 7.45%; P < 0.01). No statistically significant differences among control animals and animals undergoing decontamination were found. TNF-alpha concentration was significantly higher in samples collected at time 0 (A) and after 59 days of decontamination (B) than in those from control animals (A, 46.06 ± 3.44%, P < 0.001; B, 35.30 ± 4.13%, P < 0.05), or from samplings taken after 125 (C) and 188 (D) days of decontamination (A, 41.46 ± 3.08%, P < 0.001; B, 29.77 ± 3.69%, P < 0.05) (Fig. 4). Values from control cows and from samplings C and D did not significantly differ. Further, Hpt concentration was correlated with total TEQ of DL-PCBs + PCDD/Fs (Fig. 5) and with NDL-PCBs (Supplementary Fig. S5) in both sampling A (DL-PCBs + PCDD/Fs, r = -0.735, P = 0.038, Fig. 5, panel A; NDL-PCBs, r = -0.913, P = 0.004, Supplementary Fig. S5, panel A), and in sampling B (DL-PCBs + PCDD/Fs, r = -0.946, P = 0.0004, Fig. 5, panel B; NDL-PCBs, r = -0.763, P = 0.028, Supplementary Fig. S5,panel B). Similarly, TNF-alpha level correlated with total TEQ of DL-PCBs + PCDD/Fs (Fig. 5) and with NDL-PCBs (Supplementary Fig. S6) in both sampling A (DL-PCBs + PCDD/Fs, r = 0.881, P = 0.004, Fig. 5, panel A; NDL-PCBs, r = 0.895, P = 0.007, Supplementary Fig. S6,panel A) and in sampling B (DL-PCBs + PCDD/Fs, r = 0.791, P = 0.019, Fig. 5, panel B; NDL-PCBs, r = 0.756, P = 0.03, Supplementary Fig. S6,panel B).

Discussion ___________________________________________________________________

This study was carried out on a group of Limousine heifers that were accidentally exposed to environmental pollutants by the ingestion of forages contaminated by the emissions of an industrial plant, specialized in treatment and decontamination of equipments, transformers, and oils contaminated by PCBs. The pollution was identified as "PCBs contamination" by a control performed by the local prefecture. The heifers were removed from the contaminated area and housed inan experimental facility located far away this area for a decontamination attempt.

In this context it is worth mentioning that DL-PCBs and NDL-PCBs exert numerous and different toxic effects. DL-PCBs act essentially via binding to AhR [START_REF] Alsharif | Oxidative stress induced by 2,3,7,8tetrachlorodibenzo-p-dioxin is mediated by the aryl hydrocarbon (Ah) receptor complex[END_REF][START_REF] Mandal | Dioxin: a review of its environmental effects and its aryl hydrocarbon receptor biology[END_REF], that leads to activation of CYP1A and CYP1B genes [START_REF] Safe | Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans[END_REF][START_REF] Whitlock | Genetic and molecular aspects of 2,3,7,8-tetrachlorodibenzop-dioxin action[END_REF][START_REF] Machala | Effects of chronic exposure to PCBs on cytochrome P450 systems and steroidogenesis in liver and testis of bulls (Bos taurus)[END_REF][START_REF] Matsumura | On the significance of the role of cellular stress response reactions in the toxic actions of dioxin[END_REF][START_REF] Guruge | Differential induction of cytochrome P450 1A1 and 1B1 mRNA in primary cultured bovine hepatocytes treated with TCDD, PBD D/Fs and feed ingredients[END_REF]. NDL-PCBs have little or no affinity to the AhR, bind to or activate the constitutive androstane receptor and/or the pregnane X receptor, leading to induction of other CYP enzymes [START_REF] Waxman | P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, and PPAR[END_REF][START_REF] Masahiko | Induction of drug metabolism by nuclear receptor CAR: molecular mechanisms and implications for drug research[END_REF][START_REF] Kliewer | The nuclear pregnane X receptor regulates xenobiotic detoxification[END_REF], and display a different toxicological profile [START_REF] Westerink | Modulation of cell viability, oxidative stress, calcium homeostasis, and voltage-and ligand-gated ion channels as common mechanisms of action of (mixtures of) non-dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers[END_REF]. Despite the differences in toxicological profile and mechanisms of action, both DL-PCBs and NDL-PCBs are known to induce oxidative stress and inflammatory response [START_REF] Dutta | Differential gene expression and a functional analysis of PCB-exposed children: understanding disease and disorder development[END_REF][START_REF] Wens | Transcriptomics identifies differences between ultrapure non-dioxin-like polychlorinated biphenyls (PCBs) and dioxin-like PCB126 in cultured peripheral blood mononuclear cells[END_REF][START_REF] Ferrante | Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes[END_REF][START_REF] Westerink | Modulation of cell viability, oxidative stress, calcium homeostasis, and voltage-and ligand-gated ion channels as common mechanisms of action of (mixtures of) non-dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers[END_REF][START_REF] Abella | Nondioxin-like polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) induce chondrocyte cell death through multiple pathways[END_REF].

Our goal was to evaluate the changes in specific markers of blood redox homeostasis and inflammation during the decontamination process. To this aim, we carried out a longitudinal investigation by analysing serum and fat samples collected during a six month period (days 0, 59,125 and 188). Serum samples were used for assessing oxidative and inflammatory status, fat samples for defining the extent of contamination at each time point. We also characterized redox and inflammatory status of eight Charolaise heifers reared in the experimental farm where the decontamination took place. Although Charolaise differ from Limousine heifers for general characteristics, we regarded them as "control" group only because they never ingested contaminated forages.

At the start of experimental period (day 0), TEQ values of DL-PCBs + PCDD/PCDF and NDL-PCB amount in pericaudal fat were 6 and 4 fold higher, respectively, than those legally admitted, so bovine were classified as "exposed" to PCBs. DL-PCBs represented about 96% of the total TEQ, and PCB 126 was the more abundant congener detected, with an estimated half life of 43 days. The highest contribution to NDL-PCB contamination was found to be from PCB 153, PCB 138, and PCB 180, whose half lives were 47, 49 and 56 days respectively. The initial value of both DL-and NDL-PCB progressively decreased during the examined period (3 fold at day 59, 5 fold at day 125, 8 fold at day 188), thus indicating that the procedure of decontamination was effective. Threshold values of DL-PCB + PCDD/Fs and NDL-PCBs were reached within 148 days and 120 days respectively, therefore at the end of the experimental period (188 days) bovine were considered decontaminated. In the experimental period a significant weight gain occurred (184.0 ± 9.82 kg, P b 0.0001), thus suggesting that decontamination depended on both a dilution process and the excretion of the pollutants from the animal organism.

The blood redox status of the cows was characterized by evaluating specific indices of the antioxidant defence system and oxidative damage to lipids and proteins. In line with previously published data [START_REF] Hassoun | Induction of oxidative stress in the tissues of rats after chronic exposure to TCDD, 2,3,4,7,8pentachlorodibenzofuran, and 3,3′,4,4′,5-pentachlorobiphenyl[END_REF][START_REF] Slezak | Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)[END_REF][START_REF] Lai | Acute toxicity of 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) in male Sprague-Dawley rats: effects on hepatic oxidative stress, glutathione and metals status[END_REF][START_REF] Spagnuolo | Analysis of plasma indices of redox homeostasis in dairy cows reared in polluted areas of Piedmont (northern Italy)[END_REF], serum concentrations of Ret, Toc and Asc, as well as the TAC and the activities of both SOD and GPx were found significantly lower (about 47%, 26%, 36%, 55%, 53% and 83%) in samples collected at day 0 from PCB contaminated heifers than in control heifers. The levels of antioxidants significantly increased (about 30%) as decontamination goes on, and were found similar to those of unexposed bovine just after 59 days.

Ret and Toc are dietary liposoluble antioxidants, and their serum concentrations were expected to be correlated [START_REF] Olmedilla | Refer ence values for retinol, tocopherol, and main carotenoids in serum of control and insulin-dependent diabetic Spanish subjects[END_REF][START_REF] Cigliano | Characterization of blood redox status of early and mid-late lactating dairy cows[END_REF]. Actually, a correlation was found only in serum samples collected in the last phase of the experimental period (days 125 and 188), when both DL-and NDL-PCB concentrations in fat were within the legal limits, and namely 5-8 fold lower than those measured at time 0. Interestingly, Asc concentration was positively correlated with both TAC and SOD activity in samples collected during decontamination, irrespective of the sampling time (day 59,125, or 188), but not in the samples obtained at time 0. Taken together, these findings suggest that asevere perturbation of the blood redox homeostasis occurred before starting the decontamination, and that the restoration of physiological concentrations of liposoluble antioxidants might occur more slowly than the other antioxidants here analysed. As expected, a greater extent of oxidative modifications to protein and lipid fractions was detected at the beginning of the experimental period than during decontamination. In particular, PC and LPO concentrations were remarkably higher at day 0 with respect to any other sampling of the decontamination period and of the control cows. As LPO are produced by the oxidative attack of free radicals on polyunsaturated fatty acids, and PC may be generated by reactions of proteins with aldehydes originated during lipid peroxidation processes [START_REF] Uchida | Covalent attachment of 4-hydroxynonenal to glyceraldehyde-3-phosphate dehydrogenase. A possible involvement of intra-and intermolecular cross-linking reaction[END_REF], our results suggest that lipid peroxidation and its intermediates might playa crucial role in determining oxidative modifications during acute exposure environmental pollutants, here assessed by the concentrations of DL-and NDL-PCBs. Interestingly, the levels of N-Tyr, which is considered the footprint of protein oxidative damage induced by peroxynitrite correlated well with the concentrations of DL-and NDL-PCBs. Indeed serum N-Tyr concentration was just reduced of about 28% after 59 days of decontamination procedure. Further, N-Tyr concentration in samples obtained at day 125 and 188, was significantly lower not only than that at day 0, but also than that at day 59, when TEQ and NDL-PCB concentrations were still over the legal limit (Table 1). Therefore, N-Tyr could represent a useful index for monitoring the decontamination procedure. Finally, TAC, PC and N-Tyr levels correlated with fat content of both DL-and NDL-PCBs only at the start of decontamination.

So we suggested that such parameters could contribute to identify animal exposed to these pollutants, to evaluate the extent of animal exposure, and to monitoring the decontamination process.

TNF-alpha is a proinflammatory multifunctional cytokine released in response to several stress signals [START_REF] Chen | TNF-R1 signaling: a beautiful pathway[END_REF][START_REF] Wajant | Tumor necrosis factor signaling[END_REF]. TNF-alpha plays an important role in acute dioxin-induced toxicity, and, although the cross-talk between TNF-alpha and dioxin signalling cascades is a complex, cell-type specific regulated process [START_REF] Haarmann-Stemmann | Growth factors, cytokines and their receptors as downstream targets of arylhydrocarbon receptor (AhR) signaling pathways[END_REF], studies in mice, sheep, and guinea pigs demonstrated that the exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin was associated with the increase of serum TNF-alpha levels [START_REF] Gasiewicz | 2,3,7,8-Tetrachlorodibenzo-p-dioxin tissue distribution, excretion, and effects on clinical chemical parameters in guinea pigs[END_REF][START_REF] Clark | Tumor necrosis factor involvement in 2,3,7,8-tetrachlorodibenzop-dioxin-mediated endotoxin hypersensitivity in C57BL/6J mice congenic at the Ah locus[END_REF][START_REF] Moos | The effects of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) on tumor necrosis factor (TNF) production by peritoneal cells[END_REF]. In addition, it was recently reported that NDL-PCBS induce TNF-alpha production [START_REF] Ferrante | Polychlorinated biphenyls (PCB 101, PCB 153 and PCB 180) alter leptin signaling and lipid metabolism in differentiated 3T3-L1 adipocytes[END_REF]. Accordingly, and similar to the trend observed for N-Tyr, higher levels of serum TNF-alpha were detected in samplings performed at days 0 (about 44%) and 59 (about 33%) as compared to those obtained at days 125 and 188, suggesting that the observed variations of this cytokine might be associated with changes of DL-and NDL-PCBs. In line with this hypothesis, TNF-alpha level correlated with DL-and NDL-PCB amounts measured at time 0 and after 59 days of decontamination.

Hpt is an acute phase protein with innate antioxidant and immunomodulatory activity, and also acts as molecular chaperone, inhibiting the inappropriate self-association of proteins induced by oxidation or heat [START_REF] Saeed | Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding[END_REF][START_REF] Quaye | Haptoglobin, inflammation and disease[END_REF]. Hpt concentration in plasma was reported to be down-regulated in humans exposed to polycyclic aromatic hydrocarbons or to 2,3,7,8,-tetrachlorodibenzo-p-dioxins [START_REF] Kim | Evaluation of biological monitoring markers using genomic and proteomic analysis for automobile emission inspectors and waste incinerating workers exposed to polycyclic aromatic hydrocarbons or 2,3,7,8,-tetracholrodedibenzo-p-dioxins[END_REF]. Our investigation revealed that Hpt concentration was significantly lower in serum samples collected at time 0 than in those collected during the decontamination. More to the point, serum Hpt was negatively correlated with fat levels of DL-and NDL-PCBs measured at day 0 and 59, thus supporting the hypothesis that the extent of down-regulation of this protein depends on the degree of exposure to pollutants, as assessed by PCBs concentration in pericaudal fat. As Hpt stably binds free Haemoglobin, thus preventing Haemoglobinrelated oxidative damage, and also limits the release of haem, which exacerbates oxidative stress [START_REF] Quaye | Haptoglobin, inflammation and disease[END_REF], the lower Hpt concentration measured at time 0, when bovine are considered "exposed" to pollutants, might further contribute to the reduction of effectiveness of the antioxidant body defences.

Although the group of Charolaise represents a control only because never exposed to pollution as the Limousine heifers here examined, the finding of significant differences between the two groups, at time 0, led us to suppose that exposure to PCBs, confirmed by the measurements in fat samples, might induce remarkable physiological changes. As oxidative stress contributes to health disorders, and affects zootechnical and/or reproductive performance of dairy cows [START_REF] Lomba | Influence of dietary cation-anion and oxidative-antioxidants balances on diseases occurring around parturition in the dairy cows[END_REF][START_REF] Bernabucci | Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season[END_REF][START_REF] Bernabucci | Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows[END_REF][START_REF] Castillo | Oxidative status during late pregnancy and early lactation in dairy cows[END_REF][START_REF] Wilde | Influence of macro and micro minerals in the periparturient period on fertility in dairy cattle[END_REF][START_REF] Wathes | Associations between lipid metabolism fertility in the dairy cow[END_REF], the perturbation of oxidative status might have negative implications for animal health and reproduction, and might compromise animal welfare. Interestingly our results demonstrate that a decontamination procedure, based on the feeding a controlled diet, is effective within 148 days, and is able to restore the blood homeostasis.

It is worth to underline that the blood parameters analysed in our study may be affected by oxidative stress induced by different kinds of pollutants, such as sulphur oxides, nitrogen oxides, hydrocarbons, carbon monoxide, and transition metals. However, we propose that, in the limits of a contamination characterized by measuring DL-and NDL-PCBs, serum TAC, N-Tyr, PC, Hpt and TNF-alpha levels could represent useful tools for identifying exposed animals, because their levels correlate with PCB concentration at the starting point of investigation. We report here, for the first time, that the serum concentrations of two inflammatory markers, Hpt and TNF-alpha, correlate with TEQ value and NDL-PCB amount not only at time 0, but also during decontamination, until PCB concentrations are higher than those legally permitted (day 59). So we propose that Hpt and TNF-alpha, in our experimental conditions, could be considered reliable markers of animal exposure. Finally, we propose that N-Tyr and TNF-alpha, whose serum concentrations still differ from those of control animals until fat TEQ and N DL-PCBs are over the legal limit, could be used as biological monitoring markers of the decontamination procedure.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.10.104. Eight one year old Limousine heifers exposed to organic pollutantswere removed fromthe contaminated area and housed in an experimental farmfaraway fromany PCBs source. Analysis were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period (day 59, 125, 188; B, C, D). TEQs: toxic equivalents; PCDDs: polychlorinated dibenzo-para-dioxins; DL-PCBs: dioxin-like polychlorinated biphenyls; PCDFs: polychlorinated dibenzo-furans. Data are expressed as pg per g of pericaudal fat. Eight one year old Limousine heifers exposed to organic pollutants were removed from the contaminated area and housed in an experimental farm far away from any PCB source. Analysis were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period (days 59, 125, 1ßß; B, C, D). TEQs: toxic equivalents; PCDDs: polychlorinated dibenzo-para-dioxins; DL-PCBs: dioxin-like polychlorinated biphenyls; PCDFs: polychlorinated dibenzo-furans. Data are expressed as pg per g of pericaudal fat. The more abundant congeners are marked in bold. Eight one year old Limousine heifers exposed to organic pollutants were removed from the contaminated area and housed in an experimental farm far away from any PCBs source. Analysis were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period (days 59, 125, 188; B, C, D).

The more abundant congeners are marked in bold. Eight one year old Limousine heifers exposed to organic pollutants were removed from the contaminated area and housed in an experimental farm far away from any PCBs source. Samplings were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period (days 59, 125, 188; B, C, D). K1 and K2 are samplings, taken at different times, from Charolaise heifers reared in the same experimental farm far away from any PCB source, and regarded as controls. PC, protein-bound carbonyls (nmol per mg of protein); N-Tyr, nitro-tyrosine (nmol per mg of protein); LPO, lipid hydroperoxides (μM).

⁎⁎⁎ Avs K1 or K2, P < 0.001. ⁎⁎ Avs B, C, or D, P < 0.001.

Figure 1 .

 1 Figure 1. Decontamination kinetic of DL-PCB and PCDD/F in growing Limousine heifers. Eight one year old Limousine heifers were removed from a contaminated farm, located close to an industrial plant, and housed in an experimental facility located far away this area, for six months, for a decontamination attempt. The extent of individual contamination was evaluated by measuring total toxic equivalent (TEQ) values of DL-PCBs + PCDD/F s in pericaudal fat of each animal at the start of the decontamination process (day 0), and bimonthly during the decontamination period (days 59, 125 and 188). Data are reported as mean ± standard deviation. The threshold limit for DL-PCB + PCDD/F (4 pg WHO-TEQ/g fat; European Union Regulation No 1259/2011) is indicated by a continuous line.

Figure 2 .

 2 Figure 2. Decontamination kinetic of NDL-PCBs in growing Limousine heifers Eight one year old Limousine heiferswere removed froma contaminated farm, located close to an industrial plant, and housed in an experimental facility far away this area, for six months, for a decontamination attempt. The extent of individual contamination was evaluated by measuring NDL-PCBs amount in pericaudal fat of each animal at the start of the decontamination process (day 0), and bimonthly during the decontamination period (days 59, 125, and 188). Data are reported as mean ± standard deviation. The threshold limit for NDL-PCBs (40 ngWHO-TEQ/g fat; European Union Regulation No 1259/2011) is indicated.

Figure 3 .

 3 Figure 3. Correlation between total TEQ in pericaudal fat and specific serummarkers of redoxhomeostasis. Panel A. The total antioxidant capacity (TAC) was measured in serum samples from Limousine heifers the starting point of decontamination (day 0), and expressed as μM concentration of Trolox equivalents. Each sample was analysed in triplicate, and the average value was calculated. Deviations over 5% from the mean were not found. Total toxic equivalent (TEQ) values of DL-PCBs + PCDD/F in pericaudal fat of each animal was measured and reported as pg/g fat. The statistical programme Graph Pad Prism 5.01 performed Pearson correlation analysis and the calculation of P (r=-0.8523, P=0.007). Panel B. The level of PCwas measured, by ELISA, in serum samples collected at the starting point of decontamination (day 0), and expressed as nmol/mg of protein. The statistical programmeGraph Pad Prism5.01 performed Pearson correlation analysis and the calculation of P (r = 0.942, P = 0.0005). Panel C. The concentration of N-Tyr was measured, by ELISA, in serum samples collected at the starting point of decontamination (day 0), and expressed as nmol/mg of protein. The statistical programme Graph Pad Prism 5.01 performed Pearson correlation analysis and the calculation of P (r= 0.880, P = 0.004).

Figure 4 .

 4 Figure 4. Serum concentration of Hpt and TNF-alpha. Hpt (panel A) and TNF-alpha (panelB) concentration in serum was measured by ELISA.K1 and K2 (open bar), samplings from Charolaise heifers reared in the experimental farm far away from any PCBs source, here regarded as controls. A (fullbar), sampling from Limousine heifers exposed to organic pollutants collected at the start of the experimental period (day 0). B, C and D (bar with vertical lines), samplings performed after 59 (B), 125 (C), and 188 (D) days of decontamination. *** P < 0.001 vs A (from Dunnett's test). # P < 0.05 vs B; ## P < 0.01 vs B (from Tukey's test). ⁎⁎ P < 0.01 vs K1 and K2; ⁎⁎⁎ P < 0.001 vs K1 and K2 (from Tukey's test).

Figure 5 .

 5 Figure 5. Correlation between total TEQ in pericaudal fat and serum concentration of Hpt orTNF-alpha. Panel A. Hpt (open triangles) and TNF-alpha (full squares) concentrations were measured, by ELISA, in serum samples from heifers at the start of decontamination (day 0). Each sample was analysed in triplicate, and the average value was calculated. Deviationsover 5% from the mean were not found. Total toxic equivalent (TEQ) values of DL-PCBs + PCDD/F in pericaudal fat of each animal are reported as pg/g fat. The statistical programme Graph Pad Prism 5.01 performed Pearson correlation analysis and the calculation of P (Hpt, r = -0.735, P = 0.038; TNF-alpha, r = 0.881, P = 0.004 respectively). Panel B. Hpt (open triangles) and TNF-alpha (full squares) concentrations were measured, by ELISA, in serum samples collected 59 days after starting decontamination. The statistical programme Graph Pad Prism 5.01 performed Pearson correlation analysis and the calculation of P (Hpt, r = -0.946, P = 0.0004; TNF-alpha, r = 0.791, P = 0.019).

Table 1 . PCDD/F + DL-PCB and NDL-PCB levels in pericaudal fat of Limousine heifers under decontamination.

 1 Eight one year old Limousine heifers exposed to organic pollutants were removed from the contaminated area and housed in an experimental farm far away from any PCBs source. Analysis were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period(day 59,125, 188; B, C, D).

		A	B	C	D
	TEQs (PCDD/Fs + DL-PCBs; pg/g of NDL-PCBs (ng/g of fat) fat) Days from starting decontamination	25.34 ± 1.41 ⁎⁎⁎ 165.9 ± 14.42 ⁎⁎ 0	8.33 ± 0.35. 56.86 ± 2.88 59	4.87 ± 0.22 33.78 ± 1.50 125	3.13 ± 0.18 24.16 ± 1.42 188
	Date of sampling (mm, dd, yy)	09/15/11	11/18/11	01/23/12	03/27/12
	N	8	8	8	8
	TEQs: toxic equivalents; PCDDs: polychlorinated dibenzo-para-dioxins; DL-PCBs: dioxin-like polychlorinated biphenyls; PCDFs: polychlorinated dibenzo-furans; NDL-PCBs: non-
	dioxin-
	like polychlorinated biphenyls.
	⁎⁎⁎ P < 0.001 vs B, C, or D.
	⁎⁎ P < 0.001 vs B, C, or D.

Table 2 . Profile of fat contamination by PCDDs, PCDFs and DL-PCBs.

 2

		A		B		C		D
	Total of PCDDs Total of PCDFs OMS TEQ DL-PCBs	0.43 8 0.50 8 24.3	± 0.033 ± 0.040 ± 1.364	0.26 5 0.35 3 7.71	± 0.017 ± 0.029 ± 0.323	0.21 0 0.23 1 4.42	± 0.030 ± 0.017 ± 0.199	0.16 0.17 6 2 2.76	± 0.021 ± 0.013 ± 0.176
	Total TEQs (PCDDs/F + PCB DL)	9 25.3	± 1.405	4 8.33	± 0.353	8 4.86	± 0.215	3 3.10	± 0.184
	Days from starting decontamination	4 0		3 59		9 125		2 188
	N	8		8		8		8

Table 3 .

 3 Congeners analysis.

		A	B	C	D
	PCDD
	2,3,7,8-TCDD	0.091 ± 0.005	0.057 ± 0.009	0.054 ± 0.006	0.051 ± 0.010
	1,2,3,7,8-PeCDD	0.232 ± 0.030	0.147 ± 0.012	0.100 ± 0.027	0.070 ± 0.010
	1,2,3,4,7,8-HxCDD	0.016 ± 0.001	0.010 ± 0.001	0.013 ± 0.002	0.005 ± 0.0010
	1,2,3,6,7,8-HxCDD	0.087 ± 0.010	0.036 ± 0.003	0.029 ± 0.002	0.024 ± 0.003
	1,2,3,7,8,9-HxCDD	0.014 ± 0.001	0.009 ± 0.001	0.009 ± 0.0005	0.006 ± 0.0005
	HpCDD	0.011 ± 0.001	0.007 ± 0.0008	0.005 ± 0.0004	0.012 ± 0.003
	OCDD	0.0008 ± 0.0001	0.0007 ± 0.00008	0.0006 ± 0.00004	0.0004 ±
	Total of PCDDs	0.438 ± 0.033	0.265 ± 0.017	0.210 ± 0.030	0.00008 0.166 ± 0.021
	Days from starting decontamination	0	59	125	188
	PCDF
	2,3,7,8-TCDF	0.024 ± 0.006	0.012 ± 0.004	0.005 ± 0.0007	0.022 ± 0.003
	1,2,3,7,8-PeCDF	0.003 ± 0.0007	0.007 ± 0.0009	0.003 ± 0.0003	0.002 ± 0.0004
	2,3,4,7,8-PeCDF	0.313 ± 0.019	0.156 ± 0.017	0.106 ± 0.010	0.085 ± 0.005
	1,2,3,4,7,8-HxCDF	0.080 ± 0.011	0.092 ± 0.011	0.058 ± 0.004	0.027 ± 0.004
	1,2,3,6,7,8-HxCDF	0.039 ± 0.005	0.041 ± 0.004	0.024 ± 0.001	0.017 ± 0.002
	1,2,3,7,8,9-HxCDF	0.005 ± 0.0008	0.004 ± 0.0006	0.004 ± 0.0004	0.004 ± 0.0004
	2,3,4,6,7,8-HxCDF	0.027 ± 0.002	0.030 ± 0.003	0.019 ± 0.002	0.015 ± 0.001
	1,2,3,4,6,7,8-HpCDF	0.015 ± 0.003	0.009 ± 0.001	0.009 ± 0.0006	0.004 ± 0.001
	1,2,3,4,7,8,9-HpCDF	0.002 ± 0.0003	0.002 ± 0.0002	0.002 ± 0.0003	0.0008 ± 0.0001
	OCDF	0.0005 ± 0.00009	0.0004 ± 0.00006	0.0003 ± 0.00003	0.0002 ±
	Total of PCDFs	0.508 ± 0.040	0.353 ± 0.029	0.231 ± 0.017	0.00005 0.172 ± 0.013
	OMS TEQ PCDD/F	0.945 ± 0.063	0.619 ± 0.046	0.441 ± 0.040	0.339 ± 0.032
	Days from starting decontamination	0	59	125	188
	DL-PCB
	PCB77	0.0009 ± 0.0001	0.0007 ± 0.00009	0.0004 ± 0.00004	0.0004 ±
	PCB81	0.0007 ± 0.00009	0.0002 ± 0.00003	0.0001 ± 0.000009	0.00005 0.0001 ±
	PCB126	22.39 ± 1.236	7.033 ± 0.296	4.027 ± 0.188	0.00001 2.468 ± 0.163
	PCB169	1.330 ± 0.129	0.430 ± 0.026	0.249 ± 0.010	0.186 ± 0.012
	Total of coplanares	23.73 ± 1.327	7.465 ± 0.315	4.276 ± 0.196	2.654 ± 0.171
	PCB105	0.037 ± 0.003	0.011 ± 0.0009	0.006 ± 0.0005	0.005 ± 0.0004
	PCB114	0.006 ± 0.0006	0.003 ± 0.0001	0.002 ± 0.00006	0.001 ± 0.00008
	PCB118	0.398 ± 0.028	0.160 ± 0.006	0.099 ± 0.004	0.066 ± 0.004
	PCB123	0.003 ± 0.0001	0.0012 ± 0.00007	0.0007 ± 0.00004	0.0007 ±
	PCB156	0.122 ± 0.013	0.040 ± 0.002	0.024 ± 0.001	0.00004 0.019 ± 0.001
	PCB157	0.018 ± 0.002	0.007 ± 0.0004	0.004 ± 0.0002	0.005 ± 0.0004
	PCB167	0.062 ± 0.005	0.021 ± 0.001	0.013 ± 0.0004	0.009 ± 0.0006
	PCB189	0.0189 ± 0.002	0.006 ± 0.0005	0.003 ± 0.0001	0.002 ± 0.0002
	Total of non-coplanares	0.665 ± 0.050	0.249 ± 0.010	0.152 ± 0.006	0.109 ± 0.006
	OMS TEQ DL-PCB	24.39 ± 1.364	7.714 ± 0.323	4.428 ± 0.199	2.763 ± 0.176
	Total TEQ (PCDD/F + PCB DL)	25.34 ± 1.405	8.333 ± 0.353	4.869 ± 0.215	3.102 ± 0.184
	Days from starting decontamination	0	59	125	188

Table 4 .

 4 Congeners analysis.

		A	B		C	D
	PCB28 PCB52	0.276 ± 0.030 0.231 ± 0.039	0.20 6 0.21	± 0.035 ± 0.039	0.104 ± 0.010 0.088 ± 0.011	0.061 ± 0.010 0.118 ± 0.019
	PCB101	0.388 ± 0.044	7 0.31	± 0.057	0.143 ± 0.020	0.203 ± 0.032
	PCB138	48.57 ± 4.136	8 16.8	± 1.012	10.12 ± 0.451	6.738 ± 0.446
	PCB153	70.26 ± 5.742	0 23.9	± 1.176	14.59 ± 0.668	10.43 ± 0.567
	PCB180	46.29 ± 5.167	2 15.4	± 0.818	8.733 ± 0.441	6.614 ± 0.456
	Total of NDL-PCBs	165.9 ± 14.42	3 56.8	± 2.884	33.78 ± 1.502	24.16 ± 1.422
	Days from starting decontamination	0	6 59		125	188
	N	8	8		8	8

Table 5 . Markers of the antioxidant defence system in serum of heifers

 5

		A	B	C	D	K1	K2
	Ret (μg/ml)	0.48 ± 0.02 **	0.72 ± 0.02	0.73 ± 0.01	0.68 ± 0.01	0.71 ± 0.01 **'	0.71 ± 0.08 * * *
	Toc (μg/ml)	1.48 ± 0.04 **	1.73 ± 0.02	1.76 ± 0.03	1.77 ± 0.03	1.85 ± 0.03 * * *	1.87 ± 0.03 * * *
	Asc (μM)	6.06 ± 0.11 **	7.76 ± 0.20	7.95 ± 0.16	8.01 ± 0.12	8.00 ± 0.11 ***	8.42 ± 0.27 ***
	GPx (nmol/min/ml)	95.73 ± 4.08 * *	164.1 ± 6.7	169.2 ± 8.5	167.0 ± 8.3	170.2 ± 15 * * *	181.1 ± 14 * * *
	SOD (U/ml)	1.10 ± 0.04 **	1.55 ± 0.06	1.57 ± 0.04	1.62 ± 0.04	1.67 ± 0.06 * * *	1.69 ± 0.09 * * *
	TAC (μM)	92.24 ± 2.76 * *	133.0 ± 3.5	139.4 ± 4.2	141.0 ± 3.8	139.9 ± 3.2 ***	146.7 ± 2.6 * * *
	Days from starting decontamination	0	59	125	188
	Date of sampling (mm, dd, yy)	09/15/11	11/18/11	01/23/12	03/27/12	12/15/11	03/29/12
	N	8	8	8	7	8	6
	Ret, Retinol; Toc, α-Tocopherol; Asc, Ascorbate; TAC, total antioxidant capacity; GPx, glutathione peroxidase activity; SOD, superoxide dismutase.
	Eight one year old Limousine heifers exposed to organic pollutants were removed from the contaminated area and housed in an experimental farm far away from any PCBs
	source.
	Samplings were carried out at the start of the experimental period (day 0; A), and then bimonthly through a six months period (days 59, 125, 188; B, C, D).
	K1 and K2 are samplings, taken at different times, from Charolaise heifers reared in the same experimental farm far away from any PCBs source, and regarded as controls.
	*** Avs K1 or K2, P < 0.001.
	** Avs B, C, or D, P < 0.001

Table 6 . Markers of oxidative stress in serum of heifers

 6

		A	B	C	D	K1	K2
	PC (nmol/mgP) N-Tyr (nmol/mgP)	12.39 ± 0.17 ⁎⁎ 8.82 ± 0.09 ⁎⁎	7.29 ± 0.32 6.33 ± 0.19	7.11 ± 0.15 5.50 ± 0.21	6.48 ± 0.44 5.80 ± 0.36	6.67 ± 0.22 ⁎⁎⁎ 5.26 ± 0.18 ⁎⁎⁎	7.01 ± 0.31 ⁎⁎⁎ 5.19 ± 0.19 ⁎⁎⁎
	LPO (μM)	5.83 ± 0.16 ⁎⁎	3.16 ± 0.21	3.21 ± 0.24	3.40 ± 0.29	2.87 ± 0.18 ⁎⁎⁎	2.83 ± 0.24 ⁎⁎⁎
	Days from starting decontamination	0	59	125	188
	Date of sampling (mm, dd, yy)	09/15/11	11/18/11	01/23/12	03/27/12	12/15/11	03/29/12
	N	8	8	8	7	8	6

Acknowledgements ____________________________________________________________

The study was supported by CISIA-VARIGEAV project (Caratterizzazione e valorizzazione delle risorse genetiche animali e vegetali della Campania e della Sardegna finalizzate allo sviluppo della filiera bufalina mediante approcci multidisciplinari ed innovativi; grant number 191/2009), and by POR FESR 2007/2013 project (DEDICA: Sviluppo di tecniche biomolecolari di screening per la messa in evidenza dell'esposizione a diossine e sostanze diossino-simili in vacche da latte).