Aflatoxin Excretion Research Articles

Feed additives containing sequestrant clay minerals and inactivated yeast reduce aflatoxin excretion in milk of dairy cows

The objective was to evaluate the efficacy of 2 dietary mycotoxin sequestrants, Toxy-Nil (TN) or Unike Plus (UP), in reducing aflatoxin (AF) M1 concentrations in milk of dairy cows challenged with dietary AF. Thirty-two mid-lactation Holstein cows were blocked by parity, days in milk, and milk yield and were randomly assigned within block to receive one of the following treatments: (1) 2.8 mg of AF/cow per d (positive control, PC), (2) 2.8 mg of AF + 100 g of TN/cow per d, (3) 2.8 mg of AF + 100 g of UP/cow per d, or (4) no AF and no additives (negative control, NC). For 7 d, treatments, dispersed in 150 g of sweet feed carrier, were top-dressed twice daily by mixing into the top portion of the TMR at each feeding. After the experimental period, cows were fed the NC diet and clearance of AFM1 via milk was monitored for 7 d. Feed and water were available ad libitum throughout the trial. Treatments had no effect on feed intake, milk yield, milk composition, or milk somatic cell count. Relative intake of AF was similar among PC, TN, and UP, averaging 106.5, 107.6, and 102.5 ± 2.9 μg/kg of diet dry matter, respectively. Relative intake of mycotoxin sequestrants was similar between TN and UP, averaging 0.4 and 0.4 ± 0.1% of diet dry matter, respectively. Concentration and mass of AFM1 secreted in milk and in urine were similar between TN and UP, but were lower than PC; concentrations in milk averaged 0.2, 0.3, and 0.6 ± 0.1 μg/kg, respectively, and mass secreted in milk averaged 8.1, 9.8, and 20.5 ± 1.7 μg/d. Concentrations in urine averaged 6.9, 7.4, and 14.2 ± 1.5 μg/L, respectively, and mass secreted in urine averaged 225.7, 250.8, and 521.6 ± 53.1 μg/d. Likewise, concentration and mass of free AF excreted in feces were similar between TN and UP, but were lower than PC; concentrations averaged 7.7, 8.9, and 12.4 ± 0.6 μg/kg, respectively, and mass excreted averaged 57.8, 69.6, and 95.6 ± 4.8 μg/d. Transfer of AF from feed to AFM1 in milk was reduced by 63 and 52%, and in urine, by 57 and 52% for TN and UP, respectively. Transfer of AF from feed to free AF in feces was reduced by 38 and 26% for TN and UP, respectively. The clearance rate of AFM1 in milk did not differ among PC, TN, and UP (46.1, 66.5, and 50.0 ± 6.7%/d, respectively). Results indicate that dietary inclusion of 100 g of TN or UP significantly reduced AFM1 in milk of cows consuming TMR containing approximately 105 μg of AF/kg of diet dry matter. Results also suggest that both TN and UP reduced absorption of AF.

Effect of supplementing lactating goats fed on aflatoxin contaminated feed with calcium bentonite and activated charcoal on aflatoxin M1 concentration, excretion and carryover in milk

Aflatoxin is a collective term for a group of toxic and carcinogenic secondary metabolites produced by some strains of Aspergillus flavus and Aspergillus parasiticus during growth, on feeds and foods. The fungal spores are found worldwide, in air and soil, and infest both living and dead plants and animals. An experiment was conducted to investigate the concentration, total excretion and carry-over of Aflatoxin B1 (AFB1) into milk as Aflatoxin M1. Nine crossbred lactating goats were divided into three groups of three each, based on the level of milk production. Commercial Aflatoxin B1 (AFB1) was administered to all groups at a rate of 100 ppb in the diet. Group I served as control (T1). In group II (T2), calcium bentonite (CaB) and in group III (T3), activated charcoal (AC), were added at the rate of 1% of Dry Matter Intake (DMI). Dry matter intake was not significantly different (P>0.05) among T1 (1.22), T2 (1.14) and T3 (1.13). Daily milk yield was also not significantly different (P>0.05) among treatments T1 (0.91), T2 (0.86) and T3 (1.03) during the experimental period of 14 days. The AFM1 concentration, excretion and carry-over of AFB1 in T1 continued to increase with time, whereas, the same was seen to decline in the adsorbent fed groups T2 and T3. The results suggest that supplementation of CaB or AC at 1% of DMI for lactating goats result in a reduction in AFM1 content in milk and carryover of aflatoxin from feed to milk without causing any change in composition of milk.Key words: Activated charcoal, aflatoxin, aflatoxin excretion, bentonite, milk contamination

Genetic or Pharmacologic Activation of Nrf2 Signaling Fails to Protect Against Aflatoxin Genotoxicity in Hypersensitive GSTA3 Knockout Mice

Mice are resistant to aflatoxin hepatotoxicity, primarily due to high expression of glutathione S-transferases (GSTs), and in particular the GSTA3 subunit. Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling, which controls a broad-based cytoprotective response, was activated either genetically or pharmacologically in an attempt to rescue GSTA3 knockout mice from aflatoxin genotoxicity. Genetic activation of Nrf2 signaling was attained in a GSTA3: hepatocyte-specific Keap1 double knockout (DKO) mouse whereas pharmacologic activation of Nrf2 was achieved through pretreatment of mice with the triterpenoid 1-[2-cyano-3-,12-dioxoleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) prior to aflatoxin B1 exposure. Following oral treatment with aflatoxin, urine was collected from mice for 24 h and hepatic and urinary aflatoxin metabolites then quantified using isotope dilution-mass spectrometry. Although Nrf2 was successfully activated genetically and pharmacologically, neither means affected the response of GSTA3 knockout mice to chemical insult with aflatoxin. Hepatic aflatoxin B1-N(7)-guanine levels were elevated 120-fold in GSTA3 knockout mice compared with wild-type and levels were not attenuated by the interventions. This lack of effect was mirrored in the urinary excretion of aflatoxin B1-N(7)-guanine. By contrast, urinary excretion of aflatoxin B1-N-acetylcysteine was >200-fold higher in wild-type mice compared with the single GSTA3 knockout or DKO mouse. The inability to rescue GSTA3 knockout mice from aflatoxin genotoxicity through the Nrf2 transcriptional program indicates that Gsta3 is unilaterally responsible for the detoxication of aflatoxin in mice.

Effectiveness of modified yeast cell wall extracts to reduce aflatoxin B1 absorption in dairy ewes

This study investigated the effect of a modified yeast cell wall extract preparation (YCW) on the excretion of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in feces, urine, and milk of dairy ewes fed an aflatoxin-contaminated diet. Sixteen ewes in mid-lactation were assigned to 4 treatment groups: control, AF (60μg of AFB1/kg of feed), YCW (2g/kg of feed), and AF+YCW. The trial consisted of a short-term (3-d) exposure period followed by a long-term (21-d) exposure period. At the end of each exposure period, milk, urine, and feces were collected over 72h. The treatments did not affect feed intake, milk production, milk composition, or body weight. The presence of AFM1 was detected in all matrices, whereas AFB1 was only present in feces. Daily excretion was higher following long-term exposure and reached 26.9μg of AFB1/d in feces, 37.2μg of AFM1/d in feces, and 10.7μg of AFM1/d in urine. Supplementation with YCW was effective in increasing aflatoxin excretion in feces in the long-term exposure (up to 156% increase). The effect was accompanied by a trend of decreasing urinary excretion of AFM1. In contrast, the addition of YCW to the contaminated diet did not affect the transfer of aflatoxins from feed to milk under the present experimental conditions with low-producing ewes. The transfer rates of AFM1 in milk ranged from 0.24 to 0.54%. In conclusion, feed supplementation with YCW reduced the absorption of AFB1 and increased the elimination of AFB1 and AFM1 in ewe feces. Yeast cell wall extract could be used to protect ruminants from chronic exposure to aflatoxins present in feeds.

Effects of chlorophyll and chlorophyllin on low-dose aflatoxin B(1) pharmacokinetics in human volunteers.

Chlorophyll (Chla) and chlorophyllin (CHL) were shown previously to reduce carcinogen bioavailability, biomarker damage, and tumorigenicity in trout and rats. These findings were partially extended to humans, where CHL reduced excretion of aflatoxin B(1) (AFB(1))-DNA repair products in Chinese unavoidably exposed to dietary AFB(1). However, neither AFB(1) pharmacokinetics nor Chla effects were examined. We conducted an unblinded crossover study to establish AFB(1) pharmacokinetic parameters among four human volunteers, and to explore possible effects of CHL or Chla cotreatment in three of those volunteers. For protocol 1, fasted subjects received an Institutional Review Board-approved dose of 14C-AFB(1) (30 ng, 5 nCi) by capsule with 100 mL water, followed by normal eating and drinking after 2 hours. Blood and cumulative urine samples were collected over 72 hours, and 14C- AFB(1) equivalents were determined by accelerator mass spectrometry. Protocols 2 and 3 were similar except capsules also contained 150 mg of purified Chla or CHL, respectively. Protocols were repeated thrice for each volunteer. The study revealed rapid human AFB(1) uptake (plasma k(a), 5.05 + or - 1.10 h(-1); T(max), 1.0 hour) and urinary elimination (95% complete by 24 hours) kinetics. Chla and CHL treatment each significantly impeded AFB(1) absorption and reduced Cmax and AUCs (plasma and urine) in one or more subjects. These initial results provide AFB(1) pharmacokinetic parameters previously unavailable for humans, and suggest that Chla or CHL co-consumption may limit the bioavailability of ingested aflatoxin in humans, as they do in animal models.

Effect of Naturally Contaminated Feed with Aflatoxins on Performance of Laying Hens and the Carryover of Aflatoxin B1 Residues in Table Eggs

The aim of this study was to evaluate the effect of naturally contaminated feed with aflatoxin on performance of laying hens fed for 60 days and the carryover of AFB residues in eggs as well as the stability 1 of AFB in naturally contaminated eggs to boiling process. Forty, 30 weeks old, White Leghorn laying hens 1 were randomly assigned into four experimental groups and after 2 weeks were given naturally contaminated feed containing zero (control), 25, 50 and 100 μg aflatoxin/kg feed. Twenty eggs per treatment were collected on days (1-7); 10; 20, 30, 40, 50 and 60 and submitted to aflatoxin B analysis using ELISA. Average egg 1 production and egg weight were not affected by aflatoxin (P>0.05), while a significant decrease in feed intake (p<0.05) was appeared in the 2 groups fed on 50 and 100 aflatoxin ug/kg feed. Residues of aflatoxin B were 1 detected in eggs at levels that ranged from 0.02 to 0.09 with a mean value of 0.04, 0.05 and 0.07 μg/kg respectively. Aflatoxin B was almost stable in naturally contaminated egg for up to 20 minutes of boiling, so 1 avoiding aflatoxin B transmission into egg appears to be the only practical way to ensure their safety for 1 human consumption. Conclusively, the excretion of aflatoxin B residues in hens' eggs might occur at 1 relatively low concentrations under conditions of long term exposure of laying hens to low level of aflatoxin in naturally contaminated feed with reduction in feed intake started at 50 μg/kg.

AFLATOXIN EXCRETION THROUGH MILK

In the period January to June 2006 the samples of feed were collected from feed factories in Southern Baåka and Srem district. The samples of raw milk and full mix were taken from 5 dairy farms. A total of 50 raw milk samples was examined. The samples were examined on the presence of aflatoxin B1 using the method of thin layer chromatography (TLC) and simultaneously, using ELISA tests. Milk samples were examined using immunoenzyme tests for the presence of aflatoxin M1. Aflatoxin content in all the examined feed and mix samples was below LOD (limit of detection) of TLC method, also this content was below MRL according to ELISA method. In total of 50 samples of raw milk, aflatoxin M1 was detected in two samples originating from different farms. Aflatoxin was detected in 7.5 ng/l, i.e. 10 ng/l respectively, what is considerably lower than MRL. Based on the obtained results it is considered that obligatory control of raw milk for the presence of aflatoxin is necessary.

Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China

In vitro and in vivo studies suggest that selected strains of probiotic bacteria can form tight complexes with aflatoxin B(1) and other carcinogens. The aim of the present study was to determine whether administration of probiotic bacteria could block the intestinal absorption of aflatoxin B(1) and thereby lead to reduced urinary excretion of aflatoxin B(1)-N(7)-guanine (AFB-N(7)-guanine), a marker for a biologically effective dose of aflatoxin exposure. Elevated urinary excretion of this aflatoxin-DNA adduct is associated with an increased risk of liver cancer. Ninety healthy young men from Guangzhou, China, were randomly assigned to 2 groups; one group received a mixture of Lactobacillus rhamnosus LC705 and Propionibacterium freudenreichii subsp. shermanii strains 2 times/d for 5 wk, and the other group received a placebo preparation. The subjects provided 4 urine samples: at baseline, at 3 and 5 wk after starting the supplementation, and at the end of the 5-wk postintervention period. The percentage of samples with negative AFB-N(7)-guanine values tended to be higher in the probiotic group than in the placebo group during the 5-wk intervention period (odds ratio: 2.63, P = 0.052), and a statistically significant decrease in the concentration of urinary AFB-N(7)-guanine was observed in the probiotic group. The reduction was 36% at week 3 and 55% at week 5. The geometric means for the probiotic and placebo groups were 0.24 and 0.49 ng AFB-N(7)-guanine/mL, respectively, during the intervention period (P = 0.005). A probiotic supplement reduces the biologically effective dose of aflatoxin exposure and may thereby offer an effective dietary approach to decrease the risk of liver cancer.

Fecal and urinary excretion of aflatoxin B1 metabolites (AFQ1, AFM1 and AFB‐N7‐guanine) in young Chinese males

Our study was designed to assess the fecal and urinary excretion of 3 aflatoxin B1 (AFB1) metabolites, aflatoxins M1 (AFM1) and Q1 (AFQ1) and aflatoxin B1-N7-guanine (AFB-N7-guanine) that are produced by the predominant forms of cytochrome P450 enzymes responsible for the biotransformation of AFB1. Fecal and urinary AFM1, AFQ1 and urinary AFB-N7-guanine were assessed in 83 young Chinese males selected from a larger population (n = 300) based on detectable urinary AFM1. The concentration of fecal AFQ1 (median 137 ng/g fresh weight, IQR 9.1 to 450) was approximately 60 times higher than that of AFM1 (2.3 ng/g, IQR 0.0 to 7.3). In urine, the median AFQ1 was 10.4 ng/ml (IQR 3.4 to 23.3), and the median AFM1 and AFB-N7-guanine 0.04 ng/ml (IQR 0.01 to 0.33) and 0.38 ng/ml (IQR 0.0 to 2.15), respectively. A subgroup (n = 14) with hepatitis B virus (HBV) infection had significantly higher fecal concentrations of AFQ1 (p = 0.043) and AFM1 (p = 0.001) than those who were hepatitis B-virus antigen (HBsAg) negative, and the respective differences in urinary AFQ1 and AFM1 concentrations approached statistical significance (p = 0.054, p = 0.138). Our study demonstrates that AFQ1 is excreted in urine and feces at higher levels than AFM1, and feces are an important route of excretion of these AFB1 metabolites. AFQ1 should be further assessed for its predictive value as a marker for exposure and risk of dietary aflatoxins.

Prevalence of Aflatoxins in Blood and Urine of Egyptian Infants with Protein–Energy Malnutrition

The aim of the present work was to study the presence of aflatoxins in blood and urine of infants with protein-energy malnutrition (PEM). The study was conducted on 60 infants, 30 with kwashiorkor and 30 with marasmus, with 10 age-matched healthy infants studied as a control group. Complete blood count, liver function tests, and determination of the level of aflatoxins (B1, B2, G1, G2, M1, M2, G2a, B3, GM1, P, and aflatoxicol R0) in blood and urine were carried out in all studied infants. Serum aflatoxins were detected in more infants with kwashiorkor (80%) than in those with marasmus (46.7%). The mean serum levels of total aflatoxins, AFB1, AFG1, and AFB2a, were significantly higher in infants with kwashiorkor (p <.001). Aflatoxin B1 (AFB1) was the most commonly detected type. The prevalence of aflatoxin excretion in the urine of infants with kwashiorkor was 80%, a higher value than that in infants with marasmus (46.7%). The mean urinary concentration of total aflatoxins followed the same pattern of distribution (p < .052). There were no significant differences between groups in the mean urinary concentrations of AFB1, AFG1, AFB2a, AFM1, and AFG2a. Aflatoxins were not detected in any of the serum or urine samples of the control group. Aflatoxins are highly prevalent in this study population and show a high degree of correlation with severe PEM.

Chemoprevention with chlorophyllin in individuals exposed to dietary aflatoxin

Because of the multiplicative interaction between dietary aflatoxins and hepatitis B virus infection in the etiology of liver cancer, efforts to reduce the consequences of either chemical or viral component are likely to have substantial public health benefit. Chlorophyllin (CHL), a water-soluble form of chlorophyll, was recently evaluated as a chemopreventive agent in a population at high risk for exposure to aflatoxin and subsequent development of hepatocellular carcinoma. CHL, which is used extensively as a food colorant and has numerous medicinal applications, is an effective anticarcinogen in experimental models including aflatoxin-induced hepatocarcinogenesis. CHL is thought to form molecular complexes with carcinogens, thereby blocking their bioavailability. In the clinical trial, administration of CHL three times a day led to a 50% reduction in the median level of urinary excretion of aflatoxin- N 7-guanine compared to placebo. This excreted DNA adduct biomarker is derived from the ultimate carcinogenic metabolite of aflatoxin B 1, aflatoxin-8,9-epoxide, and is associated with increased risk of developing liver cancer in prospective epidemiologic studies. Compliance in the intervention was outstanding and no toxicities were observed. Thus, CHL has been found to be a safe and effective agent suitable for use in individuals unavoidably exposed to aflatoxins.

Enhancement of biliary excretion of aflatoxin B 1 and suppression of hepatic ornithine decarboxylase activity by 2-(allylthio)pyrazine in rats

2-(Allylthio)pyrazine (2-AP), a synthetic pyrazine derivative with an allylsulfur moiety, has protective effects against chemically-induced hepatic toxicity. Previous studies have shown that 2-AP significantly reduces the formation of preneoplastic foci in rats exposed to aflatoxin B 1 (AFB 1). The present study was designed to determine whether 2-AP could increase the biliary excretion of metabolites of AFB 1 in rats treated with this carcinogen and whether the agent could alter the activity of ornithine decarboxylase (ODC), which is considered to be associated with tumor promotion. Rats were pretreated with 2-AP (p.o.) at a daily dose of 50 mg/kg for 5 consecutive days. AFB 1 (5 mg/kg) was administered intraperitoneally 2 h after the last dose of 2-AP. Amounts of principal AFB 1 metabolites, AFB 1-glutathione and a glucuronide conjugate secreted in bile juice was increased by 56 and 50%, respectively, after the 2-AP treatment. Levels of radiolabelled AFB 1 covalently bound to calf thymus DNA catalyzed by microsomes obtained from 2-AP-treated rats (10 and 50 mg/kg, for 5 days) were reduced by 47 to 66%. ODC activity in AFB 1-treated rats was determined by the three-step medium-term hepatocarcinogenesis assay. Rats were treated with 2-AP at the daily doses of 10, 25 and 50 mg/kg for 16 consecutive days. During this period, four repeated doses of AFB 1 (1.0 mg/kg) were given to the animals. Rats were then subjected to two-third partial hepatectomy, followed by administration of phenobarbital. 2-AP inhibited AFB 1-induced ODC activity by 40 to 66%, as determined at the 44th day. Inhibition of AFB 1-induced ODC activity by 2-AP in conjunction with acceleration of AFB 1 elimination through metabolic conjugation may contribute to its chemopreventive effects against this carcinogen.

Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal reduce urinary excretion of aflatoxin M 1 in turkey poults. Lack of effect by activated charcoal on aflatoxicosis

In one experiment, the effect of inorganic sorbents on the metabolic fate of aflatoxin B 1 (AFB 1) was studied in turkey poults. At 5 weeks of age, female poults were surgically colostomized and 9 days later orally dosed with 0.75 mg AFB 1/kg BW. Hydrated sodium calcium aluminosilicate (HSCAS), acidic HSCAS, and activated charcoal (AC) were tested, by concomitant administration with AFB 1. Urine was collected up to 48 h post-dosing and analyzed for aflatoxin M 1 (AFM 1) which was the major metabolite found in all treatment groups. Hydrated sodium calcium aluminosilicate, previously proven beneficial in alleviating aflatoxicosis in farm animals, reduced urinary AFM 1 output when orally dosed simultaneous with AFB 1. Also, acidic HSCAS and AC significantly decreased AFM 1 excretion when administered concomitantly with AFB 1. A second experiment was conducted to evaluate the ability of two types of AC to modify aflatoxicosis when added to aflatoxin (AF)-contaminated (from culture material) diets of turkey poults. Although AC was able to decrease AFM 1 excretion in the first experiment, no protective effects from AF toxicity were observed in the feeding study.

Urinary aflatoxin levels, hepatitis-B virus infection and hepatocellular carcinoma in Taiwan.

Using a urinary immunoassay to measure aflatoxin metabolites, we examined the associations between exposure to aflatoxin, chronic infection with the hepatitis-B virus (HBV) and background rates of hepatocellular carcinoma (HCC) mortality in a cross-sectional survey of 250 residents from 8 areas of Taiwan with a 4-fold variation in age-adjusted HCC mortality. Specimens of fasting blood and overnight urines were used to determine HBV carrier status and excretion of aflatoxin in the subjects surveyed. While the prevalence of hepatitis-B virus carriers showed moderate variability, there was a 500-fold range in urinary aflatoxin levels. Mean log-transformed levels of aflatoxin metabolites were similar in males and females and in HBV carriers and non-carriers. In the 8 townships, HCC mortality correlated positively with both area HBV carrier prevalence and mean aflatoxin levels. The primary analyses, however, were conducted at the individual level. Each subject's aflatoxin level was treated as the response variable in a multiple regression model, and the corresponding sex-specific area HCC rate was included as a predictor along with the individual's carrier status, age and sex; alcohol consumption and cigarette smoking were also considered. In these analyses, a significant association was again observed between the marker of aflatoxin exposure and the background rate of HCC mortality. In females, the slope of the regression line was somewhat steeper in HBV carriers, but this pattern was not seen in males and formal testing yielded no statistically significant evidence of an interaction. Our findings are consistent with the hypothesis that aflatoxin plays an independent role in hepatocellular carcinoma in Taiwan.

Intestinal absorption and excretion of aflatoxin in rats

Aflatoxin B 1 (AFB 1) transfer across the gastrointestinal tract was studied in rats. The rate of biliary secretion of 3H was higher when [ 3H]AFB 1 was injected into the small intestine than when injected into the stomach. When various sites of the small intestine were perfused with the medium containing [ 3H]AFB 1, the highest rate of disappearance of 3H from the medium was noted in the duodenum. Also the rate of biliary secretion of 3H tended to be higher when the duodenum was perfused than when the other sites were perfused. These results suggest that AFB 1 is absorbed mainly from the small intestine, most efficiently from the duodenum. Uptake of AFB 1 by the everted intestine in vitro was slightly greater in the jejunum than the other sites, suggesting that the cause of the differences in the intestinal absorption among various sites may reside in the transfer process of AFB 1 from the epithelial cell layer to vascular circulation. Comparison of the AFB 1 appearance in the mesenteric venous plasma and lymph showed that AFB 1 is absorbed almost exclusively in the vein. Distribution of 3H in the mesenteric plasma on thin-layer chromatography revealed that metabolic degradation of AFB 1 takes place in the duodenal and jejunal tissues during the course of AFB 1 absorption. Examination of the appearance of AFB 1 or 3H in the intestinal perfusate after iv injection of AFB 1 or [ 3H]AFB 1 suggested that AFB 1 and its metabolites can transfer from the blood to the intestinal lumen. The rate of the AFB 1 absorption from various sites of the intestine changed with age and reproductive stage, indicating that the AFB 1 transfer across the intestinal wall is under the influence of the growth and reproductive endocrine condition.

Serum albumin adducts in the molecular epidemiology of aflatoxin carcinogenesis: correlation with aflatoxin B1 intake and urinary excretion of aflatoxin M1.

Aflatoxin-serum albumin adducts in the blood of 42 residents of Guangxi Province, People's Republic of China, were determined and compared with intake of aflatoxin B1 (AFB1) and excretion of aflatoxin M1 (AFM1) in urine. Blood specimens were obtained during the same period that urine was collected and that diet was sampled. Serum albumin was isolated from blood by affinity chromatography on Reactive Blue 2-Sepharose and subjected to enzymatic proteolysis using Pronase. Immunoreactive products were purified by immunoaffinity chromatography and quantified by competitive radioimmunoassay. A highly significant correlation (r = 0.60, P less than 0.00003) of adduct level with AFM1 excretion was observed. An equally highly significant correlation of adduct level with intake (r = 0.69, P less than 0.000001) was also observed. From the slope of the regression line for adduct level as a function of intake, it was calculated that 1.4-2.3% of ingested AFB1 becomes covalently bound to serum albumin, a value very similar to that observed when rats are administered AFB1.

Effects of single-dose and repeated-dose pretreatment with 2(3)-tert-butyl-4-hydroxyanisole (BHA) on the hepatobiliary disposition and covalent binding to DNA of aflatoxin B 1 in the rat

The effects of two distinctive BHA pretreatment regimens on the biliary excretion of aflatoxin B 1 (AFB) metabolites and the covalent binding of AFB to hepatic DNA were studied in vivo in the rat. To differentiate between enzyme induction effects and direct antioxidant effects, BHA was given to rats for 9 days (500 mg/kg/day, sc) or as a single dose (500 mg/kg, po), and [ 3H]AFB was administered ip. Repeated treatment with BHA enhanced the biliary excretion of both the glutathione conjugate of AFB and the AFP 1-glucuronide to 200% of control values, reduced the amount of AFB remaining in the liver to 53% of control and reduced the covalent binding of AFB to hepatic DNA to 16% of control. A single BHA treatment had no effect on the biliary excretion of AFB or the binding of AFB to hepatic macromolecules, even though high concentrations of BHA were present in the liver during the period of AFB metabolism. These results support the hypothesis that BHA inhibits AFB carcinogenesis via the induction of phase II biotransformation pathways such as glutathione S-transferase, which act to reduce the amount of AFB-epoxide available for binding to DNA. We found no evidence of a direct antioxidant effect of BHA in altering the hepatobiliary disposition of AFB.

Urinary excretion of thiol conjugates of aflatoxin B 1 in rats and hamsters

After intraperitoneal injection of [ 3H]aflatoxin B 1 in rats and hamsters, 24-h urinary excretion of aflatoxin B 1 metabolites was examined in these species. Total excretion was 10–15% of the injected dose with hamster excreting 50% more than the rat. In both species, 60% of the total excretion was due to glucuronide and sulfate conjugates of hydroxy metabolites. Striking differences were observed in the excretion of both aflatoxin B 1-diol and various thiol conjugates. Aflatoxin B 1-glutathione and aflatoxin B 1-cysteinylglycine were major thiol conjugates in rats and hamsters, respectively. An unknown metabolite believed to be mercapturic acid ( N-acetylcysteine conjugate) of aflatoxin B 1 was excreted by both species, hamster excreting twice as much as the rat.

Effect of manipulation of gamma-glutamyl transpeptidase levels on biliary excretion of aflatoxin B1 conjugates.

The reactive intermediate of aflatoxin B1 (AFB1) forms a glutathione conjugate (AFB1-GSH) and this has been shown to be a substrate for gamma-glutamyl transpeptidase (GGT) in vitro. This study describes the biliary excretion of AFB1-GSH and the product of GGT activity, the cysteinylglycyl conjugate (AFB1-Cys-Gly), following i.v. injection of AFB1 (5 mumol kg-1) in control male rats and in rats that had been maintained on a toxic diet containing 4 p.p.m. AFB1 for 10-12 weeks prior to the experiment. AFB1 metabolites in the bile were analyzed by reverse phase h.p.l.c. and GGT activity in the liver was assessed histochemically and by quantitative fluorimetric assay. In the control male rats (n = 6) AFB1-GSH and AFB1-Cys-Gly together were detected as 4.2 +/- 2.3% of the i.v. dose over the first two hours of bile collection (AFB1-GSH:AFB1-Cys-Gly, 5.5:1). GGT activity (38.2 +/- 7.9 nmol product formed/g liver) was located in the bile duct epithelium. The group maintained on a toxic diet (n = 2) showed higher levels of AFB1-Cys-Gly (AFB1-GSH:AFB1-Cys-Gly, 1:1). GGT activity was elevated (5-10 x control levels) and located in numerous foci throughout the liver. The involvement of GGT in the biliary excretion of AFB1-Cys-Gly was demonstrated by in vivo inhibition of GGT by administering AT125 to a group of animals (n = 3) 15 min prior to the injection of AFB1. Histochemical and quantitative estimation of GGT confirmed total inhibition throughout the liver and conversion of AFB1-GSH to AFB1-Cys-Gly was almost completely blocked. Female Fischer 344 rats (n = 3) showed slightly elevated AFB1-Cys-Gly excretion and higher GGT activity (79.3 +/- 26.3 nmol/min/g liver) compared to control male rats.

Urinary excretion of aflatoxin M1 after administration of aflatoxin B1 in sucrose- or starch-rich diets.

1. Male Sprague--Dawley rats were given 630 g/kg sucrose or starch with 2 mg/kg aflatoxin b1 for periods of 75, 145 and 200 d, and the 24 h urinary excretion of aflatoxin M1 was measured. 2. Less aflatoxin M1 was excreted by the rats fed on the sucrose-rich diet compared to those fed on the starch-rich diet. This difference was especially marked when expressed per g metabolizing tissue. 3. It is concluded that sucrose probably decreases the activity of aflatoxin B1 metabolism in a similar way to its previously found effect on the drug-metabolizing enzyme.