Monitoring of aflatoxin exposure by biomarkers.

Epidemiological studies have demonstrated a strong association between exposure to AFB1 and an increased incidence of human hepatocellular carcinoma (HCC). This association has led to a need for accurate techniques relating AF exposure to an individual's risk of developing disease. With the understanding of the progressive processes of carcinogenesis, opportunities for the identification of molecular biomarkers reflecting events from exposure through clinical disease are provided. However, the development of biomarker methods to monitor human exposure to AFs requires techniques which are sensitive, specific, and amenable to large numbers of samples. To better understand the role of AF exposure with respect to HCC incidence, immunoassays for the biological quantitation of free AFB1, its metabolites, and its adduct macromolecules have been developed. ELISA appears to offer a suitable method for use in epidemiological studies for monitoring short-term exposure to AFs, as it has the appropriate sensitivity and specificity. However, the presence of substances that are presumably not AFs and which are inhibitory in the ELISA system has necessitated the development of purification techniques, usually based on adsorption onto Sep-Pak C18 cartridges and immunoaffinity chromatography. Many protocols have been developed for the assay of soluble AF metabolites in urine, milk and blood. However, these assays only indicate recent exposure, whereas the presence of albumin-AFB1 adducts in peripheral blood could present a useful material for assessing longer-term exposure. Among the various possible biomarkers of AF exposure, the measurements of AF-DNA and -protein adducts are of major interest because they are direct products of damage to a critical cellular macromolecular target. In Thailand, AF contamination of foods was reported to be high. More recent data using biomarkers as measures of AF exposure will be discussed. The data from epidemiological studies, AF exposure assessment using AF-albumin adduct and urinary AF level as exposure markers as well as the prevalence of p53 mutation at codon 249 are all suggestive of a limited importance of AF in the etiology of HCC in this country compared to other areas, including parts of Africa and China. These results also indicate that research on other potential hepatocarcinogens should not be neglected.

Aflatoxin and outcome from acute lower respiratory infection in children in The Philippines.

Aflatoxin is immunosuppressive in experimental conditions. This study addressed its potentially contributory role in the poor outcome of acute lower respiratory infections (ALRI) in children in The Philippines. The catchment area included peri-urban slums and middle-class housing. One hundred and fifteen children (mean age 2.1, range 0.08-12 years) were enrolled and their serum and urine obtained at presentation with ALRI. Aflatoxins in serum and aflatoxin metabolites in urine were measured by previously validated ELISA tests. Using the 1986 WHO criteria for the severity of ALRI, 31% had mild, 12% moderate, 49% severe and 8% severe complicated pneumonia. Eighty of 97 (82%) chest radiographs were abnormal. Ninety per cent of the children were below average weight for age, using Filipino standards, with a mean of 79% (range 27-157%). Thirteen (11%) children died. Aflatoxin in their serum, reflecting recent ingestion, was detected in 33%, with a mean positive value of 462 pg/ml. Aflatoxin metabolites (reflecting chronic ingestion) were detected in 64 of 65 urines collected, with a mean value of 0.1-4.77ng/ml. None of the children with detectable serum aflatoxin died. Anorexia and impaired consciousness were strongly associated with a poor outcome (prolonged fever or death). There was a strong association between undetectable serum aflatoxin concentrations and death (p = 0.004), perhaps reflecting anorexia. There was no relationship between the concentration of urinary aflatoxin metabolites and outcome. Serum was also obtained from 29 mothers on admission and none contained detectable aflatoxin. As virtually all the children had evidence of exposure to aflatoxin, a potentially immunosuppressive role in the context of pneumonia cannot be excluded.

PIP: Between December 1986 and January 1987 at the Research Institute for Tropical Medicine, a small hospital serving inhabitants of peri-urban slums and middle-class housing in Alabang, the Philippines, clinical researchers measured aflatoxin in the serum and urine of 115 children aged less than 13 who had a cough for less than three weeks (i.e., acute lower respiratory infection [ALRI]). They wanted to learn whether consumption of aflatoxin found in many foods in the Philippines could increase ALRI-related mortality among Filipino children. Almost all 115 children had probably been exposed to aflatoxin. 59% were admitted to the hospital. 11% of the hospitalized children died. No child died among those not admitted to the hospital. 73% of all children were severely malnourished. 82% had abnormal chest radiographs. 49% had severe ALRI, 31% mild ALRI, 12% moderate ALRI, and 8% severe-complicated ALRI. 67% of the children and none of the mothers had no detectable aflatoxin in their sera. The mean and median aflatoxin levels in the positive sera were 462 and 140 pg/ml, respectively (range, 20-5600 pg/ml). 64 of 65 sera had some aflatoxin metabolites (0.1-4.77 ng/ml). The mean aflatoxin metabolite/creatinine ratio was 1.27 (range, 0.19-4.43). Undetectable serum aflatoxin was associated with death (p = 0.006). Both anorexia and impaired consciousness level were significantly associated with death (p 0.001). The concentration of urinary aflatoxin metabolites had no apparent effect on outcomes. These findings do not support the hypothesis that aflatoxin acts as an immunosuppressant. Since almost all children tested had aflatoxin metabolites (indicating recent ingestion of aflatoxin), however, the researchers could not exclude aflatoxin's role as a potential immunosuppressant.

Impaired absorption of paracetamol in vegetarians.

1. The absorption and disposition of paracetamol was investigated in 10 healthy male Thai vegetarians and 10 similar non-vegetarians following an oral dose of 20 mg kg-1. 2. The absorption rate of paracetamol was significantly impaired in the vegetarians compared with the non-vegetarians as shown by a lower mean Cmax (11.7 +/- 1.4 vs 15.6 +/- 1.6 mg l-1; 95% confidence interval of the difference 2.49 to 5.36), increased tmax (median 1.75, range 0.75 to 3 h compared with 0.75 and 0.25 to 2 h) and an increase in the time for input of 50% of the total amount absorbed (0.54 +/- 0.38 compared with 0.20 +/- 0.10 h; 95% confidence interval of the difference 0.063 to 0.61). 3. A significantly lower total 24 h urinary recovery of paracetamol and metabolites (72.1 +/- 5.4 vs 86.4 +/- 5.4% of the dose; 95% confidence interval of the difference 8.0 to 20.6) indicated a decrease in the extent of absorption in the vegetarians also, although the total AUC values did not differ significantly between the two groups. 4. The plasma paracetamol half-life, partial metabolic clearances and fractional urinary excretion of the glucuronide, sulphate, cysteine and mercapturic acid conjugates of paracetamol were similar in the vegetarians and non-vegetarians.

Competitive ELISA.

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application in the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins.

Competitive ELISA.

Enzyme-linked immunosorbent assay (ELISA) is a very useful technique for the specific and sensitive assay of certain compounds, in which suitable antibodies, monoclonal or polyclonal, to the compounds are available. The technique has found particular application in the monitoring of environmental contaminants and toxins, either studying the primarily contaminated materials, e.g., foodstuffs, or body fluids of potentially exposed humans. The technique has been increasingly applied to monitoring the carcinogenic mycotoxins, the aflatoxins.

Investigation of the assay of AFB1-albumin adducts using proteolysis products in ELISA.

Epidemiological evidence of the involvement of aflatoxins in the aetiology of human liver cancer has led to an increasing interest in the development of appropriate techniques for monitoring human exposure. The assay for aflatoxin adducts in albumin has a better potential for assessing long-term exposure than analyses of urine samples, and several protocols for ELISA of these adducts, following proteolysis of albumin, have been examined. However, there is usually an incomplete release of a major adduct, aflatoxin-lysine, even after prolonged hydrolysis, and the adduct is very unstable under some conditions of proteolysis for unknown reasons. Therefore, before such techniques can be recommended for general application, the significance of such factors in the quantitive estimation of aflatoxin adducts needs to be evaluated. This study has detected the presence of a considerable fraction of aflatoxin-modified material, produced by proteolysis of in vivo aflatoxin-modified rat albumin or in vitro modified bovine albumin, and which is not recognized in ELISA by an anti-aflatoxin polyclonal antibody having a wide spectrum of aflatoxin metabolite detection. This fraction increases in parallel with the proteolysis protocols.

Possible effect of infection with liver fluke (Opisthorchis viverrini) on the monitoring of urine by enzyme-linked immunosorbent assay for human exposure to aflatoxins.

Several laboratories have initiated studies to assess human exposure to aflatoxin at an individual level by measuring aflatoxin metabolites in the urine by immunoassay. The fact that the antibodies recognize a variety of metabolites, albeit with differing affinities, means that any environmental factor that modifies the pattern of urinary metabolites associated with a given exposure could affect quantification in immunoassay. We have examined two such possible effects: (i) the pattern of metabolites after a dose of 14C-aflatoxin B1 in rats and (ii) the pattern of metabolites in hamsters and humans with and without exposure to liver flukes. We found no dose-related effect on the pattern of urinary metabolites over a 250-fold range, but there was a significant increase in the proportion of water-soluble aflatoxin metabolites in hamsters infected with liver fluke over that in uninfected animals. In human urine samples, there also appeared to be a difference in the metabolites in individuals infected with liver fluke from those in uninfected persons. These observations are relevant to both mechanistic and monitoring aspects of research into aflatoxins.

Ethnic differences in the renal sodium dopamine relationship. A possible explanation for regional variations in the prevalence of hypertension?

Over the last three years we have carried out studies on the urine output of both sodium and dopamine in five different ethnic groups: whites, Ghanaians, Zimbabweans, Iranians and Thais. Sodium was measured by ion specific electrode and dopamine by HPLC with electrochemical detection (using epinine as an internal standard). In several groups salt loading studies were also carried out. The five ethnic groups differed substantially with regard to the correlation between their urinary sodium and dopamine outputs. Three groups (whites, Thais and Zimbabweans) showed a strong positive correlation (P less than .001) and this may reflect their traditionally salt rich diet. In two groups (Ghanaians and Iranians) there was no correlation and this may reflect a salt scarce environment. Taken together with our previously reported studies showing that normotensive Ghanaians do not mobilize dopamine on salt loading, this would suggest that certain ethnic groups are predisposed to develop hypertension on salt loading--that is, they are 'salt sensitive.' This genetic trait may have passed from the West Coast of Africa, with the slaves, to America and the Caribbean. Other workers have reported deficiencies in vasodilator systems in the American black, such as dopamine, kallikrein and the renal prostaglandins. These defects may lead to the nosologic entity of 'low renin' hypertension, well described in American blacks, and could open up avenues of therapy based either on DA1 activators (such as fenoldopam) or on renal prodrugs (such as gludopa).

Ethnic differences in the renal sodium-dopamine relationship: a possible explanation for regional variation in the prevalence of hypertension?

Twenty-four-h urinary sodium and dopamine output by normotensive adults from 5 different ethnic groups have been measured. The groups differed substantially in the correlation between the urinary output to sodium and dopamine. Those with a traditionally salt rich diet (Thais, Caucasians, Zimbabweans) showed a strong positive correlation (p less than 0.001), whereas no such relationship was found in West Africans and Iranians, who come from traditionally salt scarce environments. It is hypothesised that in some races the lack of or uncoupling of the renal sodium-dopamine relationship, possibly as a mechanism to help conserve dietary sodium, predisposes to the development of hypertension when the individuals encounter a salt rich diet.

Further ethnic differences in the renal sodium-dopamine relationship: its uncoupling in Iranian but not in Thai normotensive subjects.

Dopamine is a natriuretic hormone and is synthesized in the kidney in response to a sodium load. This relationship results in a positive correlation between urinary sodium and dopamine outputs. Uncoupling of the renal sodium-dopamine relationship is reflected in a loss of this correlation and will result in the sluggish excretion of a sodium load. We measured 24-h urinary sodium and dopamine outputs in Thais and Iranians, who traditionally have very different dietary salt environments (salt-rich and salt-scarce, respectively). There was a highly significant positive correlation between sodium and dopamine in the Thais (r = 0.53, P less than 0.001) but no suggestion of such a correlation in the Iranians (r = 0.03). We hypothesize that in some races the uncoupling of the renal sodium-dopamine relationship, possibly as a mechanism to help conserve dietary sodium, predisposes the race to the development of hypertension when the individuals encounter a salt-rich diet.

Changes in plasma proteins in rats treated for short periods with hepatotoxins or with agents which induce cytochrome P450 isoenzymes.

We have compared the alterations in plasma proteins following treatment of rats with the centrilobular hepatotoxin carbon tetrachloride, the periportal hepatotoxin allyl alcohol and two inducers of hepatic microsomal drug metabolising enzymes, phenobarbitone and a mixture of polychlorinated biphenyls (Aroclor 1254). The results are compared with changes observed in animals treated with agents which caused proliferation of rat liver peroxisomes and described in an earlier publication. Our results indicate that: There is a marked induction of a minor glycoprotein which migrates as a prealbumin following treatment with both inducers of microsomal mixed function oxidases and inducers of peroxisome proliferation. There is a marked increase in a minor alpha 1-glycoprotein when there is chemically-induced mitosis in the liver but not in mitosis following liver damage. Two major alpha 1-glycoprotein are depressed in all forms of liver damage. There are indications of specific protein responses to allyl alcohol, to inducers of microsomal mixed function oxidases and to inducers of peroxisome proliferation.