Effects of dietary milk thistle on blood parameters, liver pathology, and hepatobiliary scintigraphy in white Carneaux pigeons (Columba livia) challenged with [B.sub.1] aflatoxin.
Article Type: Report
Subject: Liver diseases (Risk factors)
Liver diseases (Research)
Liver diseases (Diagnosis)
Liver diseases (Care and treatment)
Milk thistle (Health aspects)
Milk thistle (Research)
Radioisotope scanning (Usage)
Authors: Grizzle, Judith
Hadley, Tarah L.
Rotstein, David S.
Perrin, Shannon L.
Gerhardt, Lillian E.
Beam, James D.
Saxton, Arnold M.
Jones, Michael P.
Daniel, Gregory B.
Pub Date: 06/01/2009
Publication: Name: Journal of Avian Medicine and Surgery Publisher: Association of Avian Veterinarians Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2009 Association of Avian Veterinarians ISSN: 1082-6742
Issue: Date: June, 2009 Source Volume: 23 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 252006951
Full Text: Abstract: Milk thistle (Silybum marianum) has been used in humans for the treatment of liver disease because of its antioxidant properties and its ability to stabilize cell membranes and regulate cell permeability. To investigate possible hepatoprotective effects in birds, standardized extracts (80%) of silymarin from milk thistle were tested in white Carneaux pigeons (Columba livia). Pigeons were separated into 3 groups and fed diets formulated to provide milk thistle at a level of 0, 10, or 100 mg/kg body weight per day. After acclimation, the birds were challenged with [B.sub.1] aflatoxin (3 mg/kg body weight for 2 consecutive days) by oral gavage. Liver function then was assessed by hematologic testing and plasma biochemical analysis, liver histopathology, and hepatobiliary scintigraphy. Results of histopathology and hepatobiliary scintigraphy showed no protective effects from milk-thistle administration. Aflatoxin challenge resulted in hepatic infammation and necrosis, biliary-duct hyperplasia, and lymphocyte infiltration. All hepatobiliary scintigraphy elements increased significantly after aflatoxin challenge. Bile acid levels and plasma enzyme concentrations of aspartate aminotransferase, lactate dehydrogenase, alanine aminotransferase, and creatine phosphokinase all increased after aflatoxin exposure and were mostly unchanged with consumption of milk thistle. Only birds fed 10 mg/kg body weight milk thistle showed significant reductions in lactate dehydrogenase, alanine aminotransferase, and creatine phosphokinase concentrations after aflatoxin exposure. Our results show that consumption of milk thistle is not associated with hepatoprotective effects against acute [B.sub.1] aflatoxin exposure in pigeons.

Key words. aflatoxin, milk thistle, silymarin, scintigraphy, histology, bile acids, liver enzymes, avian, pigeon, Columba livia

Introduction

Mycotoxins, highly toxic fungal metabolites, are commonly produced by Aspergillus, Fusarium, or Penicillium species. These compounds induce a wide range of clinical syndromes, including hepatocellular carcinoma, immunosuppression, damage to multiple organs, hormonal disorders, and edema of the body cavity? (1,2) [B.sub.1] aflatoxin, T-2 toxin, ochratoxin A, and deoxynivalenol are all important threats to birds? (1,3-5) Of the identified hepatotoxins, [B.sub.1] aflatoxin is the best known and studied. It is produced during the harvest, processing, and storage of feed grains and nuts. Cytochrome P-450 monooxygenases metabolize

aflatoxin to [B.sub.1]-8,9-epoxide, the reactive form of the compound, which binds to cellular macromolecules and causes periportal hepatic injury. (6) Clinical signs of aflatoxicosis are depression, poor growth, and weight loss, and necropsy findings include an enlarged pale liver commonly infiltrated with fat, splenomegaly, and pancreatic enlargement? Histologic examination reveals bile duct hyperplasia, hyperchromasia that involves periportal hepatocytes, hepatocytic vacuolization, and portal fibrosis. (4-7) Acute exposure results in biliary hyperplasia, massive hepatocyte necrosis, and hemorrhage. (4.6) Liver cirrhosis is a long-term result of either acute or chronic exposure to aflatoxins.

Noninvasive diagnostic tests used to evaluate the avian liver, such as serum or plasma biochemical analysis or radiography, are often not specific or sensitive enough to determine the extent of liver damage. (8) Radiography is limited to assessment of liver size and shape, as well as general evaluation of concurrent problems. Plasma enzymatic tests that evaluate concentrations of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total protein, and albumins can provide information on liver damage. However, measurement of circulating bile acid levels is currently considered the most sensitive indicator of avian liver function. (8-10) Although high bile acid levels are deemed a benchmark of liver disease in general, serum levels of AST, serum alanine aminotransferase (ALT), and LDH have also been reported to increase dramatically in aflatoxin-intoxicated chickens, quail (Coturnix coturnix japonica), pigeons (Columba livia), and cockatiels (Nymphicus hollandicus). (4,11-13) In pet bird species, increases in serum levels of AST were reported in 75% of pigeons (n = 24) and 38% of cockatiels (n = 24) challenged with 3 mg/kg BW [B.sub.1] aflatoxin for up to 10 days until death. (13) Similarly, ALT concentrations increased in 75% of pigeons and 85% of cockatiels. In the same study, LDH increased in 92% of cockatiels.

Quantitative hepatobiliary scintigraphy has been used extensively in mammals as a noninvasive tool for evaluating liver function. (14,15) In the mammalian model, scintigraphy is used to evaluate hepatocellular function, hepatic morphology, and biliary tract patency; [sup.99m]Technetium (Tc) mebrofenin is injected intravenously or intraosseously, and the hepatic extraction fraction is measured. (14) The hepatic extraction fraction can be used as a measure of hepatic function because it relates to the percentage of [sup.99m]Tc-mebrofenin removed by the liver on each circulatory pass. Recently, hepatobiliary scintigraphy was used to assess liver damage in white Carneaux pigeons challenged with ethylene glycol. (16) Good correlation was found between the overall histologic score and scintigraphic measurements, particularly with reference to the area under the heart-time activity curve. Increased levels of histologic indicators of damage to the liver correlated with worsening cellular function as measured by hepatobiliary scintigraphy? (16)

There currently is an interest in vitamins and herbal products used to support avian immune function. In Western countries, people use herbal remedies predominantly as an alternative to synthetic drugs. However, most herbs have not been tested for safety or efficacy when used as marketed, and regulatory oversight for these products is minimal to absent. (17-19) Numerous herbal products are purported to support the immune system, reduce inflammatory responses by antioxidant action, and aid in fighting bacterial, viral, and other infectious or toxic agents. (17,20) Milk thistle (Silybum marianum) has been used in humans for the treatment of liver disease associated with degeneration and necrosis; it acts as an antioxidant, stabilizes cell membranes, and regulates cell permeability. (17,21,22) Silymarin, which is extracted from the seeds of milk thistle, upregulates the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione Stransferase. Antioxidants offer protection against reactive oxygen species in erythrocytes. (23) Silybin, the active ingredient of silymarin, has been shown to have hepatoprotective effects against mushroom poisoning if administered within 48 hours of consumption. (24) Reviews by Tamayo and Diamond (25) and Rambaldi et al (26) report higher survival rates among patients with alcohol-induced liver cirrhosis when treated with the silymarin extract of milk thistle; mortality was reduced by 50% in patients with liver disease and without concurrent hepatitis C virus. (26) Among patients with chronic hepatitis, milk thistle has not been reported to alleviate complications of the hepatitis C virus, such as high serum ALT levels and the liver fibrosis marker hyaluronic acid; however, general symptoms and quality of life were improved by either self-medicated or controlled dosages of silymarin (373.5 mg/d). (27,28) Interestingly, in the controlled study, no progression of disease was detected after 24 months of continuous treatment. (29) Silymarin has also been shown to have both antiangiogenic and antimetastatic activity and to suppress the proliferation of tumor cells in lung, breast, ovarian, prostate, and bladder cancer. (30) In a phase III clinical trial of men with prostate cancer, consumption of silymarin (160mg/d) plus soy isoflavones and antioxidants delayed increases of prostate-specific antigen levels when compared with placebo controls that experienced a 2.6-fold increase in the antigen. (31) Thus, silymarin may potentially be useful in disease control and in immune system support in lieu of conventional drugs.

The objectives of this study were twofold: 1) to determine if milk thistle could be used to either prevent or alleviate liver damage in pigeons acutely challenged with dietary aflatoxin, and 2) to determine if hepatobiliary scintigraphy could be used as a diagnostic tool for avian liver disease.

Materials and Methods

Birds

White Carneaux pigeons (n = 21), (9-12) months of age, were purchased from a regional breeder (Palmetto Pigeon Plant, Sumpter, SC, USA). Birds were housed in an environmentally controlled room in individual battery grower cages that supplied a space of 0.37 m2/bird. During the study, the birds were isolated from all other animals at the facility. On arrival, all the birds were weighed and deemed healthy on the basis of physical examination findings. As a precautionary measure, all birds were treated with an anthelmintic (fenbendazole; 10 mg/kg PO for 5 days) and treated for trichomoniasis (metronidazole; 15 mg/kg PO for 5 days) and coccidiosis (sulfadimethoxine; 50 mg/kg PO for 5 days). The birds were then allowed to acclimate for 4 weeks, which served as a quarantine period before the beginning of the study. During that time, the birds were fed a basal pigeon maintenance diet, which contained 2750 Kcal/kg feed metabolizable energy, 15.0% crude protein, 0.9% calcium, and 0.65% total phosphorus. During the last week of the acclimation period, the birds were weighed again and daily feed consumption was measured. Throughout the experiment, feed and water were supplied ad libitum, and artificial light was supplied for 12 hours a day. Live-animal experiments were conducted after protocol approval of the University of Tennessee Animal Care and Use Committee.

Study protocol

Based on an average daily feed consumption of 25 g/bird per day and an average BW of 600 g, birds were assigned to 1 of 3 dietary treatments (n = 7 for each treatment group). Group 1 was fed a diet that contained no milk thistle. For groups 2 and 3, milk thistle was added to the diet based on the manufacturer's recommended dosage of milk thistle for people (10 mg/kg BW per day; 80% silymarin from Silybum marianum; Natural Factors, Everett, WA, USA). Milk thistle was added to the diet at 10 mg/kg BW per day for group 2 and at 100 mg/kg BW per day for group 3. The birds were fed experimental diets for 21 days before collecting baseline samples and then continuously until the completion of the experiment on day 60.

After the initial 21-day period, the pigeons were transported to the University of Tennessee College of Veterinary Medicine. The birds were anesthetized with 2% isoflurane delivered in oxygen by face mask. Once anesthetized, each bird was intubated with a 2-mm uncuffed endotracheal tube and maintained on 1.5%-2.5% isoflurane. Blood samples were collected from the left or right basilic vein and submitted for a complete blood cell (CBC) count, as well as plasma biochemical analysis and bile acid measurement (Comparative Pathology Laboratory, Miller School of Medicine, University of Miami, Miami, FL, USA).

Liver function was then evaluated by hepatobiliary scintigraphy as previously described. (16) Briefly, pigeons received a bolus intravenous injection of 1.5 2.0 mCi [sup.99m]Tc-mebrofenin and were placed dorsally over a large field-of-view gamma-ray detecting camera (GE 400 Gamma Camera, Fairfield, CT, USA). The camera was fitted with a low-energy, all-purpose parallel hole collimator and interfaced with a dedicated imaging computer (NucLear Mac, Scientific Imaging Inc, Littleton, CO, USA). Scintigraphic measurements were collected before injection of the radionucleotide. Data were collected at a rate of 1 frame every 5 seconds for the first 5 minutes, followed by 1 frame every 30 seconds for 35 minutes for a total data collection period of 40 minutes.

After the scintigraphy procedure, liver biopsy samples were obtained. A ventral midline incision was made into the hepatic peritoneal cavity immediately caudal to the sternum. Cup-shaped biopsy forceps were used to obtain 2 full-thickness specimens of the right liver lobe; most samples were collected from the caudal border of the liver. Deep liver biopsies were not done before aflatoxin exposure because of the risk of severe hemorrhage. Biopsy samples were submitted for histopathologic evaluation, and the percentage of tissue that exhibited degeneration, necrosis, inflammation, and hemorrhage was assessed. Numerical scores were assigned as follows: 0, no change; 1, 1%-10% cells affected; 2, 11%-25% cells affected; 3, 26%-74% cells affected; and 4, 75%-100% cells affected.

After a minimum 18-day recovery period, the birds were challenged for 2 consecutive days with [B.sub.1] aflatoxin (3 mg/kg BW delivered by crop gavage tube), as previously described. (32) Lyophilized [B.sub.1] aflatoxin powder (Sigma Chemical Company, St Louis, MO, USA) was dissolved in 99.7% pure dimethylsulfoxide (Sigma) to make a stock solution of 10 mg/ml. Tolerance to dimethylsulfoxide had been confirmed in a previous study. (32)

The morning after the second dose of aflatoxin, equal numbers of birds from each treatment group again underwent hepatobiliary scintigraphy and blood sampling. Postaflatoxin measurements were obtained during two 5-day work periods (days 46-60). After scintigraphy, birds were euthanatized by intravenous injection of 0.3 ml pentobarbital sodium. The entire right lobe of the liver was removed, and an approximately 3-cm-long deep-tissue sample was obtained and submitted for histopathologic examination.

Evaluation of scintigraphy data

Hepatobiliary scan data were converted to numerical data suitable for statistical analysis. Regions of interest were drawn over a portion of the liver that did not overlap a major biliary duct and also over the heart. The count density (radioactive counts/pixel) of both liver and heart regions were used to generate liver/heart-time activity curves. Data were imported into an Excel spreadsheet program (Microsoft Corp, Redmond, WA, USA) for further manipulation and then into a mathematical program (IgorPro 3.2, Wavemetrics, Oswego, OR, USA) for deconvolutional analysis. (16,33) Data were collected on time to maximum liver uptake of the radionucleotide, half-time ([T.sub.1/2]) plasma clearance rate for fast and slow phases, [T.sub.1/2] liver clearance rate, hepatic extraction rate of the radionucleotide, hepatic excretion, and the area under the heart-time activity curve. At minimum, for avian patients, the area under the heart-time activity curve was considered useful for detecting parenchymal cell function. (16)

Data Analysis

All data were analyzed by analysis of variance as a randomized block design by using the MIXED procedure with commercial statistical software (SAS v 9.1.3, SAS Institute Inc, Cary, NC, USA). Means were separated by the Fisher least significant difference test. Significance was set at P < .05.

Results

Postaflatoxin liver degeneration, necrosis, inflammation, hemorrhage, and total score did not differ significantly among groups 1-3 (Table 1). Photomicrographs of the liver samples clearly showed parenchymal-tissue degeneration, as evidenced by fatty changes and cellular vacuolization (Fig 1). Bile duct hyperplasia with subsequent infiltration of lymphocytes was also evident. (7,13,34,35) Liver injury was not significantly affected by consumption of milk thistle.

Similarly, postaflatoxin hepatobiliary-response elements did not differ significantly among groups 1-3 (Table 2). With the exception of the hepatic-extraction fraction of the [sup.99m]Tc-mebrofenin radionucleotide, all elements were significantly increased because of exposure to [B.sub.1] aflatoxin. After aflatoxin challenge, the trapezoidal measurement of the heart-time activity curve ([AUC.sub.h-trap]) increased by a minimum factor of 4, and the [T.sub.1/2] liver clearance of the radionucleotide was slowed by a factor of 10. Similarly, fast-phase clearance of the radionucleotide slowed by a factor of 3, and slow-phase clearance was delayed by a factor of 5. Consumption of milk thistle before challenge with aflatoxin had no significant effect on either baseline or postaflatoxin hepatobiliary-response elements.

Postaflatoxin changes in some CBC counts and biochemical parameters were apparent among groups 1-3 (Tables 3 and 4). Values significantly affected by aflatoxin administration in groups 1-3 but not influenced by milk thistle consumption included % heterophils; blood urea nitrogen (BUN), creatinine, C[O.sub.2], and lipase concentrations (increased after aflatoxin administration); and lymphocyte counts (decreased after aflatoxin administration). Mixed effects were observed in groups 1-3 among BUN:creatinine ratio and sodium concentrations (decreased postaflatoxin administration) and phosphorus and uric acid concentrations (increased postaflatoxin administration). The reduction in both the BUN:creatinine ratio and plasma sodium values was significant in groups 1 and 2 birds but not in group 3, and the increases in phosphorus and uric acid concentrations were significant in group 3 and in groups 2-3, respectively.

Feeding milk thistle appeared to result in significant changes of certain plasma biochemical analytes (Table 3). Groups 2 and 3 had a significantly smaller postaflatoxin increase in white blood cell counts compared with controls. The total protein concentration was significantly lower after exposure to aflatoxin in group 1 but was unchanged in groups 2 and 3.

Notable differences were seen in liver enzyme levels after aflatoxin administration and among groups 1-3 (Table 4). All values, with the exception of gamma glutamyl transferase (GGT), increased significantly after a 2-day exposure to aflatoxin. Among treatment birds, plasma ALT and creatine phosphokinase (CPK) values were significantly lower among group 2 birds and numerically less among group 3 birds after aflatoxin exposure. Levels of AST were increased among all groups after aflatoxin challenge and were numerically higher in groups 2 and 3 than in group 1. Both ALT and CPK levels were lower in groups 2 and 3 compared with group 1, and this difference was significantly lower in group 2 compared with either group 1 or group 3. Bile acid concentrations in all 3 groups were significantly increased after exposure to aflatoxin. However, this increase was numerically less in groups 2 and 3.

Discussion

Significant changes in liver function and pathology, as assessed by histopathologic examination of liver tissue, hepatobiliary scintigraphy, plasma biochemical analysis, and bile-acid profiles resulted from acute exposure to [B.sub.1] aflatoxin. Histopathologic examination of liver samples after aflatoxin exposure revealed fatty infiltration and vacuolization, bile-duct hyperplasia, cell necrosis, and inflammation, indicative of hepatopathy and aflatoxicosis, and consistent with findings in previous reports. (7,13,16,34,35) Pathophysiologic changes were not reduced by consumption of milk thistle. This may be the result of the high quantity of aflatoxin used in the study, as well as the short period of exposure. Protection was reported for rats (36) and chicks (35) fed milk thistle at higher doses (100 and 600 mg/kg BW for rats and chicks, respectively) and challenged with aflatoxin at relatively low doses (1 and 0.8 mg/kg BW, respectively). Rats fed 100 mg/kg BW silymarin for 7 days before challenge with a single dose of aflatoxin (1 mg/kg BW) experienced only mild liver damage compared with untreated controls. (36) Similarly, 14-day-old chicks fed milk thistle at a level of 600 mg/kg BW and challenged with aflatoxin at a dosage of 0.8 mg/kg BW for 35 days showed fewer hepatic histologic changes than the controls did. (35) After aflatoxin challenge, liver tissue in the control chicks showed tissue necrosis with multifocal portal infiltration of mononucleates, granulocytes, and eosinophils, but liver damage was less severe among the chicks fed milk thistle. Thus, milk thistle may ameliorate the toxic effects of aflatoxin when the former is fed in higher concentrations and the latter is dosed at lower levels than in the present study.

The second objective of this study was to determine if hepatobiliary scintigraphy could be used as a diagnostic tool for liver disease. We found significant increases in hepatobiliary elements related to aflatoxin exposure, and these findings were consistent with those of Hadley et al. (16-32) Among pigeons dosed with ethylene glycol as the hepatocide, strong correlations ([r.sup.2] = 0.670.74) were found between scores of liver pathology and scintigraphic elements (trapezoidal and calculated area under the heart-time activity curve, and fast-phase plasma clearance of [sup.99m]Tc-mebrofenin), (16) which indicated that the noninvasive technique of hepatobiliary scintigraphy could be used to assess liver function in birds. In the companion article to the above-mentioned study, numerical, though nonsignificant, changes were seen among hepatobiliary-response elements in pigeons challenged with aflatoxin for 2 days. (32) However, significant effects were observed in most elements after a 3- or 4-day challenge. (32)

[FIGURE 1 OMITTED]

Because there was a high mortality rate (5 of 7) in those birds scheduled to be dosed with aflatoxin for up to 6 days, the birds were reassigned to treatment groups of a shorter challenge period, and fewer birds were used. Those changes to group size and composition may have biased the results. In both the present study, and that of a companion article, (32) hepatobiliary scintigraphy was not sensitive enough to show changes in liver function among the treatment groups. Among birds challenged for 2-4 days with aflatoxin, the only response element that varied with the duration of aflatoxin exposure was the slow-phase clearance of the nucleotide. (32) In the current trial, changes in response elements were not observed for any level of dietary milk thistle.

Long-term studies with administration of much lower levels of aflatoxin may provide a better model for hepatobiliary scintigraphy. Results of this study and that of Hadley et al. (16,32) determined that scintigraphy is a viable alternative to liver biopsy for fragile animals.

In this study, histologic changes did not differ among treatment groups. This suggests that the changes in both liver morbidity and function that resulted from acute aflatoxin exposure were so severe that dietary milk thistle was not able to protect histologic architecture. In addition, even though the product used in this study was of guaranteed potency as an 80% standardized formula of silymarin, the product was not guaranteed to meet United States Pharmacopeia guidelines and may have contained a lesser amount of silymarin than advertised.

Analysis of the effects of dietary milk thistle was possible by evaluating plasma biochemical analytes, including enzymes indicative of hepatic damage. No significant changes that resulted from acute aflatoxin exposure were seen in the red blood cell, monocyte, eosinophil, or basophil counts; hematocrit; or concentrations of glucose, potassium, amylase, calcium, cholesterol, and triglycerides. Total protein levels were significantly lower after aflatoxin exposure in control birds, whereas levels remained unchanged among birds fed milk thistle at either 10 or 100 mg/kg BW per day. A decreased total protein concentration is consistent with reports of aflatoxin inhibition of protein synthesis during both transcription (37) and translation. (38) Stabilization of protein synthesis by milk thistle in the presence of aflatoxin is consistent with its properties of stabilizing membranes and stimulating protein synthesis. (17,39) The marked leukocytosis, with accompanying heterophilia and lymphopenia associated with aflatoxin exposure, was indicative of the inflammatory response to the mycotoxin and exposure to any toxic agent. (40,41) Other possible causes of the increased heterophil count are stress, starvation, tissue necrosis, or pain, (41) all of which would be expected to occur as a result of aflatoxin challenge. Compared with control birds, the increase in the white blood cell count after aflatoxin challenge was moderated in birds fed any level of milk thistle, and values were slightly above the reference range for pigeons (13-23 x l03 cells/[micro]l; Table 3). (42) Stabilization of the leukocyte-cell membrane and protection against oxidative peroxidation are likely reasons for this difference. (36)

Of primary interest were plasma liver enzyme values, because these increase after hepatic injury. (4,36) As reported by Barton et al (,6) increases in AST and ALT concentrations are consistent with hepatic parenchymal injury, whereas increased concentrations of bile acids, GGT, alkaline phosphatase, and 5'-nucleotidase are generally seen with biliary-tract alteration. In birds, however, increases in ALT concentration are not considered an accurate indicator of liver damage, because increases in hepatic levels of ALT are commonly not reflected in the plasma. (10,43) Similarly, CPK is not found in liver tissue but rather in muscle tissue, and increases in concentration of this enzyme are coincident with handling, injected substances that are irritating, or excitement? (10,43) Increases in AST, LDH, and/or CPK concentrations also occur as a consequence of starvation or, as might be expected in this study, as a result of feed refusal because of aflatoxin challenge? (10)

Studies most closely resembling this study are those of Hadley et al, (32) Campbell, (13) and Madheswaran et al. (12) All studies, with the exception of Madheswaran et al, (12) used pigeons for the research model, and all used high doses of aflatoxin as the liver hepatocide. All were acute, short-term studies, with the exception of Madheswaran

heswaran et al, (12) whose study challenged Japanese quail by administering aflatoxin at a dosage of 3 mg/kg feed for 35 days. In agreement with Campbell, (13) pigeons in the present study acutely challenged with aflatoxin had significant increases in AST and ALT concentrations but very little increase in GGT concentrations. Similarly, Hadley and Madheswarn both reported increases in AST concentration after challenge with aflatoxin. (12,32) In contrast to Campbell, (13) results of our study and that of Hadley et al (32) showed a very strong increase in LDH concentration, whereas, in the former study, only a third of the birds tested had an increased LDH concentration. Lactate dehydrogenase is not considered either a specific or sensitive indicator of liver damage; concentrations will rise after either recent liver or muscle damage, or in conjunction with hemolysis of the serum sample. (10) When considering the acute nature of the present study, recent liver damage was evident, and several of the plasma samples used in this study were hemolyzed. Concentrations of CPK were also increased in our study; however, this enzyme was not measured by Campbell. (13) Because CPK is among the first of liver enzymes to rise and birds were caught and transported to the veterinary hospital where they were held for up to 2 hours before sampling, increases in CPK concentration would not be unusual. (10) Numeric, but not significant, increases in GGT concentration were observed after aflatoxin challenge in this study, which are consistent with the data of Madheswarn et al (12) but not that of Campbell, (13) who observed an increase in only 2 of 22 birds challenged with the same dose of aflatoxin. Hadley et al (32) also found increases in GGT concentration, but these were similarly not significant. Gamma glutamyl transferase activity is low in the avian liver, and increases are inconsistent after liver damage. (10) Increases in GGT concentration are considered more useful as a means to detect hepatic carcinomas. (10) In this study, the small sample size and the fact that liver damage as measured by necrosis was less than 10% are possible reasons for not detecting a significant difference.

In contrast to the observed liver pathology and the results of hepatobiliary scintigraphy analysis, milk thistle provided some level of hepatocyte protection, as indicated by liver enzyme profiles. Increases in plasma AST levels after aflatoxin challenge were not affected at either dose of milk thistle. In fact, postaflatoxin AST values were numerically higher in pigeons fed milk thistle than in controls. In contrast, feeding milk thistle at a dose of 10 mg/kg per day resulted in a significant reduction in postaflatoxin plasma ALT levels compared with control birds; however, the reduction was not enough to return plasma ALT levels to pre-aflatoxin levels. Postaflatoxin ALT concentrations in birds fed the higher dose of milk thistle were also numerically lower than in control birds, but the difference was not significant. In rats dosed with much higher levels of silymarin than used in this experiment, complete protection against a 1-time challenge with 1 mg/ kg BW aflatoxin was afforded, and both AST and ALT levels were unchanged after mycotoxin challenge. (36) Similar protective effects, as evidenced by no change in serum ALT levels, were found with broiler chickens fed aflatoxin at the rate of 0.8 mg/kg feed for 35 days, along with silymarin, 600 mg/kg BW. (35) Therefore, higher dietary levels of milk thistle may be indicated to protect against an acute aflatoxin challenge.

Plasma CPK concentrations were increased after aflatoxin challenge. As with ALT, the postaflatoxin increase in CPK concentration was significantly less in the group fed the lower dose of milk thistle compared with control birds; in fact, the postchallenge concentration of CPK was statistically similar to the prechallenge value. In birds fed the higher milk thistle concentration, postchallenge plasma concentrations of CPK were intermediate between control birds and those fed a lower dose of milk thistle. Because CPK responds to trauma, nervousness, starvation, surgery, and other manual manipulations, the increase was not unexpected. (10) Milk thistle may simply have reduced the inflammatory response associated with capture, transport, and surgery during the experiment, as well as any inflammatory response caused by aflatoxin.

Of special interest were the changes observed in bile acids, because Baetz and McLoughlin, (44) Lumeij et al, (9) and Lumeij (45) reported that these analytes may be more sensitive indicators of aflatoxin and liver damage than changes in liver enzymes. Synthesis and excretion of bile acids, extraction from portal blood, and re-excretion depend on liver function. (10,45) Bile acids were increased 28.9-fold after the 2-day challenge with aflatoxin and would be expected to rise coincident with acute insult, hepatic lipidosis, or vacuolation. (10) However, after aflatoxin administration, bile acid concentrations among birds fed either milk thistle diet were numerically lower than those of control birds. In fact, among birds fed the higher concentration milk-thistle diet, postaflatoxin concentrations were not significantly different from prechallenge concentrations. However, even with a 40% reduction in postaflatoxin bile acid concentrations in birds fed milk thistle, the value was still over 100 [micro]mol/L, which is generally considered to be above bile acid reference ranges. (10)

The aflatoxin dose used in this trial was modeled after that used in the Campbell study. (13) In that trial, birds did not succumb to a 3 mg/kg BW dose until after 6 days of consecutive exposure. However, in the companion article to the present report, significant mortality was observed among pigeons after a 3-day exposure, hence the decision to dose for 2 consecutive days in this trial. (32) Regardless, the dose of aflatoxin used in this trial was acute and resulted in significant morbidity so that detection of subtle changes in liver histology or scintigraphic elements in association with dietary milk thistle may simply not have been possible.

Our results showed that reductions in bile acid concentrations and white blood cell counts occurred as a result of feeding milk thistle; however, this should not be interpreted as a protective effect. The fact that milk thistle supported any measure of liver function in conjunction with acute aflatoxin challenge is encouraging and warrants further study. At a lower level of aflatoxin exposure, use of dietary milk thistle may prove to have greater protective effects as has been shown in other species.

Acknowledgments. We thank the Morris Animal Foundation for financial support of this research.

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Judith Grizzle, PhD, MS, Tarah L. Hadley, DVM, Dipl ABVP (Avian), David S. Rotstein, DVM, MPVM, Dipl ACVP, Shannon L. Perrin, MS, Lillian E. Gerhardt, LVT, James D. Beam, LVT, Arnold M. Saxton, PhD, Michael P. Jones, DVM, Dipl ABVP (Avian), and Gregory B. Daniel, DVM, MS, Dipl ACVR

From the Department of Animal Science, University of Tennessee, Morgan Circle, Knoxville, TN 37996, USA (Grizzle, Perrin, Saxton); Small Animal Clinical Sciences (Hadley, Gerhardt, Beam, Jones) and the Department of Pathobiology (Rotstein), Veterinary Teaching Hospital, 2407 River Drive, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA; and Small Animal Clinical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Duck Pond Drive, Phase II, Blacksburg, VA 24061, USA (Daniel). Present address (Hadley): Atlanta Hospital for Birds and Exotics, Inc, 2274 Salem Road, no. 106-149, Conyers, GA 30013, USA.
Table 1. Histopathologic scores of liver biopsy samples collected
before (Pre) and after (Post) administration of [B.sub.1] aflatoxin
(3 mg/kg delivered by crop gavage tube) from control pigeons
(group 1) and pigeons fed milk thistle (group 2, 10 mg/kg body weight
per day; group 3, 100 mg/kg body weight per day). Numerical scores
were assigned on the basis of 0 = no change, 1 = 1%-10% cells
affected, 2 = 11%-25% affected, 3 26%-74% affected, and 4
75%-100% affected.

                     Group 1                Group 2

Parameter        Pre        Post        Pre        Post

Degeneration   0.00       1.86 (b)    0.00 (a)   2.00 (b)
Necrosis       0.00 (a)   0.57 (b)    0.00 (a)   0.86 (b)
Inflammation   1.14 (a)   2.86 (b)    1.29 (a)   2.86 (b)
Hemorrhage     0.00 (a)   0.43 (ab)   0.00 (a)   0.71 (b)
Total          1.14 (a)   5.72 (b)    1.29 (a)   6.43 (b)

                    Group 3

Parameter        Pre        Post

Degeneration   0.00 (a)   2.14 (b)
Necrosis       0.00 (a)   0.71 (b)
Inflammation   1.00 (a)   2.86 (b)
Hemorrhage     0.00 (a)   0.71 (b)
Total          1.00 (a)   6.42 (b)

(a,b) Mean values in the same row with different superscript
letters are significantly different, P < .05.

Table 2. Average hepatobiliary response elements before (Pre) and
after (Post) administration of [B.sub.1] aflatoxin (3 mg/kg delivered
by crop gavage tube) from control pigeons (group 1) and pigeons fed
milk thistle (group 2, 10 mg/kg body weight per day; group 3, 100
mg/kg body weight per day).

Parameter                                   Group 1

                                        Pre         Post

[AUC.sub.h-trap] (cts/ml x min)       4485 (a)   19 407 (b)
[AUC.sub.h-calc] (cts/ml x min)       6675 (a)   67 059 (b)
[T.sub.max] (min)                     3.11 (a)     7.16 (b)
[T.sub.1/2] liver clearance (min)     7.09 (a)    72.92 (b)
Hepatic extraction fraction (%)     107.16 (a)   100.70 (b)
Fast-phase [T.sub.1/2] plasma         0.92 (a)     2.89 (b)
  clearance (min)
Slow-phase [T.sub.1/2] plasma        15.85 (a)    73.88 (b)
  clearance (min)

Parameter                                   Group 2

                                        Pre         Post

[AUC.sub.h-trap] (cts/ml x min)       4174 (a)   21 160 (b)
[AUC.sub.h-calc] (cts/ml x min)       5941 (a)   74 739 (b)
[T.sub.max] (min)                     3.38 (a)     6.94 (b)
[T.sub.1/2] liver clearance (min)     6.53 (a)    79.77 (b)
Hepatic extraction fraction (%)     106.50 (a)    96.63 (b)
Fast-phase [T.sub.1/2] plasma         0.83 (a)     2.62 (b)
  clearance (min)
Slow-phase [T.sub.1/2] plasma        11.75 (a)    76.15 (b)
  clearance (min)

Parameter                                   Group 2

                                        Pre         Post

[AUC.sub.h-trap] (cts/ml x min)       4364 (a)   19 019 (b)
[AUC.sub.h-calc] (cts/ml x min)       6512 (a)   74 565 (b)
[T.sub.max] (min)                     3.30 (a)     9.14 (b)
[T.sub.1/2] liver clearance (min)     6.88 (a)    90.15 (b)
Hepatic extraction fraction (%)     112.23 (a)   100.37 (b)
Fast-phase [T.sub.1/2] plasma         0.93 (a)     2.45 (b)
  clearance (min)
Slow-phase [T.sub.1/2] plasma        15.07 (a)    79.92 (b)
  clearance (min)

Abbreviations: [AUC.sub.h-trap] indicates trapezoidal measurement
of the heart-time activity curve; [AUC.sub.h-calc], calculated
measurement of the heart-time activity curve; [T.sub.max], time
to maximum liver uptake.

(ab) Means in the same row with different superscript letters are
significantly different, P < .05.

Table 3. Average CBC and plasma biochemical parameters before
and after administration of [B.sub.1] aflatoxin (3 mg/kg
delivered by crop gavage tube) from control pigeons (group 1)
and pigeons fed milk thistle (group 2, 10 mg/kg body
weight per day; group 3, 100 mg/kg body weight per day).

                                               Group 1
                          Reference
Analyte                 ranges (46-48)      Pre          Post

Hematocrit (%)             56.4-58        52.9 (a)     48.4 (a)
RBC (x [10.sup.6]/pl)       3.1-4          4.3 (a)      4.0 (a)
WBC (x [10.sup.3]/pl)      13.0           16.7 (ab)    33.9 (c)
  Basophil (%)              2.61           0.0 (a)      0.0 (a)
  Eosinophil (%)            2.21           0.0 (a)      0.0 (a)
  Heterophil (%)           23.0           51.3 (b)     88.4 (a)
  Lymphocyte (%)           65.6           47.6 (b)     11.1 (c)
  Monocyte (%)              6.61           1.1 (a)      0.4 (a)
Amylase (U/L)                 --         432.0 (a)    865.1 (a)
BUN (mg/dl)                 1.1-2          2.4 (a)      8 l (b)
BUN: creatinine ratio      14.9-25        24.3 (c)     14.2 (ab)
Calcium (mg/dl)             7.6-10         8.1 (a)      7.6 (a)
Cholesterol (mg/dl)           --         227.6 (a)    207.1 (a)
C[O.sub.2] (mmol/L)           --          22.6 (ab)    25.4 (c)
Creatinine (mg/dl)          0.3-0          0.1 (a)      0.6 (b)
Glucose (mg/dl)             232-369      272.7 (a)    264.3 (a)
Lipase (U/L)                  --          75.1 (a)    507.9 (b)
Phosphorus (mg/dl)          1.7-4          2.2 (a)      3.5 (ab)
Potassium (mmol/L)          3.9-4          1.6 (a)      6.5 (a)
Sodium (mmol/L)             141-149      140.0 (c)    133.6 (ab)
Total Protein (g/dl)        2.1-3          3.5 (b)      2.9 (a)
Triglyceride (mg/dl)          --          87.0 (b)     63.4 (ab)
Uric acid (mg/dl)           2.5-12         3.0 (a)      5.0 (ab)

                                  Group 2

Analyte                    Pre          Post

Hematocrit (%)           48.6 (a)     48.9 (a)
RBC (x [10.sup.6]/pl)     4.2 (a)      3.9 (a)
WBC (x [10.sup.3]/pl)    14.1 (a)     23.1 (b)
  Basophil (%)            0.0 (a)      0.0 (a)
  Eosinophil (%)          0.0 (a)      0.0 (a)
  Heterophil (%)         29.9 (a)     77.9 (c)
  Lymphocyte (%)         69.3 (a)     20.9 (c)
  Monocyte (%)            0.9 (a)      1.3 (a)
Amylase (U/L)           339.7 (a)    545.4 (a)
BUN (mg/dl)               2.0 (a)      6.4 (b)
BUN: creatinine ratio    17.5 (b)     10.5 (a)
Calcium (mg/dl)           6.6 (a)      7.8 (a)
Cholesterol (mg/dl)     241.0 (a)    258.0 (a)
C[O.sub.2] (mmol/L)      19.8 (a)     24.6 (bc)
Creatinine (mg/dl)        0.1 (a)      0.6 (b)
Glucose (mg/dl)         263.9 (a)    267.0 (a)
Lipase (U/L)             73.7 (a)    467.6 (b)
Phosphorus (mg/dl)        2.8 (ab)     4.0 (b)
Potassium (mmol/L)        4.3 (a)      2.9 (a)
Sodium (mmol/L)         137.5 (bc)   131.3 (a)
Total Protein (g/dl)      3.3 (ab)     3 l (ab)
Triglyceride (mg/dl)     64.7 (ab)    53.1 (a)
Uric acid (mg/dl)         2.6 (a)      7.8 (b)
carbon dioxide.
                                  Group 3

Analyte                    Pre           Post

Hematocrit (%)           51.9 (a)      49.8 (a)
RBC (x [10.sup.6]/pl)     4.0 (a)       4.1 (a)
WBC (x [10.sup.3]/pl)    15.1 (a)      23.5 (b)
  Basophil (%)            0.0 (a)       0.0 (a)
  Eosinophil (%)          0.0 (a)       0.0 (a)
  Heterophil (%)         40.0 (ab)     82.3 (c)
  Lymphocyte (%)         59.6 (ab)     16.7 (c)
  Monocyte (%)            0.4 (a)       1.0 (a)
  Amylase (U/L)         351.0 (a)    1115.7 (a)
BUN (mg/dl)               2.1 (a)       8.0 (b)
BUN: creatinine ratio    15.0 (ab)     12.1 (ab)
Calcium (mg/dl)           7.5 (a)       7.8 (a)
Cholesterol (mg/dl)     225.3 (a)     240.4 (a)
C[O.sub.2] (mmol/L)      20.7 (a)      23.7 (bc)
Creatinine (mg/dl)        0.2 (a)       0.7 (b)
Glucose (mg/dl)         254.6 (a)     278.9 (a)
Lipase (U/L)             76.9 (a)     664.1 (b)
Phosphorus (mg/dl)        2.8 (ab)      5.3 (c)
Potassium (mmol/L)        1.8 (a)       3.0 (a)
Sodium (mmol/L)         141.0 (c)     135.9 (abc)
Total Protein (g/dl)      3.1 (ab)      3.1 (ab)
Triglyceride (mg/dl)     68.1 (ab)     79.6 (ab)
Uric acid (mg/dl)         2.4 (a)       8.4 (b)
carbon dioxide.

Abbreviations: CBC indicates complete blood cell; RBC, red blood
cells; WBC, white blood cells; BUN, blood urea nitrogen;
C[O.sub.2], carbon dioxide.

(abc) Mean values in the same row with different superscript
letters are significantly different, P < .05.

Table 4. Mean plasma liver enzyme and bile acid concentrations
before (Pre) and after (Post) administration of [B.sub.1]
aflatoxin (3 mg/kg delivered by crop gavage tube) from control
pigeons (group 1) and pigeons fed milk thistle (group
2, 10 mg/kg body weight per day; group 3, 100 mg/kg body
weight per day).

                                     Group 1
                  Reference
Parameter        ranges (48)      Pre         Post

AST (U/L)          45-123      162.4 (a)    770.0 (bc)
ALT (U/L)          19-48        42.7 (a)    361.7 (c)
Bile acids
([micro]mol/L)     22-60         6.8 (a)    196.4 (b)
CPK (U/L)         110-480      422.6 (a)   1865.1 (c)
GGT (mg/dl)         0-2.9        5.0 (a)      8.2 (a)
LDH (U/L)          30-205      379.4 (a)   4495.9 (b)

                    Group 2                    Group 3

Parameter           Pre          Post          Pre          Post

AST (U/L)        218.0 (ab)   1020.1 (c)    242.1 (ab)   1212.4 (c)
ALT (U/L)         44.6 (a)     178.3 (b)     48.7 (a)     262.6 (bc)
Bile acids
([micro]mol/L)     7.5 (a)     133.4 (b)      6.9 (a)     118.3 (ab)
CPK (U/L)        756.9 (a)     826.9 (ab)   558.7 (a)    1123.7 (bc)
GGT (mg/dl)        6.8 (a)      12.9 (b)      5.0 (a)       7.6 (a)
LDH (U/L)        561.9 (a)    3125.6 (ab)   460.1 (a)    4733.1 (b)

Abbreviations: AST indicates aspartate aminotransferase; ALT,
alanine aminotransferase; CPK, creatine phosphokinase; GGT,
gamma glutamyl transferase; LDH, lactate dehydrogenase.

(abc) Mean values in the same row with different superscript
letters are significantly different, P < .05.
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