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ANALYSIS OF HUMAN SERA FOR AFLATOXIN
ANALYSIS OF HUMAN SERA FOR AFLATOXIN
A.P. Wilkinson, D.W. Denning,Q H.A. Lee, C.M. Ward and M.R.A. Morgan. Department of Food Molecular Biochemistry AFRC Institute of Food Research Norwich Laboratory Colney Norwich NR4 7UA UK ()' Department of Infectious Diseases Hope Hospital Salford M6 8HD UK
1.
INTRODUCTION
The aflatoxins are a group of potent toxins formed by certain species of Aspergillus fungi, moulds that commonly infest a wide range of food components, particularly nuts and cereals. Aflatoxins are of concern because of their toxicity and carcinogenicity at very low concentrations, often parts per billion, in a large number of animal species including primates.'') Acute illness and death in humans resulting from ingestion of high levels of aflatoxin are rare, but because of their potency, it is possible that regular ingestion of aflatoxin at sub-acute levels may pose a threat to human health particularly in cancer development.n) To assess such a possibility, two strategies may be employed; (a) Test food destined for human consumption (b) Analyse human body fluids and tissues. For both approaches, the ability to rapidly and cost effectively quantify low aflatoxin levels in large sample numbers of foods, body fluids and tissues is necessary. Q
The Biorecognition and Immunotechnology group at IFR has developed enzyme-linked immunosorbent assays (ELISAS) to determine aflatoxin in a wide range of foodstuffs.@) One of these assays was adapted to measure aflatoxin in human serum.(5) The test procedure is simple, requiring the addition of methanol to samples to precipate interfering substances prior to
98
Food and Cancer Prevention: Chemical and Biological Aspects
assay. The figure below shows a standard curve for aflatoxin BI diluted with methanol-treated aflatoxin free control serum. The limit of detection of the assay, dotted line, is 1 pg.wel1-I giving an assay sensitivity of 20 pg.ml-' of original serum. Also shown in the diagram is the analysis of 27 serum samples obtained from U.K. blood donors. None of the samples had aflatoxin above 20 pg.m1-'.(6) In contrast Figure 2 shows that the blood of some Nigerian O and all of the Nepalese (') subjects studied were contaminated with aflatoxin.
Fig. 1
An ELISA standard curve for aflatoxin B, in human serum. Points are plotted 2 s.d. _+
For the Nigerian study, 19 samples (24%) had no detectable aflatoxin, whilst 59 samples (76%) were positive. The positive samples ranged from 20 pg.ml-' to 3.1 ng.ml-I with a mean of 665 pg.ml-'. All these subjects were apparently healthy. The aflatoxin levels detected in the 28 Nepalese subjects ranged from 60 pg.rn1-I to 10 ng.ml-'. Of these donors, 16 were hospitalized and 12 were healthy hospital workers. There was no correlation between the health of the donors at time of sampling and the level of aflatoxin in their blood. Though these three studies have examined a small sample size, the results reflect the risk of exposure to aflatoxin in different areas of the world. In the U.K., the most likely source of aflatoxin is from imported foods, particularly nuts, and the results seem to show that voluntary and statutory regulations
Analysb of Human Sera for Aflatoxin
- 10,000
. .
J" "lflfr Fig. 2
-
1000
- 100 - 20
Aflatoxin concentration in sera from 78 Nigerian and 28 Nepalese subjects.
governing aflatoxin levels in foods and animal foodstuffs have been effective in limiting the exposure of the U.K. population to aflatoxin. Unlike the U.K., tropical and sub-tropical countries such as Nigeria and Nepal, have climates and food storage systems that favour aflatoxin production by toxigenic moulds that commonly infest foods. Thus local produce becomes contaminated by aflatoxin particularly during or just following the wet season. Our studies of Nigerian and Nepalese subjects were made at such time and the higher risk of consuming aflatoxin contaminated food is reflected in the data. High levels of aflatoxin consumption during pregnancy might have mutagenic, carcinogenic or immunosuppressive effects on the human fetus if transplacental transmission of aflatoxin occurs. Such toxic effects have been demonstrated in other species.@ Fig. 3 shows the results of our study of blood from 35 paired samples of Thai mothers and neonates.@) The blood was collected from the cord at birth and from the mother immediately afterwards. Seventeen (48%) of the cord sera and two of the maternal sera (6%) contained detectable aflatoxin. The low contamination of the maternal blood samples possibly reflects the absence of food intake prior to delivery as well as the timing of the study which was made during the dry season when aflatoxin contamination of food has been observed to be low.('" The results show
Food and Cancer Prevention: Chemical and Biological Aspects
100
..
ffittaEa Mother S e n
Fig. 3
Cord Sera
Aflatoxin concentration in sera of paired samples of Thai mothers and neonates.
clearly transplacental transfer of aflatoxin and it would also appear that the feto-placental unit is capable of concentrating aflatoxin, as a result of excretion of aflatoxin into the amniotic fluid and reingestion of the toxin. CONCLUSIONS The ELISA is a simple and sensitive technique for monitoring aflatoxin exposure at the individual level. Aflatoxin levels in U.K. plasma is low. The high levels of aflatoxin in the sera of Nigerian and Nepalese subjects indicate that populations in tropical and sub-tropical countries can be exposed to considerable amounts of aflatoxin in their diets. Aflatoxin can cross the human placental membrane and may be concentrated by the developing fetoplacental unit. ACKNOWLEDGEMENTS This work was sponsored by the Ministry of Agriculture, Fisheries and Food.
Analysis of Human Sera for Aflatoxin
101
REFERENCES 1.
W.F.Busby and G.N.Wogan, 'Chemical Carcinogens', C.E.Searle (ed), American Chemical Society, Washington D.C., 1985, vo1.2, p.945.
2.
D.W.Denning, A d v e r s e D r u g R e a c t . A c u t e P o i s o n R e v . 1987, 4 , 175.
3.
J.D.Groopman and X.F.Donahue, 1988, 7 1 , 861.
Chem.,
J. Assoc. Off. A n a l .
4.
M.R.A.Morgan, A.S.Kang and H.W-S.Chan, J. S c i . F o o d A g r i c . , 1986, 3 7 , 908.
5.
A.P.Wilkinson, D.W.Denning, and M.R.A.Morgan, A d d . C o n t a m . , 1988, 5 , 609.
6.
A.P.Wilkinson, D.W.Denning and M.R.A.Morgan, T o x i c o l . , 1988, 7 , 353.
7.
D.W.Denning, J.X. Onwubalili, A.P.Wilkinson and M.R.A.Morgan, T r a n s . R. SOC. T r o p . Med. Hyg., 1988, 8 2 , 169.
8.
A.P.Wilkinson, D.W.Denning and M.R.A.Morgan, T o x i c o l . T o x i n R e v , 1989, 8 , 69.
9.
D.W.Denning, R.Allen, A.P.Wilkinson and M.R.A.Morgan, C a r c i n o g e n e s i s , 1990, 11, 1033.
10
R.C.Shank, J.E.Gordon, G.N.Wogan, A.Nondasuta and B.Subhamini, F o o d C o s m e t . T o x i c o l . , 1972, 1 0 , 71.
Food
Hum.
J.
A.P. Wilkinson, D.W. Denning,Q H.A. Lee, C.M. Ward and M.R.A. Morgan. Department of Food Molecular Biochemistry AFRC Institute of Food Research Norwich Laboratory Colney Norwich NR4 7UA UK ()' Department of Infectious Diseases Hope Hospital Salford M6 8HD UK
1.
INTRODUCTION
The aflatoxins are a group of potent toxins formed by certain species of Aspergillus fungi, moulds that commonly infest a wide range of food components, particularly nuts and cereals. Aflatoxins are of concern because of their toxicity and carcinogenicity at very low concentrations, often parts per billion, in a large number of animal species including primates.'') Acute illness and death in humans resulting from ingestion of high levels of aflatoxin are rare, but because of their potency, it is possible that regular ingestion of aflatoxin at sub-acute levels may pose a threat to human health particularly in cancer development.n) To assess such a possibility, two strategies may be employed; (a) Test food destined for human consumption (b) Analyse human body fluids and tissues. For both approaches, the ability to rapidly and cost effectively quantify low aflatoxin levels in large sample numbers of foods, body fluids and tissues is necessary. Q
The Biorecognition and Immunotechnology group at IFR has developed enzyme-linked immunosorbent assays (ELISAS) to determine aflatoxin in a wide range of foodstuffs.@) One of these assays was adapted to measure aflatoxin in human serum.(5) The test procedure is simple, requiring the addition of methanol to samples to precipate interfering substances prior to
98
Food and Cancer Prevention: Chemical and Biological Aspects
assay. The figure below shows a standard curve for aflatoxin BI diluted with methanol-treated aflatoxin free control serum. The limit of detection of the assay, dotted line, is 1 pg.wel1-I giving an assay sensitivity of 20 pg.ml-' of original serum. Also shown in the diagram is the analysis of 27 serum samples obtained from U.K. blood donors. None of the samples had aflatoxin above 20 pg.m1-'.(6) In contrast Figure 2 shows that the blood of some Nigerian O and all of the Nepalese (') subjects studied were contaminated with aflatoxin.
Fig. 1
An ELISA standard curve for aflatoxin B, in human serum. Points are plotted 2 s.d. _+
For the Nigerian study, 19 samples (24%) had no detectable aflatoxin, whilst 59 samples (76%) were positive. The positive samples ranged from 20 pg.ml-' to 3.1 ng.ml-I with a mean of 665 pg.ml-'. All these subjects were apparently healthy. The aflatoxin levels detected in the 28 Nepalese subjects ranged from 60 pg.rn1-I to 10 ng.ml-'. Of these donors, 16 were hospitalized and 12 were healthy hospital workers. There was no correlation between the health of the donors at time of sampling and the level of aflatoxin in their blood. Though these three studies have examined a small sample size, the results reflect the risk of exposure to aflatoxin in different areas of the world. In the U.K., the most likely source of aflatoxin is from imported foods, particularly nuts, and the results seem to show that voluntary and statutory regulations
Analysb of Human Sera for Aflatoxin
- 10,000
. .
J" "lflfr Fig. 2
-
1000
- 100 - 20
Aflatoxin concentration in sera from 78 Nigerian and 28 Nepalese subjects.
governing aflatoxin levels in foods and animal foodstuffs have been effective in limiting the exposure of the U.K. population to aflatoxin. Unlike the U.K., tropical and sub-tropical countries such as Nigeria and Nepal, have climates and food storage systems that favour aflatoxin production by toxigenic moulds that commonly infest foods. Thus local produce becomes contaminated by aflatoxin particularly during or just following the wet season. Our studies of Nigerian and Nepalese subjects were made at such time and the higher risk of consuming aflatoxin contaminated food is reflected in the data. High levels of aflatoxin consumption during pregnancy might have mutagenic, carcinogenic or immunosuppressive effects on the human fetus if transplacental transmission of aflatoxin occurs. Such toxic effects have been demonstrated in other species.@ Fig. 3 shows the results of our study of blood from 35 paired samples of Thai mothers and neonates.@) The blood was collected from the cord at birth and from the mother immediately afterwards. Seventeen (48%) of the cord sera and two of the maternal sera (6%) contained detectable aflatoxin. The low contamination of the maternal blood samples possibly reflects the absence of food intake prior to delivery as well as the timing of the study which was made during the dry season when aflatoxin contamination of food has been observed to be low.('" The results show
Food and Cancer Prevention: Chemical and Biological Aspects
100
..
ffittaEa Mother S e n
Fig. 3
Cord Sera
Aflatoxin concentration in sera of paired samples of Thai mothers and neonates.
clearly transplacental transfer of aflatoxin and it would also appear that the feto-placental unit is capable of concentrating aflatoxin, as a result of excretion of aflatoxin into the amniotic fluid and reingestion of the toxin. CONCLUSIONS The ELISA is a simple and sensitive technique for monitoring aflatoxin exposure at the individual level. Aflatoxin levels in U.K. plasma is low. The high levels of aflatoxin in the sera of Nigerian and Nepalese subjects indicate that populations in tropical and sub-tropical countries can be exposed to considerable amounts of aflatoxin in their diets. Aflatoxin can cross the human placental membrane and may be concentrated by the developing fetoplacental unit. ACKNOWLEDGEMENTS This work was sponsored by the Ministry of Agriculture, Fisheries and Food.
Analysis of Human Sera for Aflatoxin
101
REFERENCES 1.
W.F.Busby and G.N.Wogan, 'Chemical Carcinogens', C.E.Searle (ed), American Chemical Society, Washington D.C., 1985, vo1.2, p.945.
2.
D.W.Denning, A d v e r s e D r u g R e a c t . A c u t e P o i s o n R e v . 1987, 4 , 175.
3.
J.D.Groopman and X.F.Donahue, 1988, 7 1 , 861.
Chem.,
J. Assoc. Off. A n a l .
4.
M.R.A.Morgan, A.S.Kang and H.W-S.Chan, J. S c i . F o o d A g r i c . , 1986, 3 7 , 908.
5.
A.P.Wilkinson, D.W.Denning, and M.R.A.Morgan, A d d . C o n t a m . , 1988, 5 , 609.
6.
A.P.Wilkinson, D.W.Denning and M.R.A.Morgan, T o x i c o l . , 1988, 7 , 353.
7.
D.W.Denning, J.X. Onwubalili, A.P.Wilkinson and M.R.A.Morgan, T r a n s . R. SOC. T r o p . Med. Hyg., 1988, 8 2 , 169.
8.
A.P.Wilkinson, D.W.Denning and M.R.A.Morgan, T o x i c o l . T o x i n R e v , 1989, 8 , 69.
9.
D.W.Denning, R.Allen, A.P.Wilkinson and M.R.A.Morgan, C a r c i n o g e n e s i s , 1990, 11, 1033.
10
R.C.Shank, J.E.Gordon, G.N.Wogan, A.Nondasuta and B.Subhamini, F o o d C o s m e t . T o x i c o l . , 1972, 1 0 , 71.
Food
Hum.
J.