Determination of exposure to aflatoxins among Danish workers in animal-feed production through the analysis of aflatoxin B1 adducts to serum albumin.

Aflatoxin B1 is suspected as an etiologic factor in the increased risk for primary liver cancer among workers in animal-feed processing plants in Denmark. Aflatoxin bound to serum albumin was therefore measured for feed-processing workers. Blood samples were collected immediately after vacation and after four weeks of work, and aflatoxin was quantified by competitive enzyme-linked immunosorbant assay. Seven of 45 individuals with an estimated exposure of 64 ng aflatoxin B1.d-1.kg-1 body weight were positive. Three positive workers had been unloading a cargo with an aflatoxin B1 level of 26 micrograms.kg-1 raw material. The exposure level correlated well with the job titles. Dust samples collected at different sites showed considerable variation in the amount of aflatoxin B1 (nondetectable to 8 micrograms.kg-1 dust). The exposure to aflatoxin B1 may only partially explain the increased risk of liver cancer.

Aflatoxin H\, a potent liver carcinogen in experimental animals, is produced by Aspergillus flavus and paraciticus under warm and humid conditions. Aflatoxin HI has been classified as a group 1 carcinogen by the International Agency for Research on Cancer. A positive correlation between the dietary intake of aflatoxin HI and the incidence of liver cancer has been observed in African and Asian countries (1,2). A causative association between exposure to aflatoxins and human cancer has also been established for Dutch workers in an oil mill processing peanuts and linseed; especially the incidence of lung cancer was higher than expected (3). Circumstantial evidence has been reported for an association between the inhalation of aflatoxincontaminated grain dust and lung cancer (4). In a register-based analysis of occupational risk for primary liver cancer in Sweden, a significant excessof cases was observed for workers in grain mills, and it was postulated that potential exposure to aflatoxins may play a role (5). An increased incidence of liver cancer has been reported among Danish male workers in companies processing livestock feed. The exposure of these workers to aflatoxins through the respiratory route was estimated to be 170 ug . d-I according to conservative estimates (6). Attempts to detect aflatoxin H, or its metabolites in the urine of potentially exposed workers with enzyme-linked immunosorbant assay (ELISA) were unsuccessful due to the presence of aflatoxin-like antigenic material (7,8).
Aflatoxin H, is metabolized by the mixed function oxidase system to a number of hydroxylated metabolites and to the 8,9-epoxide. The latter is considered the ultimate carcinogenic form that reacts with cellular deoxyribonucleic acid (DNA) and proteins to form covalent adducts. Detection and quantification of these adducts have been suggested as alternative methods to detect human exposure to aflatoxins (9). With the use of ELISA and a monoclonal antibody against aflatoxin H" aflatoxin H, bound to albumin could be detected in blood samples collected in Gambia and Kenya, but not in samples collected in France. The sensitivity of the assay was 5 pg aflatoxin HI . mg"! albumin (10). In the present paper we have used a combination of affinity purification of pronase-digested albumin and ELISA to detect aflatoxin adducts to serum albumin from workers potentially exposed to aflatoxin B I .

Subjects
A total of 45 workers at two companies processing livestock feed in Esbjerg, Denmark, 15 at company A and 30 at company B, was included in the study. The workers were either working in the factory and silo (29 workers) or in the harbor unloading ships with raw material for the production (16 workers). They were all men in the age group of 25 to 62 years. None of the workers had been working in any other agribusiness for three months prior to the start of this study.

Sample collection
Blood samples (5 m!) were collected from the workers when they returned to work after at least two weeks of vacation, and then again after four weeks of work .
Dust samples were collected at selected sites in the factories of company A (3 samples) and company B (9 samples). Samples of all shipments of raw material were submitted to analysis for aflatoxin B) at government-approved laboratories.

Analysis of the blood samples
Serum was isolated from the blood samples by centrifugation, and albumin was precipitated by the addition of saturated ammonium sulfate, final concentration 50 070. After the removal of globulins by centrifugation, albumin was precipitated from the supernatant by acidification with acetic acid (11).
Albumin was redissolved in 500 ul of 0.1 M sodium phosphate buffer, pH 7.4, and digested with pronase (Fluka, Buchs, Switzerland, 25 units per 5 mg of albumin) for 18 h at 37°C. The concentration of albumin was determined by the Biuret reagent (Sigma, St Louis, Missouri, United States, kit 540-2) assay. The digest was applied to a prewet C18 Seppak column (Waters, Milford, Massachusetts, United States). The column was washed with 5 ml of 5 % methanol to remove small pep tides and amino acids, before the aflatoxin Bj-containing fraction was eluted in 80 % methanol (5 ml) . This fraction was evaporated to 0.5 ml and was diluted to make 10% methanol (I ml) . The eluate was applied to an Aflatest'i' affinity column (Vicam, Sommerville, Massachusetts, United States) (10), and the aflatoxin Bj-containing fraction was eluted with 1.5 ml of methanol in phosphate buffer 0.1 M, pH 7.4 (1:1) after the column was rinsed with phosphate buffered saline (PBS) (5 ml). With the use of albumin isolated from rats treated with radiolabeled aflatoxin B), the recovery was estimated to be 80 %.

Enzyme-linked immunosorbant assay
Polystyrene microtiter plates (Teknunc, Roskilde, Denmark) were coated with aflatoxin Bj-bovine serum albumin (Sigma) or bovine serum albumin (BSA) as previously described (8). On each plate 100~I of the eluates and five aflatoxin (25, 2.5, 0.25 , 0.025, and 0.0 ng . ml") standards were added immediately before 100~I of a solution of monoclonal antibody AW-l (12) (diluted 1:1000 with PBS) was applied to compete for binding. The plates were incubated overnight at 4°C, then washed with 0.05 % Tween 20 (polyoxyethylene sorbitan monolaurate) in PBS before incubation with the secondary antibody, goat antimouse immunoglobulin G conjugated with alkaline phosphatase (Dakopatts, Glostrup, Denmark) (diluted 1:1000 in I % BSA in PBS), for 2 h at room temperature. Following washing with 0.05 % Tween 20 in PBS and 0.1 M diethylamine buffer (pH 9), 100~I of 4-nitrophenyl phosphate substrate (Sigma (I mg/rnl) was added to each well, and the plates were incubated for 2 h at room temperature. The absorption was read at 405 nm. The intrasample variation was less than 2 % .

Analysis of the dust samples
Aflatoxin was isolated from the dust samples with a mod ification of the standard procedure provided by the manufacturers of Aflatest'" affinity columns.
Dust samples (0.9 mg) were suspended in 5 ml of 80 % methanol, 0.1 rng of sod ium chloride was added, and the samples were extracted by shaking for 30 min in the dark. The extracts were centrifuged for 20 min, and the supernatants were collected. This procedure was repeated, and the supernatants were pooled. Three milliliters of the extract was adjusted to 10 % methanol, and 10 ml of this solution was loaded onto the Aflatest" affinity column. After being washed with distilled water the aflatoxins were eluted with 100 % methanol. The quantities of aflatoxins were determined in ELISA assays as has already been described.

Results
The sensitiv ity of the analysis using Seppak prepurification and affinity chromatography prior to quantitative analysis with ELISA was 5 pg aflatoxin B• . mg"! albumin, corresponding to 30 % inhibition. It was assumed that, with the use of the AW-l antibody, the antigenicity of aflatoxin B) adducts to amino acids was similar to that of aflatoxin B 2 . , as epitope mapping indicated that modification at the 7,8 position only had minor influence on antibody recognition (8).
In an attempt to increase the sensiti vity of the assay, different concentrations of unmodified BSA were added. Albumin in the amount of 5 mg/incubation did not influence the quantification.
Blood samples were collected from workers with different levels of suspected exposure to material contaminated with aflatoxin B) according to job descriptions (table 1). Workers exposed to grain dust for more than 50 % of their workhours were classified as highly exposed, and those exposed for less than 25 % of the workday were considered to have low exposure. Two of the 45 blood samples collected from the workers upon return from vacation indicated recent exposure to aflatoxin B) (50 and 54 pg aflatoxin B I • mg " albumin), while all of the other samples were below the detection level of 5 pg alfatoxin B I . mg:' albumin. After four weeks of work 7 of 45 workers had detectable levels of aflatoxin B), a finding suggesting occupational exposure to aflatoxin B I (table 2). The mean level of the positive samples was ex-pressed as 64 ng . d-] . kg-1 body weight on the assumption that the exposure was equally distributed over the four weeks of work and the average body weight was 70 kg. All of the workers with exposure to aflatoxin B] were employed by company B, but with different job descriptions and different types of prescribed personal protection.
In the three-month period prior to the second blood sample, the level of aflatoxin B I in the raw material arriving in company A was less than 5 ug . kg". One shipment of peanut meal/coconut pills to company B had an aflatoxin content greater than 5 ug . kg-I, and another had an aflatox in B] level of 26 ug : kg-I . Three out of the five workers with the highest antigenicity helped to unload the latter cargo (table 3). One of the workers with a detectable level of aflatox in adducts to albumin but with suspected low exposure based upon his job description had a supervisory function at the grill where batches of raw material from different silos were dumped for use in production. Dust samples collected at this site had an aflatoxin B 1 content of 8 ug . kg-I dust. The aflatoxin B] content at the other sites of collection was generally low.

Discussion
Human exposure to airborne or particle-borne chemical compounds can be determined either by ambient air monitoring or by the detection of the compounds or their metabolites in body fluids. However, these measurements do not give any information on the potential biological effect of the exposure. Quantification of a carcinogenic adduct to protein or DNA is an indirect measure of the amount of the active metabolite formed in the organism (13). A linear relationship between hepatic aflatoxin B 1 adducts to DNA and the risk for developing liver cancer among rodents has been reported. In the present study the binding of aflatoxin B 1 to serum albumin has been used as the monitor. One of the advantages of using serum albumin rather than, for example, lymphocyte DNA is that the measured level represents the accumulated dose during the half-time of the serum albumin, as no repair pathways are known, whereas the half-time of aflatoxin adducts to DNA in experimental animals is less than 24 h. A good correlation between the binding of aflatoxin B] to serum albumin and liver DNA has been reported for experimental animals (14). This finding suggests that binding to serum albumin is a good monitor for the binding to target cell DNA even at low doses of aflatoxin B I . It has been estimated that approximately 5 0,70 of the administered dose binds to albumin (10). The major aflatoxin B 1 adduct to albumin has been identified as being formed between the active form of aflatoxin B 1 and lysine (15). With the use of affinity purification of albumin hydrolysate of aflatoxin B] in combination with radioimmunoassay, a correlation was observed between the intake of aflatoxin, as measured by food analysis, and aflatoxin adduct to lysine in a Chinese population (16). However, this assay gave an overestimate in comparison with affinity chromatography combined with high-pressure liquid chromatograph y with fluorescence detection (17). Aflatoxin B(  in the last 10 years so that the measurements made today are not representative of the period in which the liver cancer patients were exposed. As only a few of the trade union members are currently exposed, a lower number than I.7 cases/lO years is to be expected. In addition to its carcinogenic effect, aflatoxin B I has been suggested to influence the immune system (22). The efforts made by the companies to reduce the level of aflatoxin B I in the raw material, as well as encapsuling the work process, will remove some of the health hazards associated with work in the animal-feed manufacturing companies. Company adducts to lysine have not been detected in people living in Western Europe. In this paper, we report the presence of aflatoxin B I adducts to lysine in the serum albumin of some workers who have been unloading aflatoxin BI-contaminated raw material for animal-feed production. Aflatoxins are frequently found in respirable grain dusts that are common in grain silos and are produced by the handling of the raw material for animal feed pellets (3,18). Two of the workers had a detectable level of aflatoxin B I adducts to serum albumin upon return from vacation, but the adducts disappeared or their concentration dropped slightly in the subsequent four weeks. The source of this aflatoxin B I exposure is unknown, as the workers were neither involved in any other type of agribusiness during vacation nor had their own farms. It should be noted that one of these workers had only had two weeks of vacation and that the detectable level of aflatoxin Bj-albumin could have been caused by exposure prior to vacation. The half-time of albumin is more than three weeks. Only three out of five workers unloading the heavy contaminated cargos had a detectable level of exposure, although the same type of personal protection was in use . However, these workers implemented different job functions. Another explanation for the different adduct levels with the same exposure could be an interindividual difference in the metabolism of aflatoxin B I to biologically active metabolites or detoxified products (19). One of the workers in the silo at factory B had a fairly high level of aflatoxin B I adducts to albumin although his estimated exposure to dust particles was low, less than 25 070 of the workhours. He had a supervisory function controlling the ramp for unloading the raw material and grain for transport to the factory. Dust samples collected at this site showed a fairly high level of aflatoxin B, (8 ug . kg-I) compared with the I ugkg-lor nondetectable level at the other collection sites in the silo or factory . As such, there appears to be a good association between the level of aflatoxin B I adducts to albumin and exposure, as measured by the analysis of dust or raw material. An excess number of liver cancer cases has been reported in this group of workers (6), and attempts were made to explain the number of cases on the basis of the level of exposure to aflatoxin B I. The mean level of exposure, only including the individuals with detectable levels, was 64 ng aflatoxin B I . kg-I. d-I on the assumption that 5 % of the dose binds to albumin and that the exposure was equally distributed over the