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.

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.

Clinical and light microscopic studies of the conjunctival tissues of dogs with bilateral keratoconjunctivitis sicca before and after treatment with topical 2% cyclosporine.

We determined the concentrations of goblet and immune cells in conjunctival imprints and tissues of canines with keratoconjunctivitis sicca (KCS) before and after cyclosporine A (CsA) treatment. Twelve dogs with bilateral KCS were assigned to three groups: untreated, treatment group 1, and treatment group 2. The treatment groups were treated topically with 2% ophthalmic CsA solution for 45 days; CsA treatment group 2 was followed for an additional 30 days after discontinuation of the drug. Schirmer tear test (STT) scores were recorded prior to CsA treatment and on alternate days throughout the experiment. CsA treatment improved the STT scores, restored conjunctival histology, increased goblet and epithelial cell numbers, and decreased numbers of inflammatory cells. Although the STT scores regressed slightly at day 30 after discontinuing the treatment, the scores were higher than the baseline values. Topical CsA treatment resolved clinical signs of KCS, improved STT scores and restored normal conjunctival histology.

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.

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.

Investigation of α-naphthyl acetate esterase and acid phosphatase in the peripheral blood leukocytes of greyhounds.

The purpose of our study was to determine the percentages of α-naphthyl acetate esterase (ANAE)- and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL), the presence of the ANAE and ACP enzymes in leukocytes, and the proportion of PBL in greyhounds. Peripheral blood samples were collected from the cephalic antebrachial vein of 14 (7 animals of each sex) healthy 1-2-year-old greyhounds. Mean percentages of ANAE-positive PBL were found to be 73.29 ± 0.95% in female and 74.29 ± 2.21% in male dogs. The difference between mean values of the genders was not statistically significant. The ACP values were 36.00 ± 2.94% for females and 33.57 ± 2.15% for males. No significant differences were found with regard to gender. For both enzymes, although monocytes and eosinophilic granulocytes displayed a positive reaction, neutrophils gave negative reactions. The proportion of PBL was 36.29 ± 5.31% and 33.00 ± 2.38 % in female and male dogs, respectively. The differences were not significant.

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.

Hematology and enzyme histochemistry of the peripheral blood leucocytes in rock partridges (Alectoris graeca).

The purpose of this study was to determine the percentages of alpha-naphthyl acetate esterase (ANAE)-positive and acid phosphatase (ACP)-positive peripheral blood lymphocytes (PBL) and the percentages of leukocytes in the rock partridge at different ages. Blood samples obtained from the vena basilica of 18 healthy rock partridges (Alectoris graeca) at 1 d, 5 wk, and 12 wk of age were used. Mean percentages of ANAE-positive PBL for the 3 age groups were 37, 29.83, and 47.83% for 1 d, 5 wk, and 12 wk of age, respectively. Heterophils also gave ANAE-positive reactions. Mean percentages of ACP-positive PBL for the 3 age groups were 70, 81, and 86.1% for 1 d, 5 wk, and 12 wk of age, respectively. Although monocytes showed a diffuse granular staining pattern, heterophils displayed a weak positive reaction for ACP. Thrombocytes showed a small granular staining pattern. This study contributes by broadening the hematological research on avian species and provides a guideline for identifying blood cells in the rock partridge.

Effects of aflatoxin B1 on the development of the bursa of Fabricius and blood lymphocyte acid phosphatase of the chicken.

1. In this study, embryotoxicity and effects of in ovo administration of aflatoxin B1 (AFB1) on the embryonic development of the bursa of Fabricius were determined in fertilised chicken eggs by histological methods and histochemical demonstration of acid phosphatase (ACP-ase) enzyme. Peripheral blood lymphocyte percentages were also estimated. 2. Embryonic stages were determined according to the Hamburger-Hamilton (H-H) scale. Mortality rates increased in AFB1-injected groups in a dose-dependent manner. Embryonic deaths were concentrated at H-H 31 in the 5 ng AFB1/egg group while the deaths were highest at H-H 25 in the 10 and 20 ng AFB1/egg groups. In the 40ng AFB1/egg group, embryonic deaths mostly occurred during the first 70 to 72 h of incubation (H-H 20). 3. Bursal development was quite similar at d 7 of incubation in control and all experimental groups although development had been substantially impaired and retarded at d 10 of incubation in 10, 20 and 40 ng AFB1/egg groups. A gradual decrease of ACP-ase positive lymphocyte numbers was observed with increasing AFB1 doses. The chicks in AFB1-treated groups hatched with poorly developed bursae compared with those of the controls. However, proportions of ACP-ase positive lymphocytes substantially increased at hatch and at 21 d of incubation all groups had similar numbers. 4. The results revealed that breeder diets should be investigated for aflatoxins and specifically AFB1, in cases of low hatchability and flock immunity, because low concentration of AFB1 transferred into the fertilised eggs might be the cause of serious problems.

Mechanisms contributing to the vaso-active effects of prilocaine in human skin.

We investigated the roles of the endothelial nitric oxide and cyclo-oxygenase pathways in mediating the vasoactivity of prilocaine in the skin. We injected prilocaine 1% intradermally into forearm skin of 10 healthy, male subjects. Nitric oxide synthesis was inhibited at a second site by co-injecting prilocaine with l-NAME 1%. We then repeated the injections while blocking the cyclo-oxygenase pathway with aspirin (4 x 600 mg). We measured blood flow responses to the injections using laser Doppler imaging. We found that, after the traumatic effects of injection had subsided, l-NAME reduced the vascular response to prilocaine by a third (p = 0.012), indicating an influence specifically on the drug response. Aspirin had no effect on the response (p = 0.588). We conclude that the vasoactive effects of prilocaine in human skin are mediated partly through the release of endothelial nitric oxide and, although other mechanisms might also be involved, the cyclo-oxygenase pathway does not appear to play a role.

Embryotoxicity assay of aflatoxin produced by Aspergillus parasiticus NRRL 2999.

1. The embryotoxicity of mixed aflatoxins (AF) and aflatoxin B1 (AFB1) were evaluated by a modified chick embryotoxicity screening test (CHEST). Adverse effects on the early embryonic development of thymus and bursa of Fabricius were also investigated by light microscopy. AF consisted of 83.06% AFB1, 12.98% AFB2, 2.84% AFG1 and 1.12% AFG2. 2. A total of 448 fertilised laying hens' eggs were used. AF and AFB1 were injected into the eggs at doses of 10, 100 and 1000 ng/egg. Embryonic developmental stages were evaluated according to the Hamburger-Hamilton scale (HH-scale). 3. The results showed that AFB1 given at 10 ng/egg had a significantly (P<0.05) greater embryotoxic effect than AF given at a similar dose. The higher doses of both AF and AFB1 caused higher embryonic mortality and also an increase in early deaths. 4. In the groups receiving 100 ng/egg AF and AFB1 an abnormal development was seen, with a protruded central region, corresponding to the area pellucida of the blastoderm. No other developmental abnormality attributable to AF or AFB1 was found.

Efficacy of polyvinylpolypyrrolidone in reducing the immunotoxicity of aflatoxin in growing broilers.

1. Protective action of an enzyme-linked polyvinylpolypyrrolidone (PVPP, Mycofix Plus) against the immunosuppressive effect of afatoxins (AF) was evaluated by determination of peripheral blood T-lymphocyte proportions and splenic plasma cell counts. Histological changes in lymphoid organs were also investigated by light microscopy. One-d-old broiler chicks (Hybro) received 2.5 mg/kg diet AF (83.06% AFB1, 12.98% AFB2, 2.84% AFG1, 1.12% AFG2) with or without PVPP (3g/kg diet) until 21 d of age. When compared with controls, AF treatment significantly decreased peripheral T-lymphocyte counts. AF caused a slight decrease in splenic plasma cell counts. The addition of PVPP to an AF-containing diet significantly increased T-lymphocyte counts. Splenic plasma cell counts were numerically intermediate between control and AF groups. 3. The dietary addition of PVPP to AF-free diet did not significantly alter either T-lymphocyte or splenic plasma cell counts.