hit. J . Cancer: 17, 167-176 (1976)

DIETARY AFLATOXINS AND HUMAN LIVER CANCER. A STUDY IN SWAZILAND F. G . PEERS 1 * 3, G. A. GILMAN 1 * and C . A. LINSELL Tropical Products Institute, 56-62, Grays Inn Road, London, England: and International Agency for Research on Cancer, P.O. Box 46831, Nairobi, Kenya Summary. A study in Swaziland to assess the possible relationship of ajlatoxin contamination and the incidence of primary liver cancer is reported. Ajatoxin ingestion levels have been determined in “ f o o d from the plate” samples collected over a I-year period. A significant correlation between the calculated ingested daily dose and the adult male incidence of primary liver cancer in diferent parts of Swaziland has been established. Samples of foodstuffs other than the plate samples also reflected the correlation of ajlatoxin contamination and liver cancer. This study extends and amplifies the findings of an earlier study in the Murang’a district of Kenya and supports the hypothesis that aJlatoxin ingestion is a factor in the genesis of primary liver cancer in Africa.

Nairobi Research Centre,

was approximately the same as in the Kenya study area (Murang’a 344,858 and Swaziland 362,367) although the area of the latter is 10 times greater. Additional a.dvantages of Swaziland as the site of the second study were that the country is naturally divided into four well-defined altitude areas which, it was suspected, would reflect varying levels of aflatoxin contamination of food staples and the distribution of liver cancer cases as reported in the independent study by Keen and Martin (1971a) between these areas was significantly different from that expected on a population basis. MATERIAL AND METHODS

Topography

Swaziland is a landlocked, independent kingdom An evaluation of the contamination of diets by of 17,422 km2 in southern Africa bounded on the aflatoxin and the primary liver cancer incidence in north, south and west by the Transwaal province the African population of the Murang’a district of the Republic of South Africa and on the east by of Kenya established a statistically significant correlation (Peers and Linsell, 1973). The dis- Mozambique and Natal province of South Africa. The country is divided naturally into four welltribution of medical facilities in the Murang’a study defined altitude regions which extend north and and the low number of cancer cases recorded in some of the altitude areas of the district might have south in roughly parallel belts. The highveld, in contributed a bias to the correlation. A further the west, the middleveld and the lowveld are more study in Africa was therefore carried out to test or less of equal breadth, while the Lebombo, in altitude similar to the middleveld, is a markedly the strength and consistency of the association. narrower strip along the eastern border. In Swaziland it had already been reported (Keen The location of the medical facilities available in and Martin, 1971a) that the risk of developing 1964, shown in Figure 1 , were obtained from the liver cancer varied with altitude as in Kenya. Stored groundnuts, a popular food in Swaziland, were Swaziland Ministry of Health. The highveld averages 1,000-1,350 m in altitude often contaminated with aflatoxin and the occurrence of such contamination was associated with and occupies some 4,900 km2. It is mountainous an increased liver cancer frequency (Keen and and split by numerous river valleys and gorges. Martin, 1971a, b ) . It was not possible, however, There are good pastures for summer grazing and to infer dietary exposure levels of aflatoxin from considerable areas are covered by pine and eucalyptus forests. Arable production is generally conthese data. fined to the gentler slopes and valleys. The main medical facilities in Swaziland are The middleveld averages 650-750 m in altitude located in the areas of lower liver cancer frequency where it was assumed that cancer case catchment and is undulating to hilly, occupying an area of would be more effective: the reverse of the potential bias present in the Kenya study. Primary liver cancer was also more frequent, 4.9/100,000 crude Received: October 27, 1975. rate for all ages in Swaziland, 1964-1968, compared On detached duty with Nairobi Research Centre of with 3.3/100,000crude rate for all ages, in Murang’a, the International Agency for Research on Cancer. 1967-1970. The African population of Swaziland On detached duty in Swaziland.

168

PEERS ET AL.

Cliiiiate

The highveld in general has a near-temperate climate with warm summers while the lowveld is sub-tropical and semi-humid. Rainfall decreases from an average of 1,270mm in the highveld to some 660mm in the lowveld, higher in the north than the south, and most of this falls in the summer between early October and the end of March. The lowveld has a mean maximum temperature of 29" C, which is some 6" C higher than that for the highveld. Frosts are common in the winter in the highveld and occur, to a lesser extent, in the lowveld. Population

The Report on the 1966 census enumerated a total resident population of 374,571 of which 93.9% were Swazis living predominantly in rural areas. In the northern lowveld and Lebombo there are some Shangaans from the eastern Transvaal who work mainly on estates. The middleveld is the most densely populated area with 41.6 ?( of the population or a n average of 338 persons per km2. Crops, storage methods and Jlrngal deterioration

--

INIIRNAIIONAL ~OUNDARIII

0

IOWNS

... -. ....

ADMINlSlRATIVI

BOUNDARIII

0

______

ALIITUDI ARIA

lOUNDARltl

0 CLINICS

A-8

ARBIlRARY

HOSPITALI

L I N E D I V I D I N G NORlH A N D S O U T H

FIGURE 1

Swaziland-medical facilities, 1964.

about 4,700 km2. Although the soil fertility is often not particularly good, the physical aspects of the land render it suitable for arable production. There is also much natural grassland and the grazing is good in summer. The gently undulating lowveld averages 150300 m in altitude and covers more than 6,500 km3. It is seldom a true plain as isolated knolls and ridges rise above the general level. It has a distinctive " bush " vegetation which ranges from dense, thorny thicket, to more open parkland savannah with large trees. The underlying grass has a high food value which makes it excellent for cattle ranching. Apart from some irrigated areas, arable production is often sparse, especially in the lowrainfall areas. The Lebombo range in the extreme east of the country has a n impressive western escarpment. This region approximates to the middleveld in altitude although it resembles the lowveld in many places with respect to vegetation. There is good, mixed farming country but the chief type of individual holding is the cattle ranch.

Maize is the staple diet but sorghum, groundnuts, jug0 beans (Voandzeia subterranea) and various pulses including the cowpea ( Vigna sinensis), the kidney bean (Phaseolus vulgaris) and the mung bean (Phaseolus aureus) are other durable food crops grown for home consumption. The areas under these crops vary with altitude. A recent agricultural Sample Census (1971/2) has shown that the area under maize is over 62,000 hectares, of which a little less than half is in the middleveld. Areas under other durable food crops of lesser importance are groundnuts (5,000 hectares), sorghum (2,500 hectares), jug0 beans (2,800 hectares) and beans (2,000 hectares). In the lower areas especially, maize and sorghum dry out considerably before harvest which is why cribs or grain stores are seldom seen there. Open storage platforms are observed mainly in the lowveld but crude cribs inside huts with open-structured walls are more common in the high- and middlevelds. No mould damage was observed to occur in these structures, but if rain falls on the mature crop prior to harvest, considerable mould damage occurs in the field, especially to maize. Most of this damage is caused by Fusarium moniliforme and other typical " field " fungi. Aspergillus flavus and other storage species are not common at this time. After 3-4 months, maize and sorghum are shelled and stored as grain in a variety of traditional or imported containers. One traditional method that might be expected to encourage mould growth in stored grain is the

AFLATOXIN AND LIVER CANCER IN SWAZILAND

underground pit. Pits are found mainly in the lowveld and on the Lebombo range, but they were once more widely distributed. They represent, in fact, a method of great antiquity used in one form or another in many parts of Africa. Those in Swaziland are narrow-necked, beehive-shaped, and are usually situated in the cattle kraal. Those examined held between 3 and 30 bags of maize and sorghum. As the pits are not lined except with a thin layer of cattle dung, water rapidly penetrates and is absorbed by the grain. Some mould damage usually occurs in the neck region, if this is filled with grain, or at the rim of surface layers, if the pit is not completely filled. Small quantities of grain in large pits frequently become caked with fungal mycelium. Mouldy grain from pits is sometimes washed and eaten, a factor which could be of some significance in this survey. In full pits the bulk of the grain is reasonably well preserved although it undergoes a partial ensilagement, while changes in colour, flavour and texture, especially in grain near the walls and floor of the pit, are usual. Insect damage is generally absent from pitstored maize but in above-ground stores, especially in the lowveld, this is a serious problem, especially as insect-damaged grain is more easily invaded by storage fungi than undamaged seeds. Other traditional stores with a more general distribution are clay pots and spherical, woven grass baskets. These are used for all crops and are kept under cover. In certain middle-veld areas of northern Swaziland mud and wattle structures are used to store shelled grain but the method is relatively uncommon. More recent introductions, gradually replacing the traditional methods, are small tins, 200 I drums, sacks and metal silos. The silos are cylindrical and made from corrugated metal, resembling water tanks. Mould damage was seen occasionally in metal silos exposed to direct sunlight. The groundnut is an important source of protein in Swaziland. The crop is dried in a variety of ways including windrows, platforms and small stacks. After hand picking, the pods are given a further period of sun drying prior to storage. A more detailed account of storage and handling methods used in Swaziland and adjoining territories, particularly in respect of deterioration by fungi, has been published elsewhere (Gilman, 1971; Martin et al., 1971). Cancer registration During the 5-year period 1964-1968 a total of 500 primary liver cancers in Africans was registered

by the Swaziland Cancer Registry. Using the 1966

169

Census data this means a crude cancer rate of 27.6 per 100,00O/year compared with 19.9 per 100,000/year recorded for the Murang’a district of Kenya. Details of cancer data used in this study were derived from Keen and Martin (1971) and further information from Dr. Keen (personal communications, 1974) on the location, sex and age of previously recorded cases. Experimental design of the study Apart from the four topographical zones of Swaziland already described, the country is also divided administratively into four Districts. These are Hhohho (A) in the northwest, Manzini (B) in the centre and west of the country, Shiselweni ( C ) in the south and Lubombo (D) in the east. The latter district includes the Lebombo range and a large proportion of the lowveld. The study was so designed that, if necessary, a comparison of the administrative districts could also be made. For sampling, Swaziland was divided as follows into eleven main arable areas to include a range of climates, altitudes, crops and storage and handling methods : Highveld-Oshoek (A), Mankanyane (B) and Hlatikulu (C); Middleveld-Lomati Valley (A), Manzini (B) and Hluti (C); Lowveld-Belgane (A), Ngcina (D), Big Bend/Sipofaneni (D) ; Lebombo range-Lomashasha (D) and Siteki (D). In the absence of tax lists and identity numbers, which were available in the Murang’a District of Kenya, a random individual or family sampling was not possible. However, lists of Chiefs and Indunas, (sub-chiefs) living in the above areas were obtained with the assistance of the Ministry of Local Administration and from these lists Indunas were selected at random, two from each area, to form the focal points of 22 sampling clusters. It was thought that the socio-economic status of the Indunas used as cluster centres might bias the sampling and to test this, seven householders living progressively further away from the Indunas’ homes were chosen to complete each cluster. As holdings are generally well scattered this involved distances of up to 2.4 km from the cluster centre to the last sample in a cluster. The clusters were sampled every 2 months, though not necessarily in the same order, for a period of 1-year. The 2-month periods constitute arbitrary “ seasons ” to test seasonal variation within the year of study. This provided a total of 1,056 diets, according to the factorial design: 11 (areas) x 2 (clusters) x 6 (“ seasons ”) x 8 (samples). In addition to the diets one sample each of beer (Tswala), sour porridge (Incwancwa), sour drink (Amahhewu) and groundnuts were, if possible, collected separately at each visit as these foodstuffs are often not consumed with the main meal.

170

PEERS ET AL.

Collection of samples

The maize crop in Swaziland is harvested in April/May and shelled in July/August while groundnuts are harvested sooner and are therefore usually in store by the beginning of June. The collection of samples was started in October 1972 at the beginning of the rains and was completed in September 1973. When this survey commenced, therefore, maize had only recently been stored as shelled grain whereas groundnuts had been in store for approximately 6 months. It was established that the main meal of the day was usually consumed in the morning. This consisted of maize porridge with one or more of the following: vegetables, meat, spinach, groundnuts, sourmilk, pumpkin, sweet potatoes and various kinds of beans and other pulses. Groundnuts are frequently consumed as a relish in powder form, often with a " spinach " prepared from the leaves of a variety of wild and cultivated plants. Occasionally sour porridge or sorghum may be substituted for the normal maize porridge. The collector visited each cluster the night before a collection was due to take place. This was necessary as extra food had to be cooked. Periodic checks were made to ensure that special meals were not being prepared for the collector. During these preliminary visits certain households were asked to prepare sour drinks, sour porridge and powdered groundnuts if they were not already available. Beer was obtained from the brewing point nearest to the cluster concerned. Collections were made in the early morning, visiting each household in turn. If any food remained from the meal of the previous evening this was included in the diet sample. The collector was instructed to purchase a '' man-sized " portion of each meal. The dietary components of the meal, sociological and other data were recorded for each household sampled. Safeguards, such as a record of the altitude, were included to ensure that each cluster was actually sampled and that the diets were not obtained from easier sources. Also on the first visit to the cluster centre an appropriate number of serially numbered tickets were handed to the lndunas and at every subsequent food collection these were matched with the duplicates. Inability to collect a particular sample due to illness or absence occurred on a average three times in a " season ". In these instances, adjacent households, not normally included in the cluster, were sampled instead. The diets were placed in plastic bags, the fluids in 1-litre plastic bottles, and the groundnuts in

o o o o o o

!?

171

AFLATOXIN AND LIVER CANCER IN SWAZILAND

small plastic cartons. All samples were deep frozen as soon as possible after collection, almost invariably on the same day, insulated cold boxes and " scotchice " coolant packs being provided to the field team, On return to the laboratory the samples were deep frozen at -20" C until processed.

RESULTS

Table I illustrates the exposure to aflatoxin of the study population of Swaziland using the mean contamination levels of plate samples, including all negative samples, and the frequency of aflatoxin positivity by cluster and individual diet samples. The data have been divided into four altitude areas and six " seasons " of collection. Table I1 shows the frequency and mean level of contamination of samples other than whole diet plate samples which were also collected as part of the survey. These dietary items are freely available to the Swazis but it was not possible to assess accurately individual or group consumption. A total of 126 sour drink samples were also collected but all proved negative for aflatoxins at a detection level of 1 pg/litre. Although the dietary data with a large number of negative results can be fitted to a Gamma function curve (Berry and Day, 1973) it was decided to express the statistics of the results by more simple methods. In Table 111 the various frequencies of positivity have been compared by the x2 tests and in Table IV the mean contamination levels of the diets have been evaluated for significance using Student's t tests. Overall, the principal significant factor is that of altitude and this is true for both frequency and mean contamination levels. Since the administrative areas of Swaziland correspond to a large degree to the altitude areas, as opposed to the situation in Murang'a, a significant effect is observed when the frequency distribution of aflatoxin positivity is evaluated by this parameter. The data have also been evaluated on a northsouth division in order to test the conclusion of Keen and Martin ( 1 9 7 1 ~ )that liver cancer frequency differed significantly between these two

Sample processing In order to obtain representative sub-samples for analysis the diets were homogenized. However, the porridge which formed the bulk of the samples in most cases, assumed a spongy consistency on thawing and could not be easily homogenized. It was therefore often necessary to add water. This was always a known amount, either 25% or 50% of the wet weight of the sample. A potato masher was then used to break up the diet prior to homogenizing. With the beers, sour porridges and sour drinks, homogenization was not necessary but they were shaken vigorously before sampling. The diet samples were freeze-dried in shallow containers at a shelf temperature of 50" C for 24 h. to enable mycological studies to be made on the beers and sour foods and to prevent sugar inversion these were dried at a shelf temperature of 35" C for 36-48 h. The samples were stored in plastic containers and air freighted to Nairobi for aflatoxin analysis. Aflatoxin analysis

Aflatoxin analyses were carried out on the lyophilized diet samples by methods previously described (Peers and Linsell, 1973) except that no preliminary drying over silica gel was necessary. The dry residues from the lyophilization of the beer, sour drink and sour porridge samples were processed in the same way. The powdered groundnut samples were analysed by the methods described by Jones (1972).

TABLE I1 FREQUENCIES A N D MEAN LEVELS OF AFLATOXIN CONTAMINATION OF SAMPLES COLLECTED OTHER THAN DIETS ~

Sour drinks

Sour porridges

Season n'

A B C D E F

011 8 0121 0122 0121 0122 0122

Totals

01126

- 2

0.00

n

-

X

0.37

91122

0.21

0.00

811 14

0.50 0.00

n

X

0.61

0.00

0.05

Groundnuts

-

0.07 0.33 0.23

0.56 0.61

0.00

n

4/18 1/20 0122 1/22 2/20 1/20

118 3/19 1/20 2/23 0122 1/22

0.00 0.00 0.00

Beers

0.67 0.08

2/19 2/22 112 2/14 2/14 2/22

0.00

~

' n = frequency expressed as number of positives/total analysed. negatives).

-X

18.00 20.09 0.75 3.43 5.54 3.41

~

11/93

1 = mean aflatoxin in pg/kg of material

10.60

as collected (including

172

PEERS ET AL. TABLE 111 STATISTICS O F FREQUENCIES O F POSITIVE CLUSTERS, INDIVIDUAL DIET SAMPLES A N D SAMPLES OTHER THAN DIETS Statistic

Degrees of freedom

x *-dusters

x *-diets

x Z-other samples

Altitude areas Administration areas Seasons Between samples within clusters Within clusters from binomial North vs South A vs B cluster

3 3 5 I

7.04 10.53 *

17.09 ***

13.63 ** 7.13 * 2.83 for 2 d.f.

2 1 1

6.61 -

8.23 ** 0.50

14.49 ** 4.97 3.50 0.74

4.51 * 0.06

*

-

-

3.88 * 0.10

1 * 0.05;-p>0.01; ** 0.01 >p>O.OOI; *** piO.001. - * The low cell expectation for individual seasons was < 5 and hence the seasons have been combined in pairs ( L e . A+B. C + D and E + F ) t o evaluate this statistic. - Starting with the cluster centre (Induna) the set of eight samples was collected progressively further away and sequentially marked stroke I to stroke 8. This statistic shows no bias for contamination of the first or any other sample within a cluster. - Using an overall positivity of 6.7?( (71/1056) the expectations of multiple contaminated samples within a cluster have been calculated from the binominal expansion.

regions of Swaziland. The north-south division was defined by a n arbitrary line, based on the 1966 census, which avoided passing through any collection area or site of a cancer case. The frequency of aflatoxin positivity from this present study does indeed show significant differences between the north and the south. However, the mean contamination levels of the diet samples including all positives do not show a significant difference. This would imply that the more frequent exposure in the north is at a lower dose level. The principal results obtained in this study are summarized in Table V and in Figure 2 the calculated exposure data for males (four areas) and females (three areas) have been plotted against the adult liver cancer rates recorded by Keen (personal communications, 1974). The sex ratio (M/F) of the cancer incidence in Swaziland is approximately 5:1 as compared with 2:l in Kenya and with the small population in the Lebornbo area no female cases of liver cancer were recorded in the 5 years of registration, whereas 4 adult male cases were recorded. Hence the two sexes have been treated

separately with respect to correlation exercises. The male aflatoxin ingestion data have been obtained assuming 2 kg wet food and 2 litres of beer per day and a mean body weight of 70 kg and this has been evaluated for the four altitude sub-areds. The regression line, y = 23.84 log,, x--13.66, has been drawn in Figure 2 together with the 95 % confidence limits: the correlation coefkient for this line is 0.988 for two degrees of freedom (0.02>p>0.01). For females the Middleveld and Lebombo data have been combined as one area and the aflatoxin

TABLE IV STATISTICS OF THE MEAN LEVELS OF DIETARY AFLATOXINS Mean values compared

Season A VY season D (widest difference between two seasons) Highveld vs Middleveld Highveld vs Lowveld Highveld vs Lebornbo Middleveld vs Lowveld Middleveld YS Lebombo Lowveld vs Lebombo North vs South

t value

Probability

1.23 1.17 3.33

0.3 > p >0.2 0.3 > p >0.2

2.96

pp>0.05 0.01 >p>O.oOl

0.99 1.86 1.31

0.4>0>0.3 0.1 >p>0.05 0.2>p >0.1

1.68

' Means include all negative results.

Log,o aflatoxin(ng1kg bodyweight/day)

FIGURE 2 Correlation of liver cancer with aflatoxin ingestion.

AFLATOXIN AND LIVER CANCER IN SWAZILAND

a 5

-91 .rBn

E

173

ingestion levels have been calculated assuming a 2 kg wet food intake per day and a 70 kg mean body weight. The correlation line y = 4.68 log,, x -2.35 has been calculated but the correlation coefficient (r = 0.957) is not significant for only 1 degree of freedom. DISCUSSION

a D 0

iz

3

E

a 9

P

6

el

E

-P

a

5

s 5 V

3

E

This study was undertaken to ascertain whether the relationship between dietary aflatoxin levels and primary liver cancer incidence established in the Murang'a district of Kenya was valid for another area of Africa, specifically where the range of liver cancer incidence was higher than that recorded in Murang'a and where groundnuts were known to be incorporated into the diet. The foodstuffs referred to in Table 111 all form part of the Swaziland diet but they were collected separately from the diet plate samples, and it was not possible to assess ingestion levels due to the widely varying quantities and frequencies of consumption. The contamination data from Table 111 therefore have not been included in the calculation of ingestion levels. Any aflatoxin exposure levels quoted are thus almost certainly minimal ingestion levels. Our experience and that of others (Shank et al., 1972; van Rensburg et al., 1974) would suggest that it is the chronic ingestion levels that more closely parallel liver cancer incidence rather than occasional high exposures. As in other studies the anomaly of relating current exposure to a suspect carcinogen with retrospective rather than prospective data has been ignored. It has been assumed that the dietary habits and customs of the Swazi people have changed little over the last few decades. As aflatoxin contamination is mainly related to poor storage the lack of a seasonal effect on the aflatoxin content of the. diets was surprising. The data for the groundnut samples in Table 111 suggest that November to January is the period when high levels of aflatoxins may be encountered in this dietary component but the relative constancy of the exposure from the diet samples suggests that a discriminatory sorting is effected by the housewife as detected in Murang'a (Peers and Linsell, 1973) and Mozambique (van Rensburg ef al., 1975). A comparison of the components of the positive diets with those of the negative diets indicated that groundnuts, beans and cultivated vegetables are included more frequently in the positive diets. Since 98.4% of the plate samples collected contained maize it was not possible to incriminate specifically this diet component. The incrimination of ground-

1 74

PEERS ET AL. TABLE VI 1965 A N D 1966 TUBERCULOSIS RECORDS FOR SWAZILAND Altitude area

Administrative area

Highveld

Middleveld

Lowveld

Lebombo

Totals

Obs.

Exp.

Obs.

Exp.

Obs.

Exp.

Obs.

Exp.

Hhohho Manzini Shiselweni Lubombo

62 119 198

8 530 36 0

103.6 170.5 140.3 8.5

5 12 32

41.8 13.0 40.1 160.5

-

-

111.1 87.5 82.7 -

10

52.4

75 661 234 42

Totals

379

281.3

574

422.9

49

255.4

10

52.4

1,012

nuts was suspected from the work of Keen and Martin (1971~)and is confirmed by the data in Tables II/III. In Swaziland, tribal custom does not inhibit women from drinking home-brewed beers (tswala) in contrast to the Kikuyu women of Murang'a. These Swazi beers are very popular with the men and the more mature women, but younger women frequently abstain. For the purposes of evaluating minimal aflatoxin ingestion data, however, we have assumed a male preponderance in beer consumption. Although the trend to increasing incidence of liver cancer with decreasing altitude is reflected in the female data (Table V) the significance of these differing incidences rates cannot be tested statistically as with the male cases because of the small number of cases (Table VII). Experimental work in rats has also shown a sex differential in the toxic and carcinogenic effects of the aflatoxins (Butler, 1964, 1971; Wogan and Newberne, 1967). It is postulated that a sex-linked protection of some kind, among the other factors which play a part in the aetiology of liver cancer, may be present. Whether there is an individual variation, perhaps sex-linked, in the ability of microsomal enzymes of the liver to hydroxylate or otherwise metabolize the aflatoxin B, molecule to the proximal carcinogen remains to be proved. An evaluation of the incidence, by administrative areas or altitude areas, of cancers other than liver cancer was not possible from the Swaziland Cancer

0

-

Obs.

Exp.

256.5 271.0 263.1 221.4

-

Registry as exact home addresses had only been determined for liver, skin and bladder cancers which were of particular interest to the Registry in 1966. Records of tuberculosis patients for 1965 and 1966 were made available by the WHO Epidemiology Centre in Nairobi and these were examined to assess the availability of medical care for patients suffering from a chronic disease. The data were recorded by centre of first diagnosis rather than home address. In Table VI tuberculosis patients are shown by administrative and altitude areas together with the expected values on a population basis. The large deviations from probability are due to the siting of the national TB centre in the Middleveld area of Manzini and because the main hospitals obviously drain tuberculosis patients from further afield than the altitude or administrative areas in which they are sited. In Table VII we have evaluated the significance of the distribution of the liver cancer cases as allocated to the altitude areas. The major contributions to these x' values derive from the low numbers of cases recorded in the highveld area with more developed medical facilities and the large number of cases recorded in the lowveld area with less comprehensive medical facilities. This can be assessed from Figure 1 and by the details of 1964 medical facilities given in Table VIII. With such a distribution of medical facilities case catchment is indeed likely to be more efficient in

TABLE VII SIGNIFICANCE OF THE DISTRIBUTION OF LIVER CANCER CASES, BY ALTITUDE AREA Highveld

Total cases Expected from total populations Adult male cases Expected from adult male population

11 24.8 9 20.9

Middleveld

34 41.8 28 29.8

Lowveld

44 22.5 35 21.3

Totals

89 -

72 -

Ya

-

29.7 *** 15.7 ***

a

The Lebombo and Middleveld have been combined in this Table due to the low expectation from the small population in the Lebombo. -

' ***pp1>0.1). I n view of the possible biases mentioned and the low numbers involved, an increased risk for liver cancer in the Shangaans cannot be accepted from these data. Keen and Martin (1971) noted a lower liver cancer frequency in the south as compared with the north of Swaziland and that this was particularly marked for the High- and Middleveld areas. The aflatoxin ingestion levels north and south of the arbitrary line shown on Figure 1 have been evaluated and whilst a significantly increased frequency of contamination of the diets in the north is recorded, the mean level of contamination is not significant. Using information provided by Keen (personal communication), the 72 adult male liver cancer cases have been allocated to the two areas as defined by this arbitrary line and the distribution tested statistically in Table X. Hence, although there may be an increased frequency of the disease in the northern as compared with the southern areas of the High- and Middleveld areas, this is not true of the country taken as a whole. It has been demonstrated in this study that, at least in males, a log relationship exists (Fig. 2) between the liver cancer incidence and the ingestion of aflatoxin (Fig. 2). The range of the observations in Africa of dietary aflatoxin levels and primary liver cancer incidence has been extended to adult male liver cancer rates of 26 per 100,OOO per year. The data are consistent with the hypothesis that chronic aflatoxin exposure is related to the incidence of liver cancer within an African population.

Other Africans

ACKNOWLEDGEMENTS

Adult male population 215

80,369

Percentage actively employed Liver cases 1964-1968 Expected from population Crude adult male rate per 100,000 per year

3,872

4,426

44.9 64

83.6

68.0

3.3

3.1

15.9

36.2

18.1

I

67.9 4

TABLE X

SWAZILAND LIVER CANCER INCIDENCE ON A NORTH-SOUTH BASIS North

South

1964-68 adult male cases 48 24 52,834 35,833 Adult male population Expected No. of cases 42.9 29.1 1964-68 aduft female cases 7 7 Adult female population 59,230 44,569 8.0 6.0 Expected No. of cases

x' -

1.5 0.29

The considerable assistance and co-operation of many members of the Swaziland Ministries of Health, Agriculture and Local Administration are acknowledged. We are deeply indebted to Dr. Paul Keen of the Cancer Research Unit of the South African Institute for Medical Research for access to unpublished data without which we would not have been able to prepare this manuscript. We are particularly grateful to Mr. Paul Dlamini, our sample collector, and Mr. R. W. Bell and Mr. P. Jones for laboratory space at the Malkerns Agricultural Research Station. We would also like to thank Mr. Sven Christensen of the WHO Epidemiology Centre, Nairobi, Kenya, for access to the 1965-1966 Swaziland Tuberculosis records, Dr. J. Nabney and Dr. N. R. Jones of the Tropical Products Institute for helpful comments during the preparation of this manuscript, and the Directors of TPL and IARC for permission to publish this paper.

176

PEERS ET AL.

AFLATOXINE DANS L’ALIMENTATION ET CANCER D U FOIE. UNE ETUDE EFFECTUEE A U SWAZILAND

I1 est rendu compte d’ime Prude effectuPe au Swaziland pour apprecier l’eventualiti d’une relation entre la contamination par I’apatoxine et I’incidence du cancer primitif du foie. On a determine les teneurs en aflatoxine d’ichantillons d’aliments ‘‘ prPIevPs dans les assiettes ” au cours d’une piriode d’un an. Une corrPIation significative a P t P Ptablie entre la dose journali2re ingPrPe ainsi calculPe et I’incidence du cancer primitif du foie chez les hommes adultes au Swaziland, Les Pchaiitillons d’aliments autres qite ceux prtlevPs d a m les assiettes ont Pgalement fait apparaitre cette corrilation entre la contamination par I’aflatoxine et le cancer du foie. Cette etude Ptend et amplifie les conclusions d’une Ptirde menee aiittrieurement d a m le district de Murang’a, au Kenya: e l k corrobore I’hypothhe selori Iaquelle l’ingestion d’aflatoxine est un facteur Priologique du cancer primitif du foie en Afrique. REFERENCES

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