Aflatoxicosis in Chickens (Gallus gallus): An Example of Hormesis?

Aflatoxicosis in Chickens (Gallus gallus): An Example of Hormesis? G. J. Diaz,*1 E. Calabrese,† and R. Blain† *Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogota´, D. C., Colombia; and †University of Massachusetts, Department of Public Health and Health Sciences, Amherst 01003

Key words: aflatoxin, hormesis, aflatoxicosis, chicken 2008 Poultry Science 87:727–732 doi:10.3382/ps.2007-00403

to aflatoxins and quails show intermediate sensitivity, whereas chickens are the most resistant (Leeson et al., 1995). Body and relative liver weight are severely affected in turkeys fed doses as low as 0.4 ppm aflatoxin B1 in their diet, whereas chickens were not affected at this dietary concentration (Ostrowski, 1984). In fact, some studies have reported a modest enhancement in the body weight of chickens exposed to aflatoxins in their diet. Hormesis is a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition. The low-dose stimulation is typically maximal at only about 30 to 60% greater than controls (Calabrese, 2002). This type of response has been described in the past as biphasic, U-shaped, J-shaped, reverse, dual, overcompensation, stimulatory-inhibitory, and others (Calabrese and Baldwin, 2003b). Hormesis has been noted in regards to changes in body weight in numerous studies, including those performed for the US National Toxicology Program, with over 50 chemicals (see Table 1 to see chemicals tested; Calabrese and Baldwin, 2003a; Calabrese and Blain, 2005). The majority of the dose responses associated with changes in body weight have had maximum stimulatory responses between 110 and 150% of the control (Table 2). It should be noted that the hormesis database does not include studies with increases below 110% unless the results are statistically significant (Calabrese and Blain, 2005).

INTRODUCTION Aflatoxins are a group of heterocyclic metabolites synthesized predominantly by the fungi Aspergillus flavus Link and Aspergillus parasiticus Speare. Aflatoxins were first identified as the causative agent of the severe outbreak of Turkey X disease, a toxicosis that killed over 100,000 turkey poults in England in 1960 (Asplin and Carnaghan, 1961). Aflatoxins are a major concern because they are human hepatocarcinogens and are considered to play an important role in the high incidence of human hepatocellular carcinoma in certain areas of the world (CAST, 2003). In poultry, intake of feed contaminated with aflatoxins may result in poor performance, decreased organ weight, immunosupression, irreversible liver damage, morbidity, and mortality (Ostrowski, 1984; Leeson et al., 1995). Due to these effects, poultry has commonly been considered highly susceptible to aflatoxins. However, among domestic fowl there is wide variability in specific species sensitivity to this mycotoxin. Comparative toxicological studies in avian species have shown that ducklings and turkey poults are the most sensitive species

©2008 Poultry Science Association Inc. Received September 28, 2007. Accepted December 15, 2007. 1 Corresponding author: [email protected]

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body weight in numerous studies, including those performed for the US National Toxicology Program, with over 50 chemicals. The present paper assesses how relatively low levels of aflatoxin consumption in feed may affect the growth rate of chickens. In general, multiple independent investigations have shown that such aflatoxin consumption affects growth in a hormetic-like biphasic manner with a low dose stimulation and a high dose inhibition. Such observations were then generalized to other toxic agents and animal models, suggesting that low doses of stressor agents induce adaptive responses as reflected in accelerated growth rates. The implications of such hormetic dose responses are briefly discussed.

ABSTRACT Poultry has commonly been considered highly susceptible to aflatoxins. However, among domestic fowl there is wide variability in specific species sensitivity to these mycotoxins. Comparative toxicological studies in avian species have shown that ducklings and turkey poults are the most sensitive species to aflatoxins, quails show intermediate sensitivity, whereas chickens are the most resistant. Hormesis is a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition. The low-dose stimulation is typically maximal at only about 30 to 60% greater than controls. Hormesis has been noted in regards to changes in

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Table 1. Summary of the chemical and model information in studies observing hormetic effects on body weight parameters Citation

Chemicals

NTP, 1991 to 2000 (see Calabrese and Baldwin, 2003a)

Gee et al., 2002 Cruzan et al., 1998 Cunningham and Bucher, 1998 Gould et al., 1997 Til et al., 1997 Vergouwen et al., 1995 Biagini et al., 1993 Daston et al., 1991

Giridhar and Isom, 1990 Marks et al., 1989 Johnston et al., 1986 Mayes et al., 1984 Johnson and Damron, 1982 Gruger et al., 1976 Clement and Okey, 1974 Decker et al., 1958

The objective of the present article is to summarize and analyze the scientific evidence that indicates that the body weight data in chickens (Gallus gallus) exposed to dietary aflatoxins fit into the hormesis dose-response paradigm and to evaluate the possible implications of this phenomenon.

AFLATOXINS AND HORMESIS IN CHICKENS A biphasic low-dose stimulation high-dose inhibition response curve for body weight in chickens receiving

2-Butoxyethanol 2-Ethoxyethanol Methyl ethyl ketone peroxide 1,6-Hexanediamine dihydrochloride t-Butyl perbenzoate Benzoic acid Glyphosate Formic acid Diethanolamine 2-Hydroxy-4-methoxybenzophenone N,N-Dimethylformamide Monochloroacetic acid Castor oil Antimony potassium Tartrate p-Cresol o-Cresol m-Cresol Trinitrofluorenone Tris(2-chloroethyl)phosphate Probenecid D&C Yellow No. 11 Cobalt sulfate heptahydrate acetone 1,2-Dichloroethane Pentachlorobenzene 1,2,4,5-Tetrachlorobenzene Hexachloro-1,3-butadiene 2-Methoxyethanol

F344/N rats B6C3F1 mice

Sprague-Dawley rats CD-1 mice Sprague-Dawley rats Wistar rats Sprague-Dawley rats F344 rats Chickens Wistar rats Inbred CBA/P mice F344/N rats CD-1 mice

Sprague-Dawley rats CD-1 mice Sprague-Dawley rats Fathead minnows White Chinese geese Coho salmon Wistar rats Sprague-Dawley rats

graded levels of dietary aflatoxin has been extensively reported in the literature. Table 3 summarizes the results of several studies conducted with dietary aflatoxins in chickens, which observed a low stimulation at the lowdose level tested. In a study conducted in broiler chickens (Huff, 1980) birds receiving dietary total aflatoxin levels of 625 and 1,250 ␮g/kg had an average body weight of 528 ± 24 g and 515 ± 24 g, respectively. The average body weight of the control chickens was 511 ± 23 g. The body weight stimulation in this experiment was low, with a 3.3% increase at 625 ␮g/kg of aflatoxins and about 1.0% for 1,250 ␮g/kg. Huff and coworkers

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Kato et al., 2006 Newbold et al., 2004 Masutomi et al., 2003

Methacrylonitrile Carisoprodol Benzyltrimethylammonium Chloride Chloral hydrate Methyl ethyl ketoxime Sodium fluoride 3,3′,4,4′-Tetrachloroazobenzene t-Butanol Cyclohexanone oxime Urethane Cadmium oxide 1,3-Diphenylguanidine o-Chloroaniline m-Chloroaniline Magnetic field Isoprene β-Bromo-β-nitrostyrene Sodium selenate Sodium cyanide Acetaminophen Glutaraldehyde Riddelliine Tetrachlorophthalic anhydride cupric sulfate Methylene bis(thiocyanate) Pesticide/fertilizer mixture 2-Chloronitrobenzene 4-Chloronitrobenzene Bisphenol A DES (diethylstilbestrol) Methoxychlor Genistein Diisononyl phthalate Quercetin Styrene Oxazepam Aroclor 1242 Potassium nitrite X-rays Alachlor Bromodeoxyuridine Cadmium chloride Congo red Dinocap Diphenylhydantoin Ethylenethiourea Retinoic acid Trypan blue Lead PCB 77 (polychlorinated byphenyl) 1,2-Dibromo-3-chloropropane Triclopyr triethylamine Lead acetate Chlorobiphenyl DDT (dichlorodiphenyltricholoethane) Cadmium chloride

Model

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AFLATOXICOSIS IN CHICKENS Table 2. Breakdown of the dose-response relationships for changes in body weight in the hormesis database by maximum stimulatory response Maximum stimulatory response (% control) ≥ ≥ ≥ ≥ ≥ ≥

100 < 110 < 150 < 200 < 500 < 1,000

1

110 150 200 500 1,000

Number of dose-response relationships

Percent

1 135 16 7 1 1

0.6 84 10 4 0.6 0.6

1 Database criteria were established such that responses of less than 110% of the control were only included in the database when they were statistically different from the control (Calabrese and Blain, 2005).

DISCUSSION Several studies designed to evaluate the effect of aflatoxins in chickens have shown the characteristic lowdose stimulation high-dose inhibition pattern of the hormetic dose-response. A study conducted with a large number of doses, spaced at low intervals, has shown the typical inverted J-shaped curve observed in studies where the end-point is adversely affected by the expo-

Figure 1. Examples of the inverted J-shaped dose-response relationship of body weight vs. dietary aflatoxin level. Graphs constructed with data from Huff (1980) and Huff et al. (1986).

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(1986) found that broiler chickens receiving a diet containing 1,250 ␮g/kg of total aflatoxins for 21 d had an average body weight of 557 ± 28 g compared with 520 ± 20 g in the control chickens. This stimulation in body weight represented a 7.1% increase over the control group. Greater levels of aflatoxin in the diet (2,500 and 5,000 ␮g/kg) resulted in a significant decrease in body weight (89.2 and 77.1% of the control, respectively). Figure 1 shows plots of the data published in the above cited articles (Huff, 1980; Huff et al., 1986) where the typical hormetic biphasic response of low-dose stimulation high-dose inhibition is observed. In a study conducted by Diaz and Sugahara (1995), growing broiler chicks fed 3,000 ␮g/kg of purified aflatoxin B1 from d 4 to 11 of age had and average body weight of 89.1 ± 5.2 g compared with 85.3 ± 3.6 g for the corresponding controls. Day-old male White Leghorn chicks fed 1,000 ␮g/kg dietary total aflatoxin for 3 wk had a final body weight of 219.7 g compared with 210.3 g for the control group (Dixon et al., 1982). Although the low-dose stimulation of aflatoxins on body weight in these studies was always below 10%, there was a consistency to indicate a real effect. The hormetic stimulation is usually low

(normally not more than 30 to 60%; Calabrese and Blain, 2005), therefore causing it to be often overlooked. However, for a broiler chicken producer, any increase in body weight represents a large increase in income. The above-cited studies provide evidence for a lowdose stimulation and high-dose inhibition response in aflatoxins in chickens; however, the most relevant evidence for the hormetic response of dietary aflatoxins on body weight in chickens comes from the study of Richardson et al. (1987) where 15 doses at very small increments were tested. To properly study the hormetic response a large number of properly spaced doses are required (Calabrese, 2002). In this study, a typical hormetic inverted J-shaped dose-response curve was obtained for body weight vs. aflatoxin dietary level. Further, a quadratic polynomial model was fitted to the curve (Figure 2). The hormetic response of aflatoxins in body weight in chickens has not been observed in other commercial poultry species such as ducks and turkeys. For example, turkey poults receiving diets containing 125, 250, or 500 ␮g/kg for 21 d had body weights corresponding to 89.0, 81.2, and 65.4% of the control body weight, respectively (Hamilton et al., 1972). Even more sensitive to the effects of dietary aflatoxins are ducks. For example, 2-wk-old Alabio ducks fed dietary levels of 50, 100, and 200 ␮g/ kg of total aflatoxins for 14 d had body weights corresponding to 78.1, 54.3, and 40.3% of the control group, respectively (Ostrowski-Meissner, 1983).

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sure to the xenobiotic (Figure 2). It is still speculative why chickens respond in this way to dietary exposure to aflatoxins. Hormesis represents a strategy for the animal to optimize resource allocation and may occur through 2 different mechanisms of action: a direct stimulation and an indirect stimulation resulting from over-

compensation to an initial disruption in an attempt to assure that homeostasis is maintained (Calabrese, 2002). Either way, the result is the same: a biphasic response characterized by a modest stimulation compared with controls. It is possible that aflatoxins, being acutely toxic to the liver, stimulate an overcompensation response in

Table 3. Summary of experiments conducted with dietary aflatoxins in chickens showing the stimulatory effect observed at the low doses tested

Aflatoxin dietary level (␮g/kg) 625 1,000 1,250 3,000

Days of exposure

Percent body weight compared with control

21 21 21 7

103.3 104.5 107.1 104.5

Reference Huff, 1980 Dixon et al., 1982 Huff et al., 1986 Diaz and Sugahara, 1995

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Figure 2. Inverted-J dose-response for body weight against the log2 of dietary aflatoxin in chickens. Each clear circle corresponds to the mean body weight of 6 groups of 15 birds per treatment. The lines outside the curve correspond to the 95% confidence limits. The minimum effective dose (MED) of aflatoxin on body weight was calculated to be 1,370 ␮g/kg for the 2% fat diet (low fat diet) and 1,410 ␮g/kg for the 4% fat diet (normal diet). Reproduced with permission from the Poultry Science Association from Richardson et al. (1987).

AFLATOXICOSIS IN CHICKENS Table 4. Breakdown of the dose-response relationships for changes in body weight in the hormesis database by width of stimulation range

Width (-fold) ≥ ≥ ≥ ≥

1 < 10 10 < 100 100 < 1,000 1,000

Number of dose-response relationships

Percent of total dose-response relationships (108)

72 30 6 0

67 28 6 0

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the animal that manifests itself as an increase in body mass. The mechanism of action of aflatoxins on the hormetic response in chickens needs to be investigated. An analysis of the hormesis database (Calabrese and Blain, 2005) concerning parameters relating to weight gain indicates that numerous agents (Table 1) induce a hormetic-like biphasic dose response in regard to body weight gain. The parameters of the dose responses are those typically observed in hormesis with a maximum stimulatory response between 110 and 150% of the control (Table 2) and the width of the stimulatory response usually less than 100-fold with the majority less than 10fold (Table 4). These findings are generally supportive of the examples presented for aflatoxin in chickens and argue for the response being of a more general nature. The realization that low doses of dietary aflatoxins and numerous other toxic substances may stimulate growth at low doses may have implications for the poultry industry. For example, the use of feed additives intended to decrease toxicant adsorption in the gastrointestinal tract could be reevaluated within the risk-benefit context. In view of the important economic and public health implications of the hormetic response, it is necessary to investigate the potential occurrence of hormetic responses on commercial animal production in general.

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