Aflatoxin levels in sera of healthy first time rural blood donors: preliminary report

780 TRANSACTIONS

OF THE ROYAL

Aflatoxin

SOCIETY

OF

TROPICAL MEDICINE

AND HYGIENE,

VOL.

75, No.

6, 1981

levels in sera of healthy first time rural blood donors: preliminary report G. C. ONYEMELUKWEAND G. OGBADU

Depts. of Medicine and Biochemistry,

Ahmadu Belle University

Summary

Serum levels of aflatoxin Bl, B2, Gl and G2 in 20 farmers (first time blood donors at Ahmadu Bello University Teaching Hospital, Zaria, Nigeria) were determined and compared with 15 patients of varied diagnoses. 25% of the farmers had levels of Bl above 0 *2pg per ml and correspondingly the highest values of other aflatoxins. Except for one patient with nasopharyngeal carcinoma who had a Bl level of 0*13pg per ml, other sick control values ranged from 0.005 to 0*09lpg per ml. None of the farmers with high levels of Bl hadpositive serum hepatitis B surface antigen (HbsAg) as determined by counter-immunoelectrophoresis (CIE). A continuous prospective study may define the role of aflaxtoxin z&&is hepatitis B virus (Hbv) in the pathogenesis of the high incidence of liver disease prevalent the Guinea Savannah North of Nigeria. Introduction

Staple foods in the Savannah and forest regions of Nigeria are contaminated by mycotoxins (MACDONALD & HARKNESS, 1967; NWOKOLO& OKONKWO, 1978). In the sub-Saharan Africa and particularly in the Guinea Savannah of Nigeria, there is a high incidence of primary liver cell carcinoma (ANTHONY, 1977; FAKUNLEet at., 1977) and in Zaria, the HbsAg carrier rate was 9.8% (ISAACSet al., 1974). Epidemiological studies of primary liver cell carcinoma (PLCC) in relation to environmental mycotoxin contamination correlated their association in Uganda, Kenya, Thailand and Swaziland (LINSELL & PEERS, 1977) but the precise role of aflatoxins either as co-carcinogen with Hbv or as a permissive agent

Table I-Aflatoxin --A

Zaria,

Methods

20 farmers, who farm and subsist on local staple foods-millet, sorghum, rice, yams-who were donating blood for the first time at ABUH were interviewed to exclude any with a past history of liver diseases. Their agesranged from 25 to 40 years. Serum samples were taken and stored at -20°C until analysed. Aliquots of sera were screened for HbsAg by CIE and, in all cases, AST, ALT alkaline phosphatase and serum proteins were found to be normal. Nine out-patients and six in-patients, all from the surrounding villages, served as controls. All the out-patients were males and, of these, seven had Schistosoma haematobium infections of the bladder and two had asthma with pulmonary aspergillosis diagnosed by chest X-ray and positive aspergillin skin tests and aspergillin gel diffusion tests (> 1: 10 titre). One adult male in-patient had nasopharyngeal carcinoma, one adult female had osteomalacia and another had a hydatiform mole. Specimens were taken on the first day of admission or contact with out-patients.

--

farmers

and sick controls

-----

Farmers (20)

Controls (15)

Bl

Mean Range

O-123& a161 *025-0.57

O-045 10.032 0~005-0~130

B2

Mean Range

0~118~0~109 0~010-0-390

0*050&0.056 0.008-0.240

Gl

Mean Range

0*118~0*163 *024&0*59

0*044&0~043 0~005-0~180

G2

Mean Range

0*064&0.060 0~012-0*192

.022&0*019 ~002-0~080

--

Nigeria

or in exerting preparative or promotive action has been the subject of much discussion (LUTWICK, 1979). In addition to cirrhosis and PLCC, mycotoxins are probably of much importance in Reye’s syndrome, renal and other human diseases(MARTIN & GILMAN, 1976). We sampled the sera of healthy rural blood donors from villages served by Ahmadu Bello University Teaching Hospital (ABUH), Zaria, for aflatoxin content and here report the results.

levels (in pg per ml) in healthy

Aflatoxin G-M-S

Hospital,

G. C. ONYEMELUKWE

Aflatoxin Bl, B2, Gl, G2 determination To 1 ml of serum samole. 9 ml of 10% trichloroacetic acid was added toprecipitate se&A proteins. 0’5 ml of the filtrate was added to a mini-column to determine aflatoxin levels according to the standard method using a Velasco fluorotoxin meter (Neotec Instruments Inc.. Marvland. USA). Microiolumn standard prepar-ations using Bl; B2, Gl and G2 were used to calibrate the fluorotoxin meter as described in the instrument brochure. Aflatoxin is detected by trapping it on a special layer of florosil in a microcolumn. Calculations of free aflatoxin, incorporating the dilution factor, were made and expressed as pg per ml. Further studies are in progress to determine the effect of trichloroacetic acid treatment on serum protein binding of aflatoxins but present results are expressed as free aflatoxin levels after trichloroacetic acid protein precipitation. Results One of 20 farmers had serum HbsAg (5%). Two cirrhosis patients out of 15 patient controls had HbsAg (13%). Fig. 1 shows the level of Bl, B2 Gl and G2 in farmers and controls. Four farmers (25%) had a Bl level above 0.2Oyg per ml and, correspondingly, the highest levels of other aflatoxins. The mean for Bl in farmers was 0*123& O-161 pg per ml (range 0.025 to O-057 pg per ml) and the mean for Bl in sick controls was 0 *045 h

Aflatoxin . Healthy farmers o S,ck controls

Fig.

1. Serumaflatoxin

levels in pg per ml.

781

AND G. OGBADU

0.032 pg per ml (range 0.005 to 0.130 pg per ml). The difference is statistically significant p = ) in the control group was obtained in the patient with nasopharyngeal carcinoma, in which Epstein-Barr virus (E-B virus) has been said to be of aetiological importance. LEWIS et al. (1967) produced nasal neoplasms in sheep fed toxin groundnut meals for five years. Aflatoxin Bl promotes and alters the expression of the cytopathogenic effect of cytomegalovirus (GARNETT, 1978) and it would be of interest to evaluate any similarity of effects on E-B virus, Hbv and cytomegalovirus-induced lesions. To implement decisions to control the ingestions of aflatoxins in tropical environments is as urgent as ever (LINSELL, 1979;

ANTHONY,

1977).

Acknowledgements We are grateful to the Blood Bank personnel of A.B.U. Hospital, Zaria, for their assistance.

782

AFLATOXINS

IN SERA OF BLOOD

References

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DONORS

Linsell, C. A. (1979). Decision on control of a dietary carcinogen-aflatoxin. LARC Science Publication, 74 (25), 11l-l 12. Linsell, C. A. & Peers, F. (1977). Aflatoxin and liver cell cancer. Transactions of the Royal Society of Tropical Medicine and Hygiene, 71, 471-473.

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Journal

of Experimental

Volume 75, No. 5 p. 743, Table I, add to footnotes: 3Tris,

maleic

acid

and final

Pathology,

48, 20-27.

Accepted for publication 11 th February, 1981.

CORRIGENDUM

addition of MgCI,;

-

Macdonald, D. & Harkness, C. (1967). Aflatoxin in nroundnut croos at harvest in NorthernNigeria. TrGpical Science, 9, 148-161. Martin, P. & Gilman, G. A. (1976). A consideration of the mycotoxin hypothesis with special reference to mycoflora of maize, sorghum, wheat and groundnut. Tropical Products Institute, G105.Newberne, P. M., Hadrrington, D. H. & Wogan, G. N. (1966). Effects of cirrhosis and other liver insults bn induction of liver tumours by aflatoxin in rats. Laboratory Investigations, 15, 962-969. Nwokolo, C. W. & Okonkwo, P. (1978). Aflatoxin load of common food in savanna and forest regions of Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 72, 329-332. Zuckerman, A. J., Tsiquaye, K. N. & F&on, F. (1967). Tissue culture of human embryo liver cells and the cytotoxicity of aflatoxin Bl. British

EDTA neutralized pH readjusted with

with NaOH.

NaOH

before