Efficacy of N-Acetylcysteine to Reduce the Effects of Aflatoxin B1Intoxication in Broiler Chickens

Introduction

› Aflatoxin B1 (AFB1) is a secondary metabolite of the Aspergillus flavus andAspergillus parasiticus fungi

› AFB1 is found in grains and other foods and feeds as a natural contaminant.

› AFB1 is the most potent of the naturally occurring mycotoxin; it is extremely toxicand a powerful carcinogen and, therefore, represents a serious risk to health inhuman populations.

› AFB1 causes various health effects in chickens in a dose-response pattern.

Cont. …

› Hamilton (1984) has proposed an estimated minimum effective dose (10 μg/kg feed)based on the safety criterion of economic loss in broiler chickens.

AFB1 Detoxification

› Hepatic mixed-function oxydase system biotransforms AFB1 and generates anaflatoxin metabolite or reactive epoxide.

› This intermediate molecule is inactivated by conjugation with reduced glutathione.

› This reaction is catalyzed by glutathione-S-transferase (GST) to form a molecule thatis eliminated as mercapturic acid-AFB1 or N-acetylcysteine (NAC) bound to AFB1.

AFB1 Pharmacodynamics

› When birds eat AFB1, it is absorbed by the intestine and distributed by thebloodstream throughout the body.

› Approximately 90% AFB1 is removed through bile and renal secretion.

AFB1 Tissue Residues

› Studies on distribution of 14C AFB1 in chickens revealed that 6.2% of radioactivitywas retained in breast and leg meats.

› In laying hens and broiler chickens, aflatoxin clearance times are 24 h for muscleand 8 d for eggs.

Study

Study Aim

› The aim of this study was to determine whether dietary supplementation with NACprevented the appearance of negative effects during subacute AFB1 intoxication inbroiler chickens.

Material and Method

› One-day-old male Hubbard broiler chickens.

› Birds were weighed, wing-banded, and placed in electrically heated cages undercontinuous lighting with access to feed and water ad libitum.

› The basal sorghum-soybean meal starter diet, without added antibiotics,coccidiostats, or growth promoters, containing or exceeding the levels of criticalnutrients recommended by NRC (1994) was purchased from Agricultural SciencesCentre Feed Mill.

› Chicks were weighed on an individual basis, and feed consumption for eachreplicate was recorded daily.

Treatment

NAC Addition

› Purified NAC5 was added daily to the basal diet (3.2 to 5.2 g/kg feed).

› Based on daily feed intake and BW records, it was assured that every birdconsumed approximately 800 mg NAC/kg BW per day.

Cont. …

NAC and AFB1

› Pure crystallized AFB1 solution in ethanol (1 mg/10 mL) was sprayed over a thinlayer of the basal diet (<1-cm thickness), and a final concentration of 3.0 mg/kg offeed was achieved.

› The ethanol was thoroughly evaporated for 72 h; feed was mixed and then packedand stored until its use.

Cont. …

› Prior to the start of experiment, basal and contaminated whole diets where analyzedfor aflatoxins by using solid phase extraction tubes, and the eluate extracted wasderivatizated according to official method 990.33 of AOAC and by HPLC withfluorescence detection7,8 (Scott, 1995).

› The basal diet did not contain detectable levels of AFB1, AFB2, AFG1, or AFG2(detection limits <10 μg/kg).

Cont. …

› A completly randomized design was used with 120 male 1-d-old chickens assignedto each of four dietary treatments.

› The groups were as follows:

1. Control group with basal diet

2. AFB1 alone in the diet (3 mg/kg of feed) for 21 d

3. NAC alone (800 mg/kg BW per day)

4. AFB1 plus NAC at the same doses as for Groups 2 and 3.

Evaluation

› Feed intake and BW were recorded daily.

› Chickens were slaughtered at 21 d of age, by cervical dislocation.

› 10 birds per group were randomly selected for biochemical assays and three morebirds per group were used for histological studies.

Results

Treatment 21-d Body weight Daily weight gain Feed:gain

AFB1 (µg/kg of

feed)

NAC (mg/kg

BW)(g)

Change from

control (%) (g)

Change from

control (%) (g)

Change from

control (%)

0 0 458 100 21.8 100 1.45 100

3 0 408 89.1 19.4 89 1.66 114

3 800 437 95.4 20.8 95.4 1.61 111

0 800 449 98 21.4 98.2 1.51 104

Results

Treatment Protein GST GSH

AFB1 (µg/kg of

feed)

NAC (mg/kg

BW)

(mg/100 mg

tissue)

Change from

control (%)

(mmol

CDNB/min per

mg protein)

Change from

control (%) (µmol/g tissue)

Change from

control (%)

0 0 49.2 100 150 100 3.1 0

3 0 21.9 44.5 297 165 14 3

3 800 40.3 81.9 312 173 9 3

0 800 64.5 131 262 146 4.8 0

Results

Treatment ALT AST AST:ALT ratio

AFB1 (µg/kg of

feed)

NAC (mg/kg

BW)(units/L)

Change from

control (%) (units/L)

Change from

control (%) (g)

Change from

control (%)

0 0 19.4 100 4.4 100 3.0 100

3 0 15.8 81.4 6.5 148 4.2 140

3 800 18.2 93.8 6.1 139 3.3 110

0 800 21.1 109 4.5 102 2.9 96.7

Results

Treatment 28-d Body weight Daily weight gain Feed conversion index

NAC (mg/kg BW) (g) Change from

control (%) (g)

Change from

control (%) (g)

Change from

control (%)

0 915 100 36.3 100 2.02 100

400 901 98.5 34.7 89 1.97 97.5

800 921 101 35.9 95.4 2.01 99.5

Conclusion

› In summary, our results suggest that NAC intake decreased the severity of AFB1toxic effects.

› This protective action was evident on

1. Body growth

2. FCI

3. Macro- and microscopic changes in liver and kidney

4. Hepatic and plasma protein concentrations

5. GSH hepatic concentration

6. ALT plasma activity.

Cont. …

› These data suggest that NAC might be used to prevent the effects of AFB1ingestion.

› NAC did not change the production parameters in control broiler chickens.