Effects of long term oral acrylamide administration on alpha naphthyl acetate esterase and acid phosphatase activities in the peripheral blood lymphocytes of rats.

Acrylamide is an important industrial chemical; it also is formed in starch-rich foodstuffs during baking, frying and roasting. Most acrylamide exposure occurs by ingestion of processed foods. We investigated possible immunotoxic effects of extended administration of low doses of acrylamide in rats. To do this, we measured alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) activities in peripheral blood lymphocytes. Male and female weanling Wistar rats were administered 2 or 5 mg acrylamide/kg/day in drinking water for 90 days. Peripheral blood was sampled at the end of the administration period. We found ANAE staining in eosinophils and T-lymphocytes, but not in monocytes, platelets, B-lymphocytes and neutrophils. ACP-ase was found in B-lymphocytes. We found a significant reduction of the ratio of ANAE:ACP-ase in lymphocytes of the experimental animals compared to controls. We found no statistically significant differences between the doses or sexes. We found that acrylamide ingested in processed foods might affect the immune system adversely by decreasing the population of mature T- and B-lymphocytes.

The effects of aflatoxin B1 on growth hormone regulated gene-1 and interaction between DNA and aflatoxin B1 in broiler chickens during hatching.

Many types of aflatoxin cause problems for both public and animal health. Aflatoxin B1 (AFB1) is the most toxic and commonly encountered fungal toxin that appears in poultry feed and in feeds stored under unsuitable conditions. AFB1 decreases feed quality, egg production and fertility of hatching eggs. Also, AFB1 alters the development of embryos by infecting eggs. We investigated using sequence analysis the changes caused by different concentrations of AFB1 on the promoter sequences of the growth hormone regulated gene-1 (GHRG-1) in chick embryo at 13, 17, 19 and 21 days incubation. DNA isolated from the liver of chick embryos treated with different concentrations of AFB1 was separated using agarose gel electrophoresis to detect apoptosis, and DNA interaction with AFB1 was investigated using plasmids to detect changes in electrophoretic mobility and their effects on DNA. Base changes of the promoter sequences of GHRG-1 in 5 ng/egg, 15 ng/egg and 40 ng/egg doses of AFB1 were increased on day 19 compared to base changes of the same AFB1 doses on day 13. We also found that AFB at different concentrations changed the mobility of DNA by binding to it, and that high doses of AFB1 destroyed DNA. The DNA interaction study using plasmid demonstrated that AFB1 at high doses was bound to plasmid DNA, slowed its mobility and inhibited restriction cuts.

Effects of bisphenol A on antioxidant system and lipid profile in rats.

We investigated the effects of bisphenol A (BPA) on antioxidant system enzymes, blood lipid profile and histologic structure of liver and pancreas in rats. We used 40 8-week-old male Wistar albino rats. The animals were divided into five groups of eight: control, vehicle, BPA-5, BPA-50 and BPA-500. BPA was dissolved in ethanol, then mixed with corn oil. The control group was untreated. The vehicle group was given the ethanol-corn oil mixture. The BPA 5, BPA 50 and BPA 500 groups were given 5, 50, and 500 µg/kg body weights/day, respectively. After 8 weeks, blood and tissue samples were obtained from the animals and plasma GSH, TBARS, SOD, GPx, CAT, NO, total cholesterol, triglyceride, HDL, LDL, insulin and glucose were measured. The sections were stained using histochemical and immunohistochemical methods. BPA significantly decreased the levels of GSH, SOD, GPx and CAT, and increased the levels of TBARS and NO in plasma. There was no significant difference among the groups in plasma insulin and glucose levels. The percentage of insulin immunoreactive cells in islets increased significantly in the BPA-500 group. The H-score of the BPA-5 and BPA-50 groups decreased significantly compared to controls. We found that BPA caused oxidative stress and disruption of pancreatic ß-cell function. Therefore, BPA is a risk factor for animal and human health.

Adverse effects of aflatoxin B1 on skeletal muscle development in broiler chickens.

Detrimental effects of in ovo administrated of aflatoxin B1 (AFB1) on the embryonic development of skeletal muscle were determined using histological methods. A total of 420 eggs of a Ross broiler parent stock were incubated and divided into 4 groups: (1) control, (2) 5 ng AFB1/egg group, (3) 15 ng AFB1/egg group, (4) 40 ng AFB1/egg group. Test solutions were injected via the air-sac, just prior to setting the eggs in the incubator. Five eggs from each group were opened on different days of incubation (11d, 13d, 17d and 21d). Developmental stages of embryos were determined according to the Hamburger-Hamilton scale and embryos were weighed. Skeletal muscle tissue samples were dissected and fixed, sectioned and stained with Crossman's trichrome and AgNOR. The mean relative embryo, leg muscle and breast muscle weights of AFB1-treated groups were lower than the control group and decreased with increasing AFB1. The nucleus area and AgNOR area of the AFB1-treated groups were also lower than the control group whereas there were no significant differences in AgNOR numbers and AgNOR area/nucleus area among the treatment groups. It was concluded that in ovo administrated of AFB1 adversely affected the embryonic development of skeletal muscle and that affected animals might therefore be more susceptible to skeletal and muscle disorders during the growing period.

Cytochemistry of peripheral blood leukocytes in thoroughbred foals.

We attempted to characterize the cytochemical staining patterns of leukocytes and to determine the percentages of alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACPase) positive lymphocytes in peripheral blood of thoroughbred foals at different ages. Blood samples were obtained from the jugular veins of 60 healthy thoroughbred foals, 1 day, 3 days and 1 year old. Each age group included 10 male and 10 female animals. Peroxidase (PO) activity was detected in neutrophils, eosinophils and monocytes. Lymphocytes were negative for PO staining. Periodic acid-Schiff (PAS) staining was observed in neutrophils and monocytes; eosinophils were negative. The majority of lymphocytes were negative, but a few cells showed granular PAS positivity. Monocytes were strongly positive for ANAE and ACPase, and the enzymatic reaction was common in lymphocytes. Neutrophils showed a weakly positive reaction for ANAE and ACPase. Eosinophil granules were negative or weakly positive for ANAE and usually negative for ACP, but intergranular areas were positive. Mean ANAE positive peripheral blood lymphocytes (PBL) were 67.70, 73.10 and 49.20% in females; 64.00, 70.53, and 50.60% in males 1 day, 3 days, and 1 year old, respectively. ACPase positive PBL were 27.33, 32.83, and 37.40% in females; 29.67, 31.67, and 38.40% in males 1 day, 3 days, and 1 year old, respectively. For both enzymes, the differences between mean values for the genders were not statistically significant, but significant differences were found with regard to age. We provide comparative hematological data as a guide for identifying blood cells in thoroughbred foals.

The effect of acrylamide on alpha-naphthyl acetate esterase enzyme in blood circulating lymphocytes and gut associated lymphoid tissues in rats.

The aim of this study is to determine the functional effects of the acrylamide (AA) administrated by oral gavage on the peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in male Sprague-Dawley rats using alpha-naphthyl acetate esterase (ANAE) demonstration. For this purpose, two separate experiments were performed with Sprague Dawley rats. In Experiment-I rats were gavaged with 0, 30, 45 and 60 mg/kgb.w. AA for five consecutive days and in Experiment-II rats were gavaged with 0, 125, 150, and 175 mg/kg/b.w. AA for single oral dose. Animals were sacrificed 24 h after the last treatments in both experiments by servical dislocations under ether anaesthesia. Blood samples were collected from the heart in heparinized (10 UI heparin/ml(-1) of the blood) tubes before sacrification and lymphoid tissue samples from the ileal Peyer's patches (IPPs) were taken and processed for histochemical demonstration of ANAE following the sacrification. The lymphoid follicles of the IPPs of animals given 125, 150 and 175 mg/kgb.w. AA were markedly reduced in size. Germinal centres (GCs) markedly regressed in AA-treated animals compared with those of controls. ANAE-positive lymphocyte depletion of IPPs was very prominent in the high doses AA-treated animals. In the animals treated with 30, 45, and 60 mg/kg b.w. AA, the IPPs had similar histology to those of the controls. ANAE-positive peripheral blood lymphocyte levels significantly decreased in AA exposed groups in a dose dependent manner (p<0.05). In conclusion, AA has detrimental effects on peripheral blood lymphocytes (PBL) and the Gut Associated Lymphoid Tissue (GALT) in rats.

Acrylamide-treatment and responses to phenylephrine and potassium in rat aorta.

Acrylamide (ACR) is a chemical used in many industries around the world and was found to form naturally in foods cooked at high temperatures. The aim of the present study is to evaluate the influence of ACR treatment on vascular responses to phenylephrine (PHE; 10-9-3×10-4 M) and potassium chloride (KCl; 5-100 mM). We also examined the role of gender in these responses. The animals in both genders were divided into three groups as follows. (1) Control animals, (2) ACR-I; ACR-treated (2 mg/kg-d for 90 days), (3) ACR-II; ACR-treated (5 mg/kg-d for 90 days). Male rat aortas were more sensitive to PHE and KCl than female aortas. ACR-treatment increased the sensitivity to PHE and KCl, in both genders. Compared to the control group, ACR treatment significantly reduced the luminal area of both male and female rat aortas. Furthermore, the responses to PHE and KCl were similar in both 2 mg/kg-d ACR-treated rat aortas with 5 mg/kg-d ACR-treated rat aortas, in both genders. The results of this study suggest that ACR treatment affects vascular contractility and morphology in both gender of rat aorta.

Determination of the effects of aflatoxin B1 given in ovo on the proximal tibial growth plate of broiler chickens: histological, histometric and immunohistochemical findings.

Detrimental effects of aflatoxin B1 (AFB1) on the embryonic development of broiler tibia and its proximal growth plate were determined by means of histological, histometric and immunohistochemical methods. For this, 420 fertile eggs from parent stocks of Ross 308 broiler chickens were divided into five groups according to the proposed treatment: a control untreated group, a group injected with 30% ethanol and three further groups to be injected with 5, 15 or 40 ng AFB1. The eggs were injected into the air space prior to incubation. Five eggs from each group were opened at 9, 11, 13, 17, 19 and 21 days of incubation and tibial tissue samples were removed, dissected of muscle and connective tissues, and processed by means of routine histological techniques. The cell proliferation rate of the epiphyseal growth plate cells was determined by immunohistochemical assay of proliferating cell nuclear antigen (PCNA) expression. The results showed that both proliferative and hypertrophic zones narrowed significantly (P<0.05), when compared with the controls, in all of the AFB1-treated groups whereas the transitional zone thickened, especially in the group given 40 ng AFB1 group. The PCNA positivity indices of 15 and 40 ng AFB1-treated groups were significantly higher (P<0.05) on days 11, 13, 17, 19 and 21 of incubation. It was concluded that in ovo-administered AFB1 adversely affected embryonic development of the tibial growth plate, and that affected hatched broilers might also be more susceptible to skeletal disorders during growth.

Hematology and enzyme histochemistry of peripheral blood lymphocytes in domestic pigeon (Columba livia f. domestica).

The purpose of our study was to determine the percentages of alpha-naphthyl acetate esterase (ANAE)-positive and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL) in both sexes of one-year-old domestic pigeons (Columba livia f. domestica). Blood samples obtained from 12 healthy domestic pigeons were used. The mean percentages of ANAE-positive PBL for females and males were 47.8% and 48.8%, respectively, whereas the mean percentages of ACP-positive PBL were 67.5% and 68.6%, respectively. The proportions of PBL were 49.3% and 48.6% in males and females, respectively.

Histological and enzyme histochemical investigation of the hemal nodes of the hair goat.

We investigated the structure of the hemal node in six healthy hair goats using histological and enzyme histochemical methods. After processing, tissue sections were stained with Crossman's trichrome, Gordon-Sweet's silver and Pappenheim's panoptic stains. Alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (ACP-ase) were demonstrated in frozen sections. Hemal nodes were encapsulated by connective tissue and few smooth muscle cells. Several trabeculae originated from the capsule and extended into the hemal node. A subcapsular sinus was present beneath the capsule and was continuous with the deeper sinuses. Subcapsular and deep sinuses were filled with erythrocytes. The parenchyma consisted of lymphoid follicles, diffuse interfollicular lymphocytes and irregular wide lymphoid cords. Cortical and medullary regions were not distinct. ANAE (+) and ACP-ase (+) cells were located mainly in the germinal centers of the lymphoid follicles and also were scattered equally in the interfollicular region and lymphoid cords. Monocytes, macrophages and reticular cells displayed a diffuse positive reaction, whereas localized granular positivity was observed in lymphocytes. We demonstrated that the general structure of the hair goat hemal nodes is similar to that of other ruminant species.

Effects of adding yeast cell walls and Yucca schidigera extract to diets of layer chicks.

This research was conducted to determine the impact of diet supplementation with yeast cell walls and Yucca schidigera extract on the growth performance, antibody titres, and intestinal tissue histology of layer chicks. White, 1-d-old, Hy-Line hybrid chicks (n = 840) were divided into 4 main groups, each comprising 7 replicates of 30 chicks (n = 210): (1) control; (2) 1000 mg/kg yeast cell walls (YCW) added; (3) 1000 mg/kg Yucca schidigera extract (YE) added; and (4) 500 mg/kg YE + 500 mg/kg YCW added. The trial lasted 60 d. Daily weight gain of the chicks was positively affected between d 45-60 in the YE and YCW + YE groups compared with the control group. Overall, feed consumption did not differ between the control and YCW, YE, YCW + YE groups during the 60 d study period. Feed efficiency was better in the YE and YCW + YE groups than in the control group between d 1-60. During the 60 d evaluation period, live weight gain, and final live weight were higher in YE and YCW + YE groups than in the control group. Antibody titres against infectious bronchitis and infectious bursal disease did not differ among the 4 treatments, but those for Newcastle disease were higher in the YE + YCW groups than in the control, YCW and YE groups on d 45. There were differences in intestinal histomorphometry between the 4 treatments. The height of the jejunal and ileal villi was greater in the YE and YCW + YE groups than in the control and YCW groups. It can be concluded that YCW and YE supplementation for layer chicks is beneficial for growth performance and intestinal histology during the 1-60 d growing period.

Effects of Echinacea extract on the performance, antibody titres, and intestinal histology of layer chicks.

1. This research was conducted to determine the effect of diet supplementation with Echinacea extract (cichoric acid) on the growth performance, antibody titres and intestinal tissue histology of layer chicks. 2. White, 1-d-old, Hy-Line hybrid chicks (n = 540) were divided into three treatments, each consisting of 6 groups of 30 chicks (n = 180): (1) control; (2) 2·5 mg/kg cichoric-acid-fed; and (3) 5 mg/kg cichoric-acid-fed. The trial lasted 60 d. 3. While the growth performance of the chicks was depressed between d 1 and 45, it was found to improve between d 45 and 60. 4. Feed consumption was lower in both of the cichoric-acid-fed groups than in the control group between d 1-15 and 15-30, but was higher between d 30 and 45. Overall, mean feed consumption did not differ between the control and cichoric-acid-fed groups during the 60 d study period. 5. During the 60 d evaluation period, live weight gain, feed utilisation rate and final live weight were higher in the control group than in both of the cichoric-acid-fed groups. 6. Antibody titres against infectious bronchitis and infectious bursal disease did not differ between the three groups, but those for Newcastle disease were higher in the 2·5 mg/kg cichoric-acid-fed group than in the control group after 45 d. 7. Height and width of the jejunal villus and the thickness of the muscle layer were lower in the 5 mg/kg cichoric-acid-fed group than in both the control and the 2·5 mg/kg cichoric-acid-fed groups. The height of the ileal villus was also lower in the 5 mg/kg cichoric-acid-fed group than in the other two groups. 8. Echinacea extract supplementation for layer chicks appears not to benefit growth performance and intestinal histology during the growing period.

Histochemical and histological evaluations of the effects of high incubation temperature on embryonic development of thymus and bursa of Fabricius in broiler chickens.

1. The effects of experimentally induced heat-stress on the embryonic development of bursa of Fabricius and thymus of the chicken were investigated by means of histological and enzyme histochemical methods. 2. In the experiments, 250 fertile eggs of the Ross 308 broiler strain were divided into two groups. The control eggs were maintained under optimal conditions (378 degrees C and 65 +/- 2% relative humidity, RH) during the whole incubation period. Heat stressed eggs were maintained under normal conditions (378 degrees C and 65 +/- 2% RH) until the 10th d of incubation and then exposed continuously (24 h per d) to high temperature (388 degrees C and 65 +/- 2% RH). Blood and tissue samples were taken from 10 animals of each group at d 13, 15, 18 and 21 of incubation and at d 2, 4 and 7 post-hatch. Tissue samples were processed for enzyme histochemical methods in addition to routine histological techniques. 3. The results revealed that egg temperatures were higher than incubator air temperature. Long-term heat-stress (401-406 degrees C egg temperature) retarded development of thymus and bursa of Fabricius. Peripheral blood ACP-ase and ANAE-positive lymphocyte levels of heat-stressed animals were lower than in the controls. 4. These results give some morphological evidence for immunosuppression induced by high temperature exposure during the embryonic development. Temperature distribution and air circulation in incubator should be questioned in the case of lower broiler flock immunity.

Expression of Fas and Fas-ligand and analysis of argyrophilic nucleolar organizer regions in squamous cell carcinoma: relationships with tumor stage and grade, and apoptosis.

The aim of this study was to investigate whether levels of Fas and Fas-ligand (Fas-L) expression in oral and oropharyngeal squamous cell carcinomas (SCCs) are associated with tumor stage and grade, and to assess whether parameters related to argyrophilic nucleolar organizer regions (AgNORs) correlate with apoptosis in SCC cells and adjacent normal epithelium. Fifty-two specimens taken from the tumoral and neighboring normal tissues of 26 patients with oral and oropharyngeal SCC were analyzed for Fas/Fas-L expression, and 24 specimens from 12 patients for AgNOR parameters. Seventeen (65%) of the tumors were Fas and/or Fas-L-positive (by immunohistochemistry). A significant positive correlation was found between Fas/Fas-L expression and clinical tumor stage (P<0.01). Mean AgNOR number per nucleus, AgNOR size and the percentage area of each nucleus occupied by AgNORs (percent of nuclear area) were significantly increased in the SCC cells (4.49+/-1.28, 4.48+/-1.42, 5.56+/-1.22, respectively) when compared with the control neighboring squamous epithelial cells (2.58+/-0.61, 1.64+/-0.59 and 4.35+/-0.62%, respectively) (P<0.01). A significant positive correlation was found between the AgNOR parameters and Fas/Fas-L expression as apoptotic markers in the tumoral cells of SCC (P<0.05). There was also a significant positive correlation between the AgNOR parameters and the grading of tumors (P<0.05). In conclusion, AgNOR count was a strong proliferation marker in patients with SCC, and Fas and Fas-L staining was useful in tumor grading.