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The Individual and Combined Toxicity of Kojic Acid and Aflatoxin in Broiler Chickens1
The Individual and Combined Toxicity of Kojic Acid and Aflatoxin in Broiler Chickens1 L. E. GIROIR,2 W. E. HUFF, L. F. KUBENA,3 R. B. HARVEY, M. H. ELISSALDE, D. A. WITZEL, A. G. YERSIN, and G. W. IVIE USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, College Station, Texas 77845 (Received for publication August 8, 1990)
1991 Poultry Science 70:1351-1356
munosuppression (Thaxton et al., 1974), coagulopathy (Doerr et al., 1976), anemia, Kojic acid is a mycotoxin produced by increased relative weights of several organs, fungi of the genera Aspergillus and Penicillium and changes in clinical chemistry and activities (Cole and Cox, 1981). In previous work, Giroir of several serum enzymes (Huff et al., 1986). et al. (1990) described the toxicity of various Because both mycotoxins are produced by the concentrations of kojic acid in young broiler same fungi, both may simultaneously appear in chickens. Kojic acid reduced growth rates, poultry feed, hi addition, kojic acid has been altered relative weights of several organs, demonstrated to be an aflatoxin synergist in possibly stimulated erythropoiesis, changed the two species of caterpillars (Dowd, 1988a). serum concentrations of several components, Therefore, the purpose of the present study disrupted some serum enzyme activities, and was to characterize the interaction between decreased colonic temperature. Another kojic acid and aflatoxin in young male broiler mycotoxin produced by the genus Aspergillus chickens. is aflatoxin. The toxicity of aflatoxin in young broiler chickens is characterized by imINTRODUCTION
MATERIALS AND METHODS
1
Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843. 'To whom correspondence should be addressed; USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, Route 5, Box 810, College Station, TX 77845.
Male broiler chicks (Peterson x Hubbard) were purchased from a local hatchery and maintained in electrically heated batteries with feed and water available for ad libitum intake. A two by two factorial arrangement of treatments was completely randomized and consisted of four treatments: 0 or 2,500 mg kojic acid/kg feed, 2.5 mg aflatoxin/kg feed, and 2,500 mg kojic acid plus 2.5 mg aflatoxin/ kg feed. The chicks were fed a commercial,
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Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on December 4, 2015
ABSTRACT The individual and combined effects of kojic acid and aflatoxin weie studied in male broiler chicks (Peterson x Hubbard). The experiment had a two by two factorial arrangement of treatments with dietary treatments of 0 and 2,500 mg kojic acid/kg feed and 0 and 2 J mg aflatoxin/kg feed. The broilers were obtained at 1 day of age and housed in electrically heated batteries with feed and water available for ad libitum intake until they reached 3 wk of age. The toxicity of kojic acid was characterized by significant (P<05) reductions in body weight, the relative weight of the bursa of Fabricius, serum cholesterol concentration, and serum alkaline phosphatase activity, and by significant (P<.05) increases in the relative weight of the pancreas, proventriculus, and gizzard, and serum concentrations of uric acid and triglycerides. Aflatoxicosis was characterized by significant (P<.05) reductions in body weight, serum concentrations of total protein, albumin, cholesterol, and inorganic phosphorus, serum glutamic oxalacetic transaminase activity, and mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Significant (P<05) increases in the relative weight of the liver, kidney, spleen, pancreas, proventriculus, and heart, and the serum pyruvic transaminase activity were also caused by aflatoxin alone. The only significant (P<.05) interaction between kojic acid and aflatoxin, which can best be described as antagonistic, was seen through an increase in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. These data indicate that kojic acid is not an aflatoxin synergist at the levels used in the present study. {Key words: kojic acid, aflatoxin, toxicity, body weight, broilers)
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GIROIR ET AL.
Red blood cell count was measured on a Coulter Model ZM counter.6 Hemoglobin was measured as cyanmethemoglobin, using a hemoglobinometer6 according to manufacturer's recommendations, and packed cell volumes were determined in microhematocrit tubes. Serum inorganic phosphorus, creatinine, total protein, albumin, cholesterol, glucose, triglycerides, blood urea nitrogen, and uric acid concentrations, serum gamma glutamyl transferase, cholinesterase, lactic dehydrogenase, glutamic oxalacetic transaminase (SGOT), glutamic pyruvic transaminase
4 Sigma Chemical Company, St. Louis, MO 63178-9916. 5 Sherwood Medical, St Louis, MO 63103. 6 Coulter Electronics, Hialeah, FL 33012. 7 Gilford Impact 400E, Ciba Coming Diagnostics Corporation, Gilford Systems, Oberlin, OH 44774.
(SGPT), creatine kinase, and alkaline phosphatase activities were determined with a clinical chemistry analyzer using manufacturer's recommended procedures. Reagents for all determinations were purchased from Gilford.7 Data (pen x) for all response variables were subjected to ANOVA (Snedecor and Cochran, 1967) as a two by two factorial using the General Linear Model procedure in the SAS® software (SAS Institute, 1982). Variable means for treatments showing significant differences were indicated using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance were based on the probability of .05. RESULTS
The combined aflatoxin and kojic acid treatment produced a lower feed efficiency for the 21-day experimental period but the other two dietary treatments had no significant effect upon feed efficiency (Table 1). Both aflatoxin and kojic acid alone decreased body weight. These reductions in body weight were first observed on Day 14 and continued through Day 21. By Day 21, the combined dietary treatment reduced body weight more than the other two dietary treatments alone. However, this reduction did not represent interaction and was merely additive. The relative weights of all eight organs examined were changed by at least one of the dietary treatments (Table 2). The liver, kidney, spleen, and heart all showed the same response to these dietary treatments. These four organs were insensitive to kojic acid at 2,500 rng/kg feed and were characterized by an increase in their relative weights due to aflatoxin at 2.5 mg/kg feed. These same organs showed an increase in relative weights to the combined treatment; however, significant interaction between the two mycotoxins was not observed. The relative weight of the gizzard was not altered by the aflatoxin dietary treatment but was increased by the kojic acid and combined treatments. The bursa weight was not altered by the aflatoxin and combined dietary treatments but was decreased by the kojic acid dietary treatment. The relative weights of the proventriculus and the pancreas were increased by the kojic acid, by the aflatoxin dietary treatments, and by the combined dietary treatment; the pancreas increased more than did the proventriculus.
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unmedicated, corn and soybean meal basal diet that contained or exceeded the levels of nutrients recommended by the National Research Council (1984). There were six replicates of 10 broilers per treatment. Broilers were maintained on dietary treatments from hatching to 3 wk of age. Feed consumption for each treatment was recorded during the study. Kojic acid4 was incorporated into the diets by mixing the required amount of mycotoxin with feed to yield 1 kg of premix. This premix was then added and mixed into the appropriate diet Aflatoxin was produced and incorporated into the diets by methods previously reported (Huff and Doerr, 1981). Broilers were weighed weekly and deaths were recorded as they occurred. When broilers reached 3 wk of age, they were weighed, bled via cardiac puncture, and killed by cervical dislocation. The bursa of Fabricius, gizzard, heart, kidney, liver, pancreas, proventriculus, and spleen were excised and weighed. Organ weights were expressed as relative weights (grams of organ per 100 g of body weight). Blood for hematological evaluations was collected by methods described by Doerr et al. (1975). The hematological components measured were red blood cell count, packed cell volume, percentage of hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Serum was collected using corvac serum separator tubes.5
TOXICITY OF COMBINED KOJIC ACID AND AFLATOXIN
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TABLE 1. Individual and combined effects of kojic acid and aflatoxin on body weight and feed efficiency1 Body weight
Kojic acid 0 2,500 0 2,500
Aflatoxin
Day 14
(mg/kg) 0 0 2.5 2.5
344 318 305 290
Feed efficiency2
Day 21 (g) 637 596 559 513
±4' ± 4b ±5bc ±5°
± ± ± ±
— (g:g) — 1.71 ± .01 b 1.77 ± .041* 1.74 ± .02»b 1.80 ± .02'
8' 8b 11° lld
a-d
The serum concentrations of total protein decreased by all three dietary treatments, but and albumin were not altered by the kojic acid no significant interaction between kojic acid dietary treatment but were lowered by the and aflatoxin was observed. No differences aflatoxin and combined dietary treatments were observed in the serum concentrations of (Table 3). The serum protein concentration calcium, creatinine, or glucose (data not was significantly decreased by the combined presented). dietary treatment, but there was no evidence of The activities of SGOT, SGPT, alkaline toxic synergy. Serum concentrations of both phosphatase, and cholinesterase were altered uric acid and triglycerides were not signifi- by the presence of kojic acid, aflatoxin, or both cantly altered by the aflatoxin or combined mycotoxins in the diet (Table 4). The activities dietary treatments, yet they were elevated by of SGOT and SGPT were altered only by the kojic acid dietary treatment. The serum aflatoxin alone or the combined dietary treatinorganic phosphorus concentration was not ment, SGPT activity was increased by the significantly altered by the kojic acid and aflatoxin and combined dietary treatments, and combined dietary treatments but was lowered SGOT activity was decreased by these same by the aflatoxin dietary treatment. The serum two treatments. The activity of alkaline phosconcentration of blood urea nitrogen was phatase was also lowered by the kojic acid and increased only by the combined dietary treat- combined dietary treatments but without sigment. The serum cholesterol concentration was nificant interaction. Cholinesterase activity was
TABLE 2. Individual and combined effects of kojic acid and aflatoxin on the relative weights of the liver, kidney, spleen, heart, gizzard, bursa, proventriculus, aid pancreas'Kojic acid
Aflatoxin
Liver
fr/inn
± ± ± ±
.06b .07b .12' .12'
0 2,500 0 2,500
0 0 2.5 2.5
3.00 3.19 3.95 4.06
0 2,500 0 2,500
0 0 2.5 2.5
Gizzard 2.60 ± .04b 2.89 ± .06* 2.71 ± .04 b 2.92 ± .04'
c
Spleen
Kidney
— (mg/kg) SI ± .01 b S3 ± .01 b .75 ± .03' .77 ± .04' Bursa of Fabricius 29 ± .01' 26 ± .01 b .28 ± .01 ab .28 ± .Ol1*
Heart
.09 .09 .13 .14
± ± ± ±
.00 b .00 b .01' .01'
Proventriculus .64 ± .01 b .68 ± .01' .69 ± .02' .71 ± .02'
Means within columns with no common superscripts differ significantly (P<05). 1= x ± SEM of six groups of 10 broilers minus mortality.
.71 .70 .79 .81
± ± ± ±
.02 b .02 b .02' .02*
Pancreas .36 ± .01 c .41 ± .01 b .42 ± .01 b .47 ± .01'
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Means within columns with no common superscripts differ significantly (P<05). x ± SEM of six groups of 10 broilers minus mortality. 2 Ratio is grams of feed consumed per gram of weight gain and is representative of a 21-day period.
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GIROIR ET AL.
TABLE 3. Individual and combined effects of kojic acid and aflatoxin on serum concentrations of total protein, albumin, uric acid, triglycerides, cholesterol, inorganic phosphorus, and blood urea nitrogen^ Kojic acid
Aflatoxin
Total protein
Albumin
2.50 254 1.90 1.75
1.14 1.13 .77 .72
Uric acid •
0 0 2.5 2.5
0 2,500 0 2,500
± .03* ± .03* ± .08 b ± .08°
Triglycerides
± .02* ± .01* ± .05b ± .04 b
Cholesterol
66.9 89.4 68.0 66.9
± ± ± ±
3.7b 4.6* 4.3 b 3.7b
137.2 127.7 99.0 87.8
± ± ± ±
2.4* 2.0b 5.9° 4.7d
_T
Blood urea nitrogen
\
5.60 5.88 4.82 5.44
± ± ± ±
.14* .14* .17b .14*
1.08 1.38 1.39 1.66
± ± ± ±
.06b .O^ .09^ .14*
*~dMeans -within columns with no common superscripts differ significantly (P<.05). *x ± SEM of six groups of eight randomly selected broilers.
decreased by only the combined dietary treatment. No differences were observed in the serum activities of creatine kinase, gamma glutamyl transferase, or lactate dehydrogenase (data not presented). The dietary treatments also affected three hematological components (Table 5). Like many other components, MCV was insensitive to kojic acid alone but was decreased by the aflatoxin and the combined dietary treatments. There was no evidence of a significant interaction between aflatoxin and kojic acid. The MCH and the MCHC were not significantly changed by kojic acid alone but were lowered by aflatoxin alone. The hemoglobin concentration of red blood cells was not altered from that of the controls by the combined dietary treatment. This combination
of the two mycotoxins was characterized by a significant interaction in which kojic acid appeared to reduce the deleterious effects of aflatoxin upon the hemoglobin concentration of red blood cells. Although they were measured, no differences were noted for red blood cell count, packed cell volume, or percentage of hemoglobin (data not presented). DISCUSSION
Commonly, poultry feed is infested with molds from the genera of Aspergillus and Penicillium, thus exposing broilers to the mycotoxins, kojic acid, and aflatoxin. Although the individual toxic effects of kojic acid (Giroir et al, 1990) and aflatoxin (Huff et al., 1986) in broilers have been studied, the
TABLE 4. Individual and combined effects of kojic acid and aflatoxin on the serum enzyme activities of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase, and cholinesteraser
Kojic acid 0 2,500 0 2,500 a-c
Aflatoxin (mg/kg) 0 0 2.5 2.5
Glutamic oxalacetic transaminase
Glutamic pyruvic transaminase
138 147 119 116
19.5 22.2 24.5 23.5
± ± ± ±
4* 9a 4b 6b
± 5b ± .8*b ± 1.1* ± .9*
Alkaline phosphatase -(IU/L) 6,333 4,436 4,967 3,391
± ± ± ±
535* 355*° 569 ab 390°
Means within columns with no common superscripts differ significantly (P<05). x ± SEM of six groups of eight randomly selected broilers.
Cholinesterase 1,656 1,609 1,411 1,290
± ± ± ±
81* 75* 79*b 65 b
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(mr n (-in 0 0 2.5 2.5
0 2,500 0 2,500
(mg/100 mL) 5.76 ± .24 b 7.39 ± .31* 5.82 ± .34b 6.33 ± .31 b Inorganic phosphate
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TOXICITY OF COMBINED KOJIC ACID AND AFLATOXTN
TABLE 5. Individual and combined effects of kojic acid and aflatoxin on mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration Kojic acid
Aflatoxin
MCVZ
MOF
MCHC4
38.0 ± .3a 37.2 ± .3a 35.1 ± .8b 36.6 ± .5ab*
30.2 ± .2a 29.6 ± .2a 28.3 ± .3b 29.8 ± .3a*
(mg/kg)
0 2,500
0 2,500
0 0 2.5 2.5
146 ± 145 ± 139 ± 139 ±
la 1" 2b lb
combined toxic effects of these two mycotoxins have not. A toxic synergism as described in caterpillars (Dowd, 1988a) did not occur in broilers. The only significant interaction between kojic acid and aflatoxin found in the present study was associated with the mean MCH and the MCHC and could best be described as antagonistic. This interaction appears to result in kojic acid's arrest of the reduction in size of red blood cells and a corresponding loss of hemoglobin caused by aflatoxin. The physiological or toxicological significance of this finding is unknown. The proposed mechanism of kojic acid's synergy with aflatoxin in caterpillars is based upon kojic acid's inhibition of oxidative enzymes (Dowd, 1988b). Previous work, demonstrating that kojic acid does inhibit some oxidative enzymes of the liver and kidney (Klein and Olsen, 1947), supports this proposed mechanism. Possibly kojic acid is inhibiting the mixed function oxidases, thereby preventing the oxidation of aflatoxin into less toxic metabolites. Kojic acid appears to be more toxic in caterpillars than in poultry. In the previous work in caterpillars (Dowd, 1988a), kojic acid was fed at 25 mg/kg diet and aflatoxin at .25 mg/kg diet, and toxic synergy was observed. In the present study kojic acid was fed at 2,500 mg/kg feed and aflatoxin at 2.5 mg/kg feed, and no toxic synergy was observed. The ratio of kojic acid to aflatoxin in the caterpillar study was 100:1 and in the poultry study was 1000:1. These differences in doses and results suggest that poultry may detoxify kojic acid more efficiently than caterpillars or that large differ-
ences exist in inherent toxicity. Kojic acid may possibly be detoxified in poultry through oxidation by alcohol dehydrogenase, and then aldehyde dehydrogenase (Hodgson and Levi, 1987); or under physiological conditions, kojic acid is spontaneously oxidized into its carboxylic acid derivative, comenic acid (2-carboxy5-hydroxy-^pyrone) (Ciegler et ah, 1971). The comenic acid would men be conjugated and excreted via the urine. The present data indicate that kojic acid does not interact in a synergistic manner with aflatoxin in broilers at the concentrations used. The occurrence of kojic acid at various concentrations in the presence of different concentrations of aflatoxin, other mycotoxins, and other contaminants prevents the dismissal of the possible interactions of kojic acid with other toxicants. REFERENCES Ciegler, A., S. Kadis, and S. J. Ajl, 1971. Page 236 in: Microbial Toxins. Vol. VI. Academic Press, New York, NY. Cole, R. J., and R. H. Cox, 1981. Page 759 in: Handbook of Toxic Fungal Metabolites. Academic Press, New York, NY. Doerr, J. A., R. D. Wyatt, and P. B. Hamilton, 1975. Investigation and standardization of prothrombin times in chickens. Poultry Sci. 54:969-980. Doerr, J. A., R. D. Wyatt, and P. B. Hamilton, 1976. Impairment of coagulation function during aflatoxicosis in young chickens. Toxicol. AppL Pharmacol. 35:437-446. Dowd, P. F., 1988a. Synergism of aflatoxin S1 toxicity with the co-occurring fungal metabolite kojic acid to two caterpillars. EntomoL Exp. AppL 47:69-71. Dowd, P. F., 1988b. Toxicological and biochemical interactions of the fungal metabolites fusaric acid and kojic acid with xenobiotics in Heliothis zea (F.)
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"•''Means within columns with no common superscripts differ significantly (P<05). J x ± SEM of six groups of five randomly selected broilers. 2 MCV = mean corpuscular volume (tun3). 3 MCH = hemoglobin (g/100 mL) X 10 + red blood cells (x 106/mm3). 4 MCHC = hemoglobin (g/100 mL) x 100 + packed cell volume (%). •Significant (P<.05) interaction between kojic acid and aflatoxin.
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GIROIR ET AL. aflatoxicosis in broiler chickens. Poultry Sci. 65: 1891-1899. Klein, J. R., and N. S. Olsen, 1947. Inhibitory effect of kojic acid upon oxidations mediated by liver and kidney. J. Biol. Chem. 170:151-157. National Research Council, 1984. Pages 11-15 in: Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Snedecor, G. W., and W. E. Cochran, 1967. Pages 258-380 in: Statistical Methods. 6th ed. The Iowa State University Press, Ames, IA. Thaxton, J. P., H. T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chickens by anatoxins. Poultry Sci. 53:721-725.
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and Spodoptera frugiperda (J. E. Smith). Pestic. Biochem. Physiol. 32:123-134. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. Giroir, L. E., W. E. Huff, L. F. Kubena, R. B. Harvey, M. H. Elissalde, D. A. Witzel, A. G. Yersin, and G. W. Ivie, 1991. Toxic effects of kojic acid in the diet of male broilers. Poultry Sci. 70:499-503. Hodgson, E., and P. E. Levi, 1987. Pages 65-67 in: A Textbook of Modem Toxicology. Elsevier Science Publishing, New York, NY. Huff, W. E., and J. A. Doerr, 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 60:550-555. Huff, W. E., L. F. Kubena, R. B. Harvey, D. E. Corner, and H. H. Mollenhauer, 1986. Progression of
1991 Poultry Science 70:1351-1356
munosuppression (Thaxton et al., 1974), coagulopathy (Doerr et al., 1976), anemia, Kojic acid is a mycotoxin produced by increased relative weights of several organs, fungi of the genera Aspergillus and Penicillium and changes in clinical chemistry and activities (Cole and Cox, 1981). In previous work, Giroir of several serum enzymes (Huff et al., 1986). et al. (1990) described the toxicity of various Because both mycotoxins are produced by the concentrations of kojic acid in young broiler same fungi, both may simultaneously appear in chickens. Kojic acid reduced growth rates, poultry feed, hi addition, kojic acid has been altered relative weights of several organs, demonstrated to be an aflatoxin synergist in possibly stimulated erythropoiesis, changed the two species of caterpillars (Dowd, 1988a). serum concentrations of several components, Therefore, the purpose of the present study disrupted some serum enzyme activities, and was to characterize the interaction between decreased colonic temperature. Another kojic acid and aflatoxin in young male broiler mycotoxin produced by the genus Aspergillus chickens. is aflatoxin. The toxicity of aflatoxin in young broiler chickens is characterized by imINTRODUCTION
MATERIALS AND METHODS
1
Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843. 'To whom correspondence should be addressed; USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, Route 5, Box 810, College Station, TX 77845.
Male broiler chicks (Peterson x Hubbard) were purchased from a local hatchery and maintained in electrically heated batteries with feed and water available for ad libitum intake. A two by two factorial arrangement of treatments was completely randomized and consisted of four treatments: 0 or 2,500 mg kojic acid/kg feed, 2.5 mg aflatoxin/kg feed, and 2,500 mg kojic acid plus 2.5 mg aflatoxin/ kg feed. The chicks were fed a commercial,
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Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on December 4, 2015
ABSTRACT The individual and combined effects of kojic acid and aflatoxin weie studied in male broiler chicks (Peterson x Hubbard). The experiment had a two by two factorial arrangement of treatments with dietary treatments of 0 and 2,500 mg kojic acid/kg feed and 0 and 2 J mg aflatoxin/kg feed. The broilers were obtained at 1 day of age and housed in electrically heated batteries with feed and water available for ad libitum intake until they reached 3 wk of age. The toxicity of kojic acid was characterized by significant (P<05) reductions in body weight, the relative weight of the bursa of Fabricius, serum cholesterol concentration, and serum alkaline phosphatase activity, and by significant (P<.05) increases in the relative weight of the pancreas, proventriculus, and gizzard, and serum concentrations of uric acid and triglycerides. Aflatoxicosis was characterized by significant (P<.05) reductions in body weight, serum concentrations of total protein, albumin, cholesterol, and inorganic phosphorus, serum glutamic oxalacetic transaminase activity, and mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Significant (P<05) increases in the relative weight of the liver, kidney, spleen, pancreas, proventriculus, and heart, and the serum pyruvic transaminase activity were also caused by aflatoxin alone. The only significant (P<.05) interaction between kojic acid and aflatoxin, which can best be described as antagonistic, was seen through an increase in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. These data indicate that kojic acid is not an aflatoxin synergist at the levels used in the present study. {Key words: kojic acid, aflatoxin, toxicity, body weight, broilers)
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GIROIR ET AL.
Red blood cell count was measured on a Coulter Model ZM counter.6 Hemoglobin was measured as cyanmethemoglobin, using a hemoglobinometer6 according to manufacturer's recommendations, and packed cell volumes were determined in microhematocrit tubes. Serum inorganic phosphorus, creatinine, total protein, albumin, cholesterol, glucose, triglycerides, blood urea nitrogen, and uric acid concentrations, serum gamma glutamyl transferase, cholinesterase, lactic dehydrogenase, glutamic oxalacetic transaminase (SGOT), glutamic pyruvic transaminase
4 Sigma Chemical Company, St. Louis, MO 63178-9916. 5 Sherwood Medical, St Louis, MO 63103. 6 Coulter Electronics, Hialeah, FL 33012. 7 Gilford Impact 400E, Ciba Coming Diagnostics Corporation, Gilford Systems, Oberlin, OH 44774.
(SGPT), creatine kinase, and alkaline phosphatase activities were determined with a clinical chemistry analyzer using manufacturer's recommended procedures. Reagents for all determinations were purchased from Gilford.7 Data (pen x) for all response variables were subjected to ANOVA (Snedecor and Cochran, 1967) as a two by two factorial using the General Linear Model procedure in the SAS® software (SAS Institute, 1982). Variable means for treatments showing significant differences were indicated using Duncan's new multiple range procedure (Duncan, 1955). All statements of significance were based on the probability of .05. RESULTS
The combined aflatoxin and kojic acid treatment produced a lower feed efficiency for the 21-day experimental period but the other two dietary treatments had no significant effect upon feed efficiency (Table 1). Both aflatoxin and kojic acid alone decreased body weight. These reductions in body weight were first observed on Day 14 and continued through Day 21. By Day 21, the combined dietary treatment reduced body weight more than the other two dietary treatments alone. However, this reduction did not represent interaction and was merely additive. The relative weights of all eight organs examined were changed by at least one of the dietary treatments (Table 2). The liver, kidney, spleen, and heart all showed the same response to these dietary treatments. These four organs were insensitive to kojic acid at 2,500 rng/kg feed and were characterized by an increase in their relative weights due to aflatoxin at 2.5 mg/kg feed. These same organs showed an increase in relative weights to the combined treatment; however, significant interaction between the two mycotoxins was not observed. The relative weight of the gizzard was not altered by the aflatoxin dietary treatment but was increased by the kojic acid and combined treatments. The bursa weight was not altered by the aflatoxin and combined dietary treatments but was decreased by the kojic acid dietary treatment. The relative weights of the proventriculus and the pancreas were increased by the kojic acid, by the aflatoxin dietary treatments, and by the combined dietary treatment; the pancreas increased more than did the proventriculus.
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on December 4, 2015
unmedicated, corn and soybean meal basal diet that contained or exceeded the levels of nutrients recommended by the National Research Council (1984). There were six replicates of 10 broilers per treatment. Broilers were maintained on dietary treatments from hatching to 3 wk of age. Feed consumption for each treatment was recorded during the study. Kojic acid4 was incorporated into the diets by mixing the required amount of mycotoxin with feed to yield 1 kg of premix. This premix was then added and mixed into the appropriate diet Aflatoxin was produced and incorporated into the diets by methods previously reported (Huff and Doerr, 1981). Broilers were weighed weekly and deaths were recorded as they occurred. When broilers reached 3 wk of age, they were weighed, bled via cardiac puncture, and killed by cervical dislocation. The bursa of Fabricius, gizzard, heart, kidney, liver, pancreas, proventriculus, and spleen were excised and weighed. Organ weights were expressed as relative weights (grams of organ per 100 g of body weight). Blood for hematological evaluations was collected by methods described by Doerr et al. (1975). The hematological components measured were red blood cell count, packed cell volume, percentage of hemoglobin, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). Serum was collected using corvac serum separator tubes.5
TOXICITY OF COMBINED KOJIC ACID AND AFLATOXIN
1353
TABLE 1. Individual and combined effects of kojic acid and aflatoxin on body weight and feed efficiency1 Body weight
Kojic acid 0 2,500 0 2,500
Aflatoxin
Day 14
(mg/kg) 0 0 2.5 2.5
344 318 305 290
Feed efficiency2
Day 21 (g) 637 596 559 513
±4' ± 4b ±5bc ±5°
± ± ± ±
— (g:g) — 1.71 ± .01 b 1.77 ± .041* 1.74 ± .02»b 1.80 ± .02'
8' 8b 11° lld
a-d
The serum concentrations of total protein decreased by all three dietary treatments, but and albumin were not altered by the kojic acid no significant interaction between kojic acid dietary treatment but were lowered by the and aflatoxin was observed. No differences aflatoxin and combined dietary treatments were observed in the serum concentrations of (Table 3). The serum protein concentration calcium, creatinine, or glucose (data not was significantly decreased by the combined presented). dietary treatment, but there was no evidence of The activities of SGOT, SGPT, alkaline toxic synergy. Serum concentrations of both phosphatase, and cholinesterase were altered uric acid and triglycerides were not signifi- by the presence of kojic acid, aflatoxin, or both cantly altered by the aflatoxin or combined mycotoxins in the diet (Table 4). The activities dietary treatments, yet they were elevated by of SGOT and SGPT were altered only by the kojic acid dietary treatment. The serum aflatoxin alone or the combined dietary treatinorganic phosphorus concentration was not ment, SGPT activity was increased by the significantly altered by the kojic acid and aflatoxin and combined dietary treatments, and combined dietary treatments but was lowered SGOT activity was decreased by these same by the aflatoxin dietary treatment. The serum two treatments. The activity of alkaline phosconcentration of blood urea nitrogen was phatase was also lowered by the kojic acid and increased only by the combined dietary treat- combined dietary treatments but without sigment. The serum cholesterol concentration was nificant interaction. Cholinesterase activity was
TABLE 2. Individual and combined effects of kojic acid and aflatoxin on the relative weights of the liver, kidney, spleen, heart, gizzard, bursa, proventriculus, aid pancreas'Kojic acid
Aflatoxin
Liver
fr/inn
± ± ± ±
.06b .07b .12' .12'
0 2,500 0 2,500
0 0 2.5 2.5
3.00 3.19 3.95 4.06
0 2,500 0 2,500
0 0 2.5 2.5
Gizzard 2.60 ± .04b 2.89 ± .06* 2.71 ± .04 b 2.92 ± .04'
c
Spleen
Kidney
— (mg/kg) SI ± .01 b S3 ± .01 b .75 ± .03' .77 ± .04' Bursa of Fabricius 29 ± .01' 26 ± .01 b .28 ± .01 ab .28 ± .Ol1*
Heart
.09 .09 .13 .14
± ± ± ±
.00 b .00 b .01' .01'
Proventriculus .64 ± .01 b .68 ± .01' .69 ± .02' .71 ± .02'
Means within columns with no common superscripts differ significantly (P<05). 1= x ± SEM of six groups of 10 broilers minus mortality.
.71 .70 .79 .81
± ± ± ±
.02 b .02 b .02' .02*
Pancreas .36 ± .01 c .41 ± .01 b .42 ± .01 b .47 ± .01'
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Means within columns with no common superscripts differ significantly (P<05). x ± SEM of six groups of 10 broilers minus mortality. 2 Ratio is grams of feed consumed per gram of weight gain and is representative of a 21-day period.
1354
GIROIR ET AL.
TABLE 3. Individual and combined effects of kojic acid and aflatoxin on serum concentrations of total protein, albumin, uric acid, triglycerides, cholesterol, inorganic phosphorus, and blood urea nitrogen^ Kojic acid
Aflatoxin
Total protein
Albumin
2.50 254 1.90 1.75
1.14 1.13 .77 .72
Uric acid •
0 0 2.5 2.5
0 2,500 0 2,500
± .03* ± .03* ± .08 b ± .08°
Triglycerides
± .02* ± .01* ± .05b ± .04 b
Cholesterol
66.9 89.4 68.0 66.9
± ± ± ±
3.7b 4.6* 4.3 b 3.7b
137.2 127.7 99.0 87.8
± ± ± ±
2.4* 2.0b 5.9° 4.7d
_T
Blood urea nitrogen
\
5.60 5.88 4.82 5.44
± ± ± ±
.14* .14* .17b .14*
1.08 1.38 1.39 1.66
± ± ± ±
.06b .O^ .09^ .14*
*~dMeans -within columns with no common superscripts differ significantly (P<.05). *x ± SEM of six groups of eight randomly selected broilers.
decreased by only the combined dietary treatment. No differences were observed in the serum activities of creatine kinase, gamma glutamyl transferase, or lactate dehydrogenase (data not presented). The dietary treatments also affected three hematological components (Table 5). Like many other components, MCV was insensitive to kojic acid alone but was decreased by the aflatoxin and the combined dietary treatments. There was no evidence of a significant interaction between aflatoxin and kojic acid. The MCH and the MCHC were not significantly changed by kojic acid alone but were lowered by aflatoxin alone. The hemoglobin concentration of red blood cells was not altered from that of the controls by the combined dietary treatment. This combination
of the two mycotoxins was characterized by a significant interaction in which kojic acid appeared to reduce the deleterious effects of aflatoxin upon the hemoglobin concentration of red blood cells. Although they were measured, no differences were noted for red blood cell count, packed cell volume, or percentage of hemoglobin (data not presented). DISCUSSION
Commonly, poultry feed is infested with molds from the genera of Aspergillus and Penicillium, thus exposing broilers to the mycotoxins, kojic acid, and aflatoxin. Although the individual toxic effects of kojic acid (Giroir et al, 1990) and aflatoxin (Huff et al., 1986) in broilers have been studied, the
TABLE 4. Individual and combined effects of kojic acid and aflatoxin on the serum enzyme activities of glutamic oxalacetic transaminase, glutamic pyruvic transaminase, alkaline phosphatase, and cholinesteraser
Kojic acid 0 2,500 0 2,500 a-c
Aflatoxin (mg/kg) 0 0 2.5 2.5
Glutamic oxalacetic transaminase
Glutamic pyruvic transaminase
138 147 119 116
19.5 22.2 24.5 23.5
± ± ± ±
4* 9a 4b 6b
± 5b ± .8*b ± 1.1* ± .9*
Alkaline phosphatase -(IU/L) 6,333 4,436 4,967 3,391
± ± ± ±
535* 355*° 569 ab 390°
Means within columns with no common superscripts differ significantly (P<05). x ± SEM of six groups of eight randomly selected broilers.
Cholinesterase 1,656 1,609 1,411 1,290
± ± ± ±
81* 75* 79*b 65 b
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(mr n (-in 0 0 2.5 2.5
0 2,500 0 2,500
(mg/100 mL) 5.76 ± .24 b 7.39 ± .31* 5.82 ± .34b 6.33 ± .31 b Inorganic phosphate
1355
TOXICITY OF COMBINED KOJIC ACID AND AFLATOXTN
TABLE 5. Individual and combined effects of kojic acid and aflatoxin on mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration Kojic acid
Aflatoxin
MCVZ
MOF
MCHC4
38.0 ± .3a 37.2 ± .3a 35.1 ± .8b 36.6 ± .5ab*
30.2 ± .2a 29.6 ± .2a 28.3 ± .3b 29.8 ± .3a*
(mg/kg)
0 2,500
0 2,500
0 0 2.5 2.5
146 ± 145 ± 139 ± 139 ±
la 1" 2b lb
combined toxic effects of these two mycotoxins have not. A toxic synergism as described in caterpillars (Dowd, 1988a) did not occur in broilers. The only significant interaction between kojic acid and aflatoxin found in the present study was associated with the mean MCH and the MCHC and could best be described as antagonistic. This interaction appears to result in kojic acid's arrest of the reduction in size of red blood cells and a corresponding loss of hemoglobin caused by aflatoxin. The physiological or toxicological significance of this finding is unknown. The proposed mechanism of kojic acid's synergy with aflatoxin in caterpillars is based upon kojic acid's inhibition of oxidative enzymes (Dowd, 1988b). Previous work, demonstrating that kojic acid does inhibit some oxidative enzymes of the liver and kidney (Klein and Olsen, 1947), supports this proposed mechanism. Possibly kojic acid is inhibiting the mixed function oxidases, thereby preventing the oxidation of aflatoxin into less toxic metabolites. Kojic acid appears to be more toxic in caterpillars than in poultry. In the previous work in caterpillars (Dowd, 1988a), kojic acid was fed at 25 mg/kg diet and aflatoxin at .25 mg/kg diet, and toxic synergy was observed. In the present study kojic acid was fed at 2,500 mg/kg feed and aflatoxin at 2.5 mg/kg feed, and no toxic synergy was observed. The ratio of kojic acid to aflatoxin in the caterpillar study was 100:1 and in the poultry study was 1000:1. These differences in doses and results suggest that poultry may detoxify kojic acid more efficiently than caterpillars or that large differ-
ences exist in inherent toxicity. Kojic acid may possibly be detoxified in poultry through oxidation by alcohol dehydrogenase, and then aldehyde dehydrogenase (Hodgson and Levi, 1987); or under physiological conditions, kojic acid is spontaneously oxidized into its carboxylic acid derivative, comenic acid (2-carboxy5-hydroxy-^pyrone) (Ciegler et ah, 1971). The comenic acid would men be conjugated and excreted via the urine. The present data indicate that kojic acid does not interact in a synergistic manner with aflatoxin in broilers at the concentrations used. The occurrence of kojic acid at various concentrations in the presence of different concentrations of aflatoxin, other mycotoxins, and other contaminants prevents the dismissal of the possible interactions of kojic acid with other toxicants. REFERENCES Ciegler, A., S. Kadis, and S. J. Ajl, 1971. Page 236 in: Microbial Toxins. Vol. VI. Academic Press, New York, NY. Cole, R. J., and R. H. Cox, 1981. Page 759 in: Handbook of Toxic Fungal Metabolites. Academic Press, New York, NY. Doerr, J. A., R. D. Wyatt, and P. B. Hamilton, 1975. Investigation and standardization of prothrombin times in chickens. Poultry Sci. 54:969-980. Doerr, J. A., R. D. Wyatt, and P. B. Hamilton, 1976. Impairment of coagulation function during aflatoxicosis in young chickens. Toxicol. AppL Pharmacol. 35:437-446. Dowd, P. F., 1988a. Synergism of aflatoxin S1 toxicity with the co-occurring fungal metabolite kojic acid to two caterpillars. EntomoL Exp. AppL 47:69-71. Dowd, P. F., 1988b. Toxicological and biochemical interactions of the fungal metabolites fusaric acid and kojic acid with xenobiotics in Heliothis zea (F.)
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"•''Means within columns with no common superscripts differ significantly (P<05). J x ± SEM of six groups of five randomly selected broilers. 2 MCV = mean corpuscular volume (tun3). 3 MCH = hemoglobin (g/100 mL) X 10 + red blood cells (x 106/mm3). 4 MCHC = hemoglobin (g/100 mL) x 100 + packed cell volume (%). •Significant (P<.05) interaction between kojic acid and aflatoxin.
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GIROIR ET AL. aflatoxicosis in broiler chickens. Poultry Sci. 65: 1891-1899. Klein, J. R., and N. S. Olsen, 1947. Inhibitory effect of kojic acid upon oxidations mediated by liver and kidney. J. Biol. Chem. 170:151-157. National Research Council, 1984. Pages 11-15 in: Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. SAS Institute, 1982. SAS® User's Guide: Statistics. SAS Institute Inc., Cary, NC. Snedecor, G. W., and W. E. Cochran, 1967. Pages 258-380 in: Statistical Methods. 6th ed. The Iowa State University Press, Ames, IA. Thaxton, J. P., H. T. Tung, and P. B. Hamilton, 1974. Immunosuppression in chickens by anatoxins. Poultry Sci. 53:721-725.
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and Spodoptera frugiperda (J. E. Smith). Pestic. Biochem. Physiol. 32:123-134. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. Giroir, L. E., W. E. Huff, L. F. Kubena, R. B. Harvey, M. H. Elissalde, D. A. Witzel, A. G. Yersin, and G. W. Ivie, 1991. Toxic effects of kojic acid in the diet of male broilers. Poultry Sci. 70:499-503. Hodgson, E., and P. E. Levi, 1987. Pages 65-67 in: A Textbook of Modem Toxicology. Elsevier Science Publishing, New York, NY. Huff, W. E., and J. A. Doerr, 1981. Synergism between aflatoxin and ochratoxin A in broiler chickens. Poultry Sci. 60:550-555. Huff, W. E., L. F. Kubena, R. B. Harvey, D. E. Corner, and H. H. Mollenhauer, 1986. Progression of