ENVIRONMENT AND HEALTH. The Toxicity of Fumonisin B 1,B 2, and B 3, Individually and in Combination, in Chicken Embryos 1

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1 ENVIRONMENT AND HEALTH The Toxicity of Fumonisin B 1,B 2, and B 3, Individually and in Combination, in Chicken Embryos 1 M. H. Henry* and R. D. Wyatt*,2 *Department of Poultry Science, The University of Georgia, Athens, Georgia ABSTRACT Three recently described and toxicologically 52.5, 77.5, and 100%, respectively. The 50% lethal dose important mycotoxins, fumonisin B 1 (FB 1 ), fumoni- sin B 2 (FB 2 ), and fumonisin B 3 (FB 3 ), produced by Fusarium moniliforme in various grains, have been associated with a number of diseases in both humans and animals. The toxicity of purified FB 1,FB 2, and FB 3, individually and in combination (3:1:1 ratio), were evaluated with regard to their embryo toxicity by injection of the toxins into the air cell of chicken eggs at 72 h of incubation. Under these conditions, FB 1 at doses of 0, 2, 4, 8, 16, 32, and 64 µg per egg resulted in embryonic mortality of 5, 12.5, 17.5, 20.0, for FB 1, when injected into the air cell of embryonating chicken eggs, was determined to be µg per egg. A comparison of the toxicity of FB 1,FB 2, and FB 3, individually and in combination (3:1:1 ratio), at doses of 16 µg of total fumonisin per egg, indicated that the toxicity of the fumonisins differed, FB 1 being the most toxic. Microscopic examination of chicken embryos exposed to fumonisin did not reveal any gross developmental abnormalities; however, severe hemorrhages of the head, neck, and thoracic area of the dead embryos were evident. (Key words: fumonisins, embryo, mycotoxin, toxicity) 2001 Poultry Science 80: INTRODUCTION Fumonisin B 1 (FB 1 ), fumonisin B 2 (FB 2 ), and fumonisin B 3 (FB 3 ) are mycotoxins produced by Fusarium moniliforme, Fusarium proliferatum, and Fusarium nygamai (Voss et al., 1990; Thiel et al., 1992). Several strains of the fumonisin-producing Fusarium species have been isolated from animal feeds and feedstuffs (Thiel et al., 1992). Fusarium moniliforme has been associated with leukoencephalomalacia (LEM) in equine species (Wilson, 1971; Haliburton et al., 1979) and white-tail deer (Howerth et al., 1989), and pulmonary edema in pigs (Kriek et al., 1981). Corn contaminated with F. moniliforme and associated with equine LEM resulted in hepatotoxicity and renal toxicity when fed to male rats (Kriek et al., 1981; Wilson et al., 1985; Voss et al., 1989). Broiler chicks fed Fusarium-contaminated culture material have been shown to exhibit increased liver, kidney, and proventriculus weights, and increased mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration (Weibking et al., 1993). Leg weakness in chickens has also been associated with the feeding of F. moniliforme-contaminated diets (Marijanovic et al., 1991). In certain geographic regions Received for publication February 4, Accpeted for publication June 30, Supported in part by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia. 2 To whom correspondence should be addressed: rwyatt@ arches.uga.edu. of China and South Africa, where there are high incidences of esophageal cancer, F. moniliforme has been implicated as the causative agent due to the high incidence of this mold in home-grown corn (Yang, 1980; Marasas et al., 1981). Furthermore, FB 1 has been associated directly with a high incidence of esophageal cancer in South Africa (Thiel et al., 1992). Fumonisin B 1, isolated from culture material contaminated with F. moniliforme, was found to be hepatocarcinogenic and hepatotoxic to rats (Voss et al., 1990; Gelderblom et al., 1991) and horses (Marasas et al., 1988). Fumonisin B 1 was identified as the noninfectious causative agent of LEM in horses (Marasas et al., 1988) and pulmonary edema in pigs (Harrison et al., 1990; Haschek et al., 1992). In vitro studies by Norred et al. (1992) indicated that FB 1 inhibits the biosynthesis of sphingolipids in hepatocytes and kills renal cells. Fumonisin B 1 is also toxic to chicken macrophages by causing a decrease in their functional activity (Qureshi and Hagler, 1992). Studies aimed at investigating the responses of chickens to the fumonisins have utilized fumonisin-contaminated material as the source of fumonisin. Studies using F. moniliforme-contaminated culture material with known concentrations of fumonisin B 1 suggest that the fumonisins are toxic to chickens and turkeys at levels above 100 mg/kg of feed (Ledoux et al., 1992; Weibking et al., 1993). Abbreviation Key: FB 1,FB 2,FB 3 = Fumonisins B 1,B 2, and B 3, respectively; LEM = leukoencephalomalacia. 401

2 402 HENRY AND WYATT Chick embryos have been used for toxicity testing of mycotoxins and numerous other toxic chemicals. Fumonisin B 1 has been demonstrated to possess moderate toxicity to chicken embryos, and the mortality associated with exposure to FB 1 is time- and dose-dependent (Javed et al., 1993). Furthermore, pathological changes included: hydrocephalus, enlarged beaks, elongated necks, and pathological changes of the liver, kidneys, heart, lungs, musculoskeletal system, intestines, testes, and brain. Bacon et al. (1995) demonstrated the toxicity of FB 1 to chick embryos, and also established a synergistic toxicological activity between FB 1 and fusaric acid. Despite the known production of three different fumonisins (FB 1,FB 2, and FB 3 )byf. moniliforme, no studies have been conducted to determine the toxicity of these fumonisins in chicken embryos. The present series of experiments was designed to investigate and compare the embryo-toxicity of FB 1,FB 2, and FB 3, administered individually and in combination, in the developing chicken embryo. MATERIALS AND METHODS Fumonisin B 1,FB 2, and FB 3, were produced and isolated according to the methodology outlined by Alberts et al. (1990) using sterile, yellow corn as the substrate and F. moniliforme MRC 826 as the organism. Purification of crude extracts was accomplished by the method of Alberts et al. (1990) with modifications (M. H. Henry and R. D. Wyatt, unpublished data). The resulting toxins were purified to a purity of >98%. A stock solution of FB 1 was made by dissolving 64 mg of the toxin in 10 ml of methanol:water (1:1 vol/vol). The FB 2 and FB 3 stock solutions were prepared by dissolving 8 mg of each toxin in 5 ml of methanol:water (1:1 vol/ vol). The 3:1:1 toxin combination, the approximate ratio of the fumonisins most often observed in culture material, was prepared by dissolving 9.6, 3.2 and 3.2 mg of FB 1, FB 2, and FB 3, respectively, in 10 ml of methanol:water (1:1 vol/vol). Further dilutions of these stock solutions were made to achieve the concentrations required for injections of the solutions into the air cells of developing chick embryos. In all experiments, the total volume of solvent used per egg was 10 µl. Broiler chicken hatching eggs (Peterson-Arbor Acres) were obtained from a local broiler breeder company and were used in all experiments. The flock age that served as the source of the hatching eggs ranged from 45 to 47 wk in Experiment 1, 52 to 53 wk in Experiment 2, and 49 to 50 wk in Experiment 3. Eggs weighing between 59.5 and 69.5 g (mean of 64.4 g) were selected and incubated at a dry bulb temperature of 37.8 C and a wet bulb temperature of 29.5 C, with automatic rotation of the eggs at 4- h intervals. 3 Embryonating eggs were assessed for viabil- FIGURE 1. The dose-response of mortality in chicken embryos exposed to fumonisin B 1 (FB 1 ) at 72 h of incubation (Experiment 1). ity by candling each egg after 72 h of incubation. Eggs with a clinically normal embryo and an air space in the normal location were selected for use in the experiments. The selected eggs were randomly divided into treatment groups, and each egg was injected with the test solution. Injection was accomplished by drilling a hole (2 to 3 mm in diameter) through the egg shell at a site in the center of the air space while simultaneously maintaining the integrity of the underlying shell membrane. The hole was created with the aid of an electric motor-tool drill 4 equipped with a circular drilling bit. All eggs were injected with a 10-µL volume of each test solution directly into the air space at a depth of 5 mm from the surface of the shell. Immediately after injection, the injection site was sealed with a drop of nontoxic white glue. 5 All eggs were labelled with a pencil and then returned to the incubator with the large end of the egg in a upward position. Experiment 1 was a dose-ranging experiment utilizing only FB 1. A total of 360 eggs was divided randomly into nine groups of 40 eggs each. To properly control the 3 Humidaire Incubator Co., Model No. 50, New Madison, OH Dremel, Model 245-5, Racine, WI Elmers, Glue-All, Borden, Inc., Columbus, OH FIGURE 2. The regression analysis used to calculate the 50% lethal dose (LD 50 ) for fumonisin B 1 (FB 1 ) in the chicken embryo (Experiment 1).

3 FUMONISIN TOXICITY IN CHICK EMBRYOS 403 TABLE 1. The response of embryonic mortality of chicken embryos exposed to fumonisin B 1 (FB 1 ) (Experiment 1) Cumulative mortality at FB 1 No. of injected eggs Day of incubation 18 d of incubation (µg) injected No. dead/total % (No. of dead embryos) Control: / f,g Drilled: / g Solvent: / f,g / e,f / d,e / d / c / b / a a g Mortality values with different superscripts differ significantly (P < 0.05). experiments, three control groups were used. The first control group consisted of eggs that were incubated as they were received from the hatching egg source (control). The second control group consisted of eggs that were drilled, the membrane was pierced with the injecting needle, no solution was injected, and then the eggs were sealed with glue (drilled). In the third control group, eggs were drilled, and 10 µl of methanol:water (1:1 vol:vol) was injected as described previously and then sealed immediately with glue (solvent). Six additional groups of 40 eggs each were injected with FB 1 at a dose of 2, 4, 8, 16, 32, or 64, µgoffb 1 per egg, respectively. Immediately after injection, all eggs were returned to the incubator and assessed for viability at 7, 10, 14, and 18 d of incubation by candling each egg. Eggs containing a dead embryo were opened, the embryo was weighed, and a visual inspection of the embryo was made. All eggs with a viable embryo on Day 18 were opened and visually assessed, and final mortality was calculated. Experiments 2 and 3 were designed to compare the toxicity of FB 1,FB 2, and FB 3. In Experiment 2, a total of 350 eggs containing viable embryos, determined by candling each egg after 72 h of incubation, were divided into seven groups of 50 eggs each. Three control groups, as described in Experiment 1, were also used. The treatments were FB 1,FB 2, and FB 3, individually, and a combination of FB 1,FB 2, and FB 3, in a 3:1:1 ratio, respectively. In Experiment 2, each test solution was injected such that each egg received 2 µg of total fumonisin. Mortality was determined by candling on 7, 10, 14, and 18 d of incubation. Eggs with dead embryos at each candling time were opened and visually inspected for the presence of gross abnormalities. Viable embryos were returned to the incubator on Day 18, and the wet bulb temperature was ad- FIGURE 3. The cumulative mortality of chicken embryos in response to graded doses of fumonisin B 1 (FB 1 ) (Experiment 1).

4 404 HENRY AND WYATT TABLE 2. The response of embryonic mortality of chicken embryos exposed to fumonisins B 1,B 2,andB 3 (FB 1,FB 2, and FB 3 ), individually and in combination, at a total dosage of 2 µg per egg (Experiment 2) Cumulative mortality at Fumonisin B 1 No. of eggs Day of incubation 18 d of incubation injected per egg injected No. dead/total % (No. of dead embryos) Control (0 µg) / b Drilled (0 µg) / b Solvent (0 µg) / a,b FB 1 (2 µg) / a FB 2 (2 µg) / b FB 3 (2 µg) / a,b FB 1 +FB 2 +FB 3 (3:1:1) (2 µg total) / b a,b Mortality values with different superscripts differ significantly (P < 0.05). justed to 32.2 C. Hatchability was determined on Day 22 of incubation. All chicks that hatched were weighed on Day 22. Experiment 3 was similar to Experiment 2, with the following modifications: The dosage in this experiment was 16 µg of total fumonisin per egg. All surviving embryos were removed on Day 18 from the embryonic membranes, weighed, and examined with the aid of a dissecting microscope for detection of potential developmental abnormalities of the head, beak, and limbs. Statistical analysis was conducted on all data. Statistically significant differences (P < 0.05) in mortality and hatchability between control and treatment groups were determined by a one-tailed Z-test for binomial population percentages using proportions to estimate standard errors of the differences (Ott, 1988). Mortality data from Experiment 1 were transformed to Probit and subjected to linear regression analysis in order to determine the 50% lethal dose of FB 1 (Fryer, 1966). Hatching weights of chicks (Experiment 1) and embryonic weights (Experiment 2) were subjected to one-way analysis of variance. Fisher s least significant difference test was used to compare the means of the treatments (Ott, 1988). RESULTS The effect of FB 1 on the mortality of chicken embryos indicated a nonlinear, dose-response relationship (Figure TABLE 3. The response of hatchability and hatching weight of chicken embryos following exposure to fumonisins B 1,B 2, and B 3 (FB 1,FB 2, and FB 3 ), individually and in combination, at a dosage of 2 µg per egg (Experiment 2) Fumonisin No. of Hatchability Hatching Treatment µg per egg eggs % 1 wt., g 1 Control ± 3.1 Drilled ± 3.9 Solvent ± 4.0 FB ± 3.4 FB ± 3.0 FB ± 3.0 FB 1 +FB 2 +FB 3 (3:1:1) ± No significant differences were observed (P > 0.05). 1). Embryonic mortality ranged from 5% in the control eggs to 100% in eggs injected with 64 µg FB 1 (Table 1). Cumulative mortalities of embryos exposed to 4, 8, 16, 32, and 64 µg offb 1 were significantly higher than those of the controls. Cumulative mortality of embryos exposed to FB 1 at 2 µg/egg was not significantly different than those of the controls. When FB 1 in methanol:water was injected into the air space of eggs, the 50% lethal dose for chicken embryos was determined to be 18.7 µg/egg (Figure 2). For embryos injected with 2, 4, and 8 µg of FB 1, most of the mortality occurred within the first7d postinjection (Day 10 of incubation). In embryos injected with 16, 32, and 64 µg fumonisin B 1, mortality was also highest within the first 7 d postinjection, and mortality gradually increased throughout the 22-d incubation period (Figure 3). Microscopic examination of embryos that died between Days 7 and 10 of incubation did not reveal any gross structural abnormalities of the head, beak, or limbs. However, all dead embryos taken from eggs on Day 10 that were injected with FB 1 at 8, 16, 32, and 64 µg/egg had extensive hemorrhages in the head, neck, and thoracic area. A similar hemorrhage pattern was also observed in embryos of eggs injected with 16, 32, and 64 µg FB 1 that died after Day 10 of incubation. There was no morphological difference between embryos from FB 1 -exposed eggs and embryos from control eggs that survived 18 d of incubation. Injection of FB 1,FB 2, and FB 3, singly and in combination, at a dose of 2 µg per fertile egg after 72 h of incubation, resulted in 14, 2, 8, and 4%, respectively (Table 2). The cumulative embryonic mortality of eggs injected with 2 µg offb 1 was significantly higher than that of the control, drilled, FB 2, or combination treatments, but was not significantly different from that of eggs injected with solvent or FB 3 (Table 2). Hatchability of control eggs was not significantly different compared with the hatchability of eggs injected with FB 1,FB 2,FB 3, and the combination treatment at a dosage level of 2 µg total fumonisin per egg (Table 3). There were no significant differences in the hatching weights of chicks from eggs injected with FB 1, FB 2,orFB 3, singly or in combination, and weights of chicks from control eggs (Table 3).

5 FUMONISIN TOXICITY IN CHICK EMBRYOS 405 TABLE 4. The response of embryonic mortality of chicken embryos following exposure to fumonisins B 1, B 2, and B 3 (FB 1,FB 2, and FB 3 ), individually and in combination, at a dosage of 16 µg per egg (Experiment 3) Cumulative embryonic No. of eggs Day of incubation mortality at 18 d Treatment used No. dead/total % (No. of dead embryos) Control (0 µg) / d Drilled (0 µg) / d Solvent (0 µg) / d FB 1 (16 µg) / a FB 2 (16 µg) / b FB 3 (16 µg) / b FB 1 +FB 2 +FB 3 (3:1:1) (16 µg total) / a a d Mortality values with different superscripts differ significantly (P < 0.05). In Experiment 3, FB 1,FB 2, and FB 3, singly and in combination at 16 µg/egg, resulted in embryonic mortality that was significantly higher than that of the controls (Table 4). The cumulative embryonic mortalities were 4.5% for the control, 56.0% for FB 1, 18.4% for FB 2, 14.6% for FB 3, and 34.0% for the fumonisin combination. Embryonic mortality of eggs injected with FB 1 was significantly higher than that of FB 2,FB 3, or the combination. The fumonisin combination resulted in significantly higher mortality than FB 2 or FB 3. There was no significant difference between mortality in the groups receiving FB 2 and FB 3. Mortality of these embryos occurred within 7 d after injection of eggs with the mycotoxins (Figure 4). Embryos that died between Days 7 and 10 from the different fumonisin-treated eggs had severe hemorrhages in the back of the head, along the neck, and in the thoracic area. No gross developmental abnormalities were observed in embryos examined after 18 d of incubation. Body weights of the 18-d-old embryos exposed to fumonisin were not significantly different from the control embryo weights (Table 5). DISCUSSION The chick embryo assay has been used as a preliminary screen to assess the toxic and teratogenic responses from mycotoxins and other toxic chemicals. These experiments show that the administration of a single air sac injection of these mycotoxins into chicken eggs after 72 h of incubation was toxic to embryos. The 50% lethal dose for FB 1 FIGURE 4. The cumulative mortality of chicken embryos when exposed to a dose of 16 µg per egg of fumonisins B 1,B 2,orB 3 (FB 1,FB 2,FB 3 ), singly and in a combination of FB 1 + FB 2 + FB 3 in a 3:1:1 ratio (Experiment 3).

6 406 HENRY AND WYATT TABLE 5. The relationship of gross morphological embryonic changes and embryonic weight in chicken embryos following exposure to fumonisins B 1,B 2, and B 3 (FB 1,FB 2, and FB 3 ), individually and in combination, at a dosage of 16 µg per egg (Experiment 3) No. of No. of malformed Embryo body Treatment eggs used embryos weight, g 1 Control (0 µg) ± 0.73 Drilled (0 µg) ± 0.60 Solvent (0 µg) ± 0.72 FB 1 (16 µg) ± 1.34 FB 2 (16 µg) ± 0.34 FB 3 (16 µg) ± 1.00 FB 1 +FB 2 +FB 3 (3:1:1) (16 µg) ± No significant differences were observed with any treatment (P > 0.05). 2 These embryos were very small, and detection of structural abnormalities was difficult. injected into the air space of chicken embryos at 72 h of incubation was 18.7 µg per egg. Embryos that survived to the final day of the experiments showed no gross developmental abnormalities of the head, beak, or limbs, suggesting that the fumonisins have no teratogenic effects. The lack of structural changes observed in this study, in contrast to the structural changes noted in other studies (Javed et al., 1993; Bacon, et al., 1995) may be due to the difference in the route of exposure of the embryos to the toxins, the vehicle used for administration of the toxins, or the age of embryonic development when exposure to the toxins occurred. Doses of the fumonisins of less than 2 µg per egg did not affect hatchability of the embryos. Most embryonic mortality occurred within 7 d after exposure with the fumonisins. These results also demonstrate that FB 1 is far more toxic to chicken embryos than either FB 2 or FB 3. Furthermore, there appears to be little difference between the embryo-toxicities of FB 2 and FB 3. The reason for the higher toxicity of FB 1 is apparently related to the absence of the hydroxyl group in FB 2 and FB 3, and may be related to the higher water solubility of FB 1 compared with the water solubility of either FB 2 or FB 3. Little information is available concerning the metabolism and the mode of action of the fumonisins in avian species. The exact causes or mechanisms involved in the hemorrhage seen in the embryos of eggs injected with fumonisin are unknown. In vitro studies using hepatocytes from rats and pig kidney epithelial cells indicate that the fumonisins inhibit the synthesis of sphingolipids, important constituents of the animal cell membrane (Wang et al., 1991). Broiler chicks fed diets containing FB 1 exhibited alterations in the sphinganine:sphingosine ratio (Weibking et al., 1993). If the fumonisins did inhibit the synthesis of sphingolipids in the chicken embryo, the hemorrhage seen in embryos injected with 16, 32, and 64 µgoffb 1 may be attributed to the action of the fumonisin on capillary permeability. The reduction of sphingolipids in the cell membrane may impair membrane integrity. 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