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(CPA) on oxidative stress markers in the liver and kidneys of broiler chicks was studied. Ten-day-old male broi- ler chicks (Ross 308) were assigned into the ...
Cyclopiazonic acid augments the hepatic and renal oxidative stress in broiler chicks

Human and Experimental Toxicology 30(8) 910–919 ª The Author(s) 2010 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327110384285 het.sagepub.com

H Malekinejad1, P Akbari1, M Allymehr2, R Hobbenaghi3 and A Rezaie1

Abstract Generation of reactive oxygen species (ROS) leads to serious tissue injuries. The effect of cyclopiazonic acid (CPA) on oxidative stress markers in the liver and kidneys of broiler chicks was studied. Ten-day-old male broiler chicks (Ross 308) were assigned into the control and test groups, which received normal saline and 10, 25, and 50 mg/kg CPA, respectively, for 28 days. Body weight gain, serum level of alkaline phosphatase (ALP), g-glutamyl transferase (GGT), uric acid, creatinine, and blood urea nitrogen (BUN) were measured after 2 and 4 weeks exposure. Moreover, the total thiol molecules (TTM) and malondialdehyde (MDA) content of the liver and kidneys were assessed. No significant differences (p > 0.05) were found in body weight gain between the control and test groups. Whereas, the hepatic weight increased significantly (p < 0.05) in animals that received 25 and 50 mg/kg CPA. Both ALP and GGT level in serum were elevated in comparison to the control group. CPA also resulted in uric acid, creatinine, and BUN enhancement in broilers. The MDA content of the liver and kidneys showed remarkable increase. By contrast, the TTM levels in the liver and kidneys were significantly (p < 0.05) attenuated. Histopathological findings confirmed the biochemical changes in either organ characterized by inflammatory cells infiltration along with severe congestion and cell swelling, suggesting an inflammatory response. These data suggest that exposure to CPA resulted in hepatic and renal disorders, which were reflected as biochemical markers alteration and pathological injuries in either organ. The biochemical alteration and pathological abnormalities may be attributed to CPA-induced oxidative stress. Keywords cyclopiazonic acid, reactive oxygen species, liver, kidney, broiler

Introduction Cyclopiazonic acid (CPA) is an indol-tetramic acid mycotoxin, which is produced by several species of fungi such as Penicillium and Aspergillus.1,2 There are reports indicating the toxicity of CPA in laboratory and farm animals. However, there has been no direct report to show any outbreak in humans due to CPA poisoning. CPA is a selective inhibitor of calcium-dependent ATPase in the sarcoplasmic reticulum (SR). The Caþþ-ATPase is a membrane protein, which contains 994 amino acids and moves calcium out of the cytoplasm.3 CPA by inhibiting the Caþþ-ATPase reduces the storage capacity of the SR for Ca2þ and inhibits contractions that depend on intracellular Ca2þ release.4 It has been shown that CPA inhibits the

Caþþ-ATPase in skeletal, smooth, and cardiac muscles.5 There are also evidences indicating that CPA increases Ca2þ influx via voltage-depending Ca2þ channels.6

1

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran 2 Department of Poultry Diseases, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran 3 Department of Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran Corresponding author: H Malekinejad, Department of Pharmacology and Toxicology, Faculty of veterinary Medicine, P.O.Box:1177, Urmia University, Urmia, Iran Email: [email protected]

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Previous toxicokinetic studies in rodents’ indicate a rapid absorption of CPA followed by fast distribution in almost entire body. In this regard, it has been shown that the largest amount of the toxin was found in skeletal muscles.7 Multiple-dose studies with CPA in rats demonstrated that CPA resulted in weight loss, liver enlargement, and also pathological damages such as coagulative necrosis and picknotic nuclei in the liver.8 CPA is also toxic in other species including swine, guinea pigs, and dogs, with affecting mainly the gastrointestinal tract, liver, heart, kidneys, and skeletal muscles.9 Oxidative stress has been implicated in the pathogenesis of numerous diseases and also in the processes of various biological events. Toxic effects of reactive oxygen species (ROS) as oxidative stress can cause damages in the cellular level such as oxidizing nucleic acids, proteins, and membrane lipids.10 Moreover, the crucial role of free radicals mediated oxidative stress in pathogenesis of degenerative diseases including Alzheimer’s disease and in the aromatic antiepileptic drugs-induced hepatotoxicity well documented.11,12 CPA toxicity in poultry is associated with immunosuppressive effect, body weight loss, hepatic enlargement, and pathological lesions in the alimentary tract, liver, and the kidneys.2,13 Although occurrence of CPA in poultry diet has been reported, little is however known about its mechanism of toxicity in this species. Therefore, the present study was designed to evaluate some biochemical and pathological changes in the liver and kidneys following subacute exposure to CPA and the role of oxidative stress on these changes.

Materials and methods Chemicals Cyclopiazonic acid standard and 5.50 -dithiobis-2nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co. (St Louis, Missouri, USA). Thiobarbituric acid, phosphoric acid (85%), dimethyl sulfoxide (DMSO), and ethanol were obtained from Merck (Germany). N-butanol was obtained from Carl Roth, GmbH Co. (Germany). Commercially available standard kits were used for the determination of alkaline phosphatase (ALP, 744, Man Inc. Tehran, Iran), g-glutamyl transferase (GGT), Uric acid, and creatinine (CK, Parsazmun Inc. Karaj, Iran) and blood urea nitrogen (BUN, Darman Kave, Isfahan, Iran). All other

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chemicals were commercial products of analytical grade. CPA was dissolved in DMSO (10 mg/mL) and diluted in normal saline to obtain the appropriate dose levels, which are stated below.

Animals and experimental design The day-old chicks (Ross 308) were purchased from a local broiler hatchery and kept under Ross companyrecommended standard conditions. During the experiment, birds received a Corn-Soya-base feed as following: from 0 to 10 days, the birds were fed with a crumb starter feed and from 11 to 28 days, they were fed with a grower pellet. Ten-day-old male broiler chickens (Ross 308) weighing between 275 and 300 g were subjected to current study. The birds were acclimated for a week at temperature of 26 C + 2 C. Feed and water were given ad libitum. The experimental protocols were approved by the ethical committee of Urmia University. The chicks were assigned into the test and control groups (N ¼ 8). The test group subdivided based on the dose levels of CPA for 28 days into 3 groups of T1 (10 mg/kg), T2 (25 mg/kg), and T3 (50 mg/ kg), and the control group received only normal saline and the same volume of the toxin solvent. All groups received the toxin or normal saline by crop gavage. The chicks were weighed weekly.

Serum preparation and tissue collection On days 15 and 29, blood samples were collected directly from the jugular vein and left to clot at room temperature for 1 hour before being centrifuged at 1500 g for 10 min to obtain the serum. The obtained serum samples were stored for maximum 2 weeks at 20 C for further biochemical analysis. On day 29 following blood collection, all the animals were sacrificed and the liver and kidney tissues were dissected immediately. The liver and kidneys were divided to 2 parts and the first part after washing with chilled normal saline, were snap frozen in liquid nitrogen and then immediately were stored at 70 C for further biochemical analyses. The second parts of the samples were preserved in 10% buffered formaldehyde for further histopathological examinations.

Determination of serum biochemical parameters Serum levels of ALP and GGT enzymes were measured using the commercially available standard kits

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and according to manufacturer’s instructions. To evaluate the effect of CPA on renal function serum level of three well-established biomarkers including creatinine, uric acid, and BUN were assessed using commercially available kits, as well.

Measurement of serum total thiol molecules (TTM) Total sulfhydryl level in the liver and kidneys was measured as described previously.14 Briefly, 0.2 mL from the liver and kidney homogenate was added to 0.6 mL Tris-EDTA buffer (Tris base 0.25 M, EDTA 20 mM, pH 8.2) and thereafter 40 mL DTNB (10 mM in pure methanol) was added in a 10 mL glass test tube. The final volume of this mixture was made up to 4.0 mL by extra addition of methanol. After 15 min incubation at room temperature, the samples were centrifuged at 3000 g for 10 min and ultimately the absorbance of the supernatant was measured at 412 nm.

Malondialdehyde determination To determine the lipid peroxidation activity in the control and test groups, the malondialdehyde (MDA) content of the liver and kidney samples was measured using the thiobarbituric acid (TBA) reaction as described previously.15 In short, 23 g of the liver and kidney samples were homogenized in ice-cooled KCl (150 mM), and then the mixture was centrifuged at 3000 g for 10 min; 0.5 mL of the supernatant was mixed with 3 mL phosphoric acid (1% V/V) and then following vortex mixing, 2 mL of 6.7 g L1 TBA was added to the samples. The samples were heated at 100 C for 45 min, chilled in ice, and after adding 3 mL N-butanol, the samples were further centrifuged at 3000 g for 10 min. The absorbance of supernatant was measured spectrophotometerically at 532 nm and the MDA concentration calculated according to the simultaneously prepared calibration curves using MDA standards. The amount of MDA was expressed as nM per mg protein of the samples. The protein content of the samples was measured according to the Lowry method.16

Histopathological investigations The liver and kidney samples, which previously had been preserved in 10% buffered formaldehyde, were embedded in paraffin, and 56 mm sections were cut using a rotary microtome and stained with

Human and Experimental Toxicology 30(8) Table 1. Effect of CPA on body weight gain (BWG) and the ratio of the hepatic weight (HW) to body weight in broilers after 28 days exposure to CPAa Groups C T1 T2 T3

BWG (g) 532 538 522 503

+ 27.46 + 34.06 + 38.24 + 35.04

(HW/BW)  100 2.070 + 0.002 2.058 + 0.011 2.343 + 0.015b 2.669 + 0.048b

Abbreviations: CPA: cyclopiazonic acid, C: control. a T1: (10 mg/kg), T2: (25 mg/kg), T3: (50 mg/kg). b Significant differences (p < 0.05) between control and CPA-treated groups at the same column.

hematoxyline and eosin for investigation under light microscope. To evaluate the level of damages following exposure to CPA, indexes such as congestion, hemorrhages, necrosis, cellular hyperplasia, and mononuclear inflammatory cells infiltration were scored numerically. The evaluation criteria were as follows: zero for no detectable lesion, 1 for mild changes, 2 for moderate changes, and 3 for severe changes. For each animal in the test and control groups, at least three slides from distinct organs were prepared and the averages of scored marks were analyzed. The histpathological studies were conducted by a pathologist who was unaware of the study purposes.

Statistical analyses For all results, numerical mean and standard deviation of the measured parameters were calculated. The results of three independent experiments for each assessment were analyzed using Graph Pad Prism software (version 2.01. Graph Pad software Inc. San Diego, California, USA). The comparisons between groups were made by analysis of variance (ANOVA) followed by Bonferoni post test. For comparing the graded degree of pathological findings between groups, the Kruskal-Wallis test was used. A value of p < 0.05 was considered as significant.

Results Effect of CPA on body and organ weight During the experimental period, no bird died in any of the treatment and control groups. Body weight gain showed no significant (p > 0.05) differences between the control and test groups after 4 weeks exposure to the toxin at the given dose levels (Table 1). As the collection of the entire kidneys from the chicken was

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Table 2. Effect of CPA on serum levels of ALP and GGTa ALP (U/L) Groups

GGT (U/L)

2w

C T1 T2 T3

469.57 461.08 521.99 868.21

4w

+ 56.02 + 15.88 + 38.59 + 91.22b

2w

482.34 + 13.65 523.84 + 82.72 716.82 + 27.57b 964.95 + 137.05b

12.73 + 13.89 + 19.68 + 23.15 +

4w 1.63 0.08 1.64b 1.39b

13.29 14.48 24.89 32.50

+ 1.71 + 1.52 + 1.37b + 0.53b

Abbreviations: ALP: alkaline phosphatase, CPA: cyclopiazonic acid, GGT: g-glutamyl transferase, 2 w: 2 weeks, 4 w: 4 weeks, C: control. a T1: 10 mg/kg, T2: 25 mg/kg, T3: 50 mg/kg. b Significant differences (p < 0.05) between control and CPA-treated groups at the same column.

found to be practically impossible, we could only determine the weight changes of the liver tissues. As shown in Table 1, following 28 days treatment, a significant (p < .05) elevation in the hepatic weight in the T2 and T3 groups was obtained. The mentioned increase appears to be in a dosedependent fashion.

Effect of CPA on enzyme activities and serum biochemical parameters Assessment of the ALP and GGT levels in the control and test groups showed that the ALP level after 2 weeks exposure to CPA was significantly (p < 0.05) elevated only at the highest given dose (50 mg/ kg), while the serum level of GGT increased at the lower dose (25 mg/kg) of the toxin in comparison with the control group. The same assay after 4 weeks exposure revealed that the levels of ALP and GGT increased non-significantly (p > 0.05) when compared to the values obtained after 2 weeks exposure in the control group (Table 2). Moreover, the serum level of ALP after 4 weeks exposure enhanced significantly, at 25 mg/kg dose level of CPA. The serum level of uric acid, creatinine and BUN were found to increase significantly (p < 0.05) in a time- and dose-dependent manner (Table 3).

Effect of cyclopiazonic acid on concentrations of MDA and TTM in the liver and kidneys Assessment of the TTM content in the liver and kidneys showed that the TTM content of the liver is approximately 11-fold higher than that in the kidney tissues. Moreover, 28 days exposure to CPA resulted in a significant (p < 0.05) reduction of the TTM content in both the liver and kidney tissues at 25 and 50 mg/kg dose levels (Figure 1). To measure the pro-oxidant effect of CPA in broilers, MDA content of the liver and kidneys were assessed. The obtained results show that MDA contents of the liver in groups of birds that received 25 and 50 mg/kg CPA for 28 days were significantly (p < 0.05) higher than that in the control group (Figure 2).

Histopatholgy examination The histopathological examination of the liver and kidneys in the control group showed a slight congestion in the liver and no pathologic changes in the kidneys (Figure 3A, and Figure 4A). The CPA-exposed animals showed dose-dependent pathologic alterations such as congestion, cell swelling, fatty degeneration, focal and multifocal inflammatory cells infiltration, necrosis, and disintegration of the cells

Table 3. Effect of CPA on serum biochemical parametersa Uric acid (mg/dL) Groups C T1 T2 T3

2w 7.43 0.50 9.13 12.1

+ 0.15 + 0.36 + 0.47b + 0.36b

Creatinine (mg/dL)

4w 7.27 + 7.60 + 10.83 + 16.23 +

2w 0.31 0.44 0.4b 0.25b

0.20 0.21 0.29 0.26

+ + + +

0.04 0.03 0.04b 0.04b

BUN (mg/dL)

4w 0.49 0.49 0.83 0.79

+ + + +

0.05 0.04 0.17b 0.02b

2w 12.34 12.65 13.26 13.60

Abbreviations: CPA: cyclopiazonic acid, BUN: blood urea nitrogen, C: control. a T1: 10 mg/kg, T2: 25 mg/kg, T3: 50 mg/kg. b Significant differences (p < 0.05) between control and CPA-treated groups at the same column.

+ 0.47 + 0.23 + 0.34 + 0.48b

4w 12.91 13.85 16.72 19.59

+ 0.23 + 0.76 + 0.63b + 1.03b

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0.8 Liver

0.7

Kidney

mmol/mg

0.6 0.5

*

0.4 *

Discussion

0.3 0.2 0.1

**

0 C

T1

T2

T3

Figure 1. Effect of cyclopiazonic acid (CPA) on total thiol molecules (TTM) content of the liver and kidneys; Stars indicate significant differences between the control and CPA-treated animals (p < 0.05). For each group n ¼ 8 and mean values + SD are given. The birds were administered CPA as T1 (10 mg/kg), T2 (25 mg/kg), and T3 (50 mg/kg), and the control group received only normal saline.

12 Liver

*

Kidney

*

10 * 8 nMol/mg

cells hyperplasia were the main pathological changes in the kidneys (Figure 4BF). Infiltration of inflammatory cells was not the dominant symptoms of the CPA in the renal system. The severity of histopathological lesions in studied organs as scrod numerical data are depicted in Tables 4 and 5.

*

6 4 2 0 C

T1

T2

T3

Figure 2. Effect of CPA on MDA content of the liver and kidneys; stars indicate significant differences between the control and CPA-treated animals (p < 0.05). For each group n ¼ 8 and mean values + SD are given. The birds were administered CPA as T1 (10 mg/kg), T2 (25 mg/kg), and T3 (50 mg/kg), and the control group received only normal saline.

in the liver. The necrotic reactions were localized largely around the bile ducts and were more applicable at the highest given dose level of the toxin (Figure 3BH). Tubular degeneration and necrosis, hydropic degeneration in the ureter, hemorrhages in the renal parenchyma, tubular cell swelling, ureter epithelial

Elevation of the pro-oxidant factors including MDA and attenuation of the antioxidant capacity as depicted by low TTM after 28 days exposure to CPA indicated that oxidative stress contributes in CPA-induced toxicity. The present study showed that sub-acute exposure of broilers to CPA up to 50 mg/kg dose does not change the body weight gain significantly although a slight decline after 4 weeks and at the highest given dose level was obtained. This observation confirmed and extended an earlier study, which showed that the body weight gain decline following exposure to 50 ppm CPA can occur after 4 weeks and at higher doses of the toxin in broilers.2 Later studies in rodents also support this finding that 13 consecutive weeks oral administration of CPA up to 4 mg/kg had no definite effects on general appearance, behavior, body weight gain, and food consumption.17,18 In this respect, our finding showed that although there were no significant differences between the control and test groups in terms of body weight gain, the hepatic weight and the ratio of the hepatic to body weight, however, were increased dose-dependently. The reasonable explanation for this finding could be the possible hepatic inflammation reactions, which CPA might have exerted in birds. Furthermore, biochemical assessments of the serum ALP level in the exposed birds support the fact that the liver weight elevation could be due to CPA-induced inflammatory reactions. ALP elevation could be counted as a compensatory reaction of the body against various inflammatory mediators as ALP is able to remove a phosphate group from pro-inflammatory compounds.19-21 Generally, the GGT is used as supplementary test to clarify that an elevated ALP is due to disease or injury to the liver or biliary tract, rather than disease affecting other organs. At the site of inflammation with the development of the inflammatory process, the activity of GGT is elevated and this elevation is counted as a novel biochemical marker in inflammation. Our results showed that

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A

B

C

D

E

F

G

H

Figure 3. Photomicrograph of chicken liver sections: (A) control group, no histological changes are detectable; (B) cyclopiazonic acid (CPA)-treated chickens at low dose (10 mg/kg), a minor infiltration of the inflammatory cells along with slight congestion are observed; (C, E, and G) CPE-treated group at medium dose (25 mg/kg), representing a focal mononuclear cells infiltration, moderate necrosis, and bile duct epithelial cells destruction, respectively; and (D,F, and H) CPA-treated animals at high dose (50 mg/kg) show multifocal inflammatory cells infiltration, severe necrosis, and bile duct epithelial cells destruction, respectively. Hematoxyline and eosin (400).

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Figure 4. Photomicrograph of chicken kidney sections: (A) Control group, no histological changes are observed; (B) cyclopiazonic acid (CPA)-treated chickens at low dose (10 mg/kg), a mild tubular necrosis is seen; (C and E) CPEtreated group at medium dose (25 mg/kg), representing a moderate tubular necrosis and transitional cells hyperplasia, respectively, and (D and F) CPA-treated animals at high dose (50 mg/kg) show a severe tubular necrosis and transitional cells hyperplasia, respectively. Hematoxyline and eosin (400).

the rate of GGT elevation (2.5-fold) was more than ALP (1.8-fold) increase in serum, suggesting that most likely the CPA-induced hepatic inflammation

causes GGT elevation in the broilers, which might be considered more applicable marker of the hepatic injuries.

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Table 4. Pathological findings in the liver following CPA-exposure; mean values + SD are givena Groups Control T1 T2 T3

C

LI b

0.0 2.4 + 0.4c 5.4 + 0.6d 7.4 + 1.8d

N b

0.0 4.2 + 1.2c 5.2 + 0.7c 6.4 + 0.6d

BDEN b

0.0 1.4 + 0.4c 4.8 + 1.4d 7.2 + 1.2d

0.0b 0.0b 3.6 + 1.2c 6.4 + 1.6c

Abbreviations: CPA: cyclopiazonic acid, C: congestion, LI: leukocyte infiltration, N: necrosis, BDEN: bile duct epithelial necrosis. a T1: 10 mg/kg, T2: 25 mg/kg, T3: 50 mg/kg. b-d Values in same column with different superscripts differ significantly (p < 0.05).

Table 5. Pathological findings in the kidneys after 28 days exposure to CPAa Groups

C

H

TN

TCH

Control T1 T2 T3

0.0b 1.8 + 0.3c 4.2 + 0.6d 6.2 + 1.2e

0.0b 1.2 + 0.4c 3.2 + 0.4d 7.8 + 1.8e

0.0b 1.8 + 0.3c 4.8 + 0.6d 6.8 + 1.4d

0.0b 1.8 + 0.6c 4.6 + 0.8d 7.8 + 1.2e

Abbreviations: CPA: cyclopiazonic acid, C: congestion, H: hemorrhages, TN: tubular necrosis, TCH: transitional cells hyperplasia, C: Control. a Mean values + SD are given. T1: 10 mg/kg, T2: 25 mg/kg, T3: 50 mg/kg. b,c,d,e Values in same column with different superscripts differ significantly (p < 0.05).

The renal system function testing assays such as BUN, creatinine, and uric acid levels in serum indicate that CPA markedly resulted in abnormalities in the kidneys. Therefore, it appears that CPA acts on various systems rather than influencing the single target organ. To clarify the possible pathway(s) of the inflammation induction due to CPA exposure in vital organs such as the liver and kidneys, the role of oxidative stress was studied. Our findings showed that in either of the examined tissues, the MDA content as an oxidative stress biomarker was elevated, suggesting that CPA exposure resulted in lipid peroxidation in the liver and kidney tissues. ROS are generated following exposure of neutrophils and macrophages to appropriate stimuli.22 The formation of potent oxidants results in reactions with proteins, lipids, carbohydrates, and nucleic acids.23 Thus, it might be suggested that the TTM reduction due to CPA exposure reported in the present study may play a crucial role in MDA augmentation in both liver and kidneys. It has been shown that cells are protected by GSH (glutathione) as intracellular antioxidant from oxidants generated by inflammatory cells. To measure the reducing power of GSH, sulfhydryl-donating capacity is one of the best established methods.24 In this study, TTM determination

demonstrated that CPA dose-dependently attenuated the TTM amount in the liver and kidneys, suggesting that CPA likely exerts its toxicity via the alteration in antioxidant capacity of target organs. The mentioned assessment also confirmed and extended that the TTM content of the liver is remarkably higher than that in kidney. This finding is in accordance with previous studies as it has been demonstrated that hepatic sinusoidal efflux is the major source of GSH and cysteine. In this respect, it is also known that the liver plays a crucial role in homeostasis of GSH.25 There is evidence indicating that the generation of ROS induces GSH depletion and ultimately leads to decline in thioldependent antioxidant capacity.26 The histopathological findings of this study showed the pathologic feature of CPA intoxication in the liver and kidneys. Moreover, these findings further confirmed the biochemical alterations in CPAexposed groups, which are reflected as the hepatic enzymes elevation and/or the renal system biomarkers changes indicating the abnormalities that occurred due to CPA intoxication. The pathological feature of the CPA intoxication demonstrated an inflammation in the liver, which accompanied with mononuclear inflammatory cells infiltration. The abnormal pathological changes, however, in the kidneys largely showed cell swelling and at the highest given dose

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level hemorrhages in the parenchyma of the kidneys. A possible explanation for these pathological findings might be related to elevation of ROS, which resulted in remarkable augmentation of lipid peroxidation. The role of mononuclear inflammatory cells in producing ROS and other inflammatory cytokines, which leads to lipid peroxidation, has been reported.27 Therefore, the remarkable infiltration of inflammatory cells in the liver as demonstrated in this study may have caused a significant augmentation of ROS and ultimately pathological injuries. Previous reports showed that CPA accumulates in edible tissues in poultry, in milk of ewes, and in eggs of laying hens.28 Therefore, humans could be exposed to CPA through the ingestion of contaminated meat, milk, egg, and / or directly through the consumption of contaminated grains and certain cheeses ripened by microorganisms that produce CPA.29 Thus, it raises concern that CPA may cause the same pathological damages and biochemical changes in humans, as well. In summary, these data showed that CPA induces both the biochemical and pathological changes in the liver and kidneys of the exposed broilers. The elevated MDA content and equally the attenuation of the antioxidant capacity due to CPA exposure may likely result in inflammation in target tissues, which could cause pathological abnormalities. Funding This research received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.

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