A. A. Frohlich

Comparison of toxicity of different mycotoxins to several species of bacteria and yeasts: use of Bacillus brevis in a disc diffusion assay

Twelve species of bacteria and two species of yeast were tested for sensitivity against 11 different mycotoxins using a disc diffusion assay. Among the bacterial species, Bacillus brevis appeared to be the most sensitive microorganism, being sensitive to eight mycotoxins (AFB1, ochratoxin A [OA], citrinin [CT], patulin [PAT], penicillic acid [PA], cyclopiazonic acid [CPA], penitrem A [PT-A], and zearalenone [ZEE]). This microorganism was not affected by a high concentration (500 μg per disc) of any of the trichothecenes (T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and deoxynivalenol). Kluyveromyces marxianus , a species of yeast, was the only microorganism that was inhibited by all four of the trichothecenes but was not inhibited by the other mycotoxins. The area of the inhibition zone produced by some of the mycotoxins such as OA with the B. brevis assay was dramatically influenced by the pH of the medium, while the toxicity of other mycotoxins such as AFB1 was relatively pH independent. The sensitivity of the B. brevis assay also tended to decrease at agar volumes above 6 ml and as the number of microbes per plate increased. The lowest amounts of the different ochratoxins; OA, OB and OC (OA ethyl ester) that could be detected under optimal conditions were 0.5, 20, and 2 μg per disc, respectively. The lowest amounts of CPA, AFB1 CT, PAT, PA, PT-A, and ZEE that were detectable were 0.5, 1, 1, 1, 1, 1, and 10 μg per disc, respectively. These results demonstrate that B. brevis can be used as a positive indicator organism to detect the presence of several common non-trichothecene mycotoxins. The results demonstrate that, used together, B. brevis and K. marxianus can be used as indicator organisms in a bioassay approach to the detection of several of the most common mycotoxins.

Effect of T-2 toxin on in vivo lipid peroxidation and vitamin E status in mice

The effects of an acute administration of T-2 toxin on vitamin E status and the corresponding degree of lipid peroxidation, as determined by the plasma and organ content of malondialdehyde (MDA), was studied in mice. The effects of T-2 toxin administration on the body weight and weights of liver, spleen and thymus were also assessed. T-2 toxin was administered in doses ranging from 1 to 6.25 mg/kg body weight, depending on the experiment, while the dietary content of vitamin E ranged from near 0 to 5000 IU/kg. There was a significant decrease in vitamin E content of plasma after the administration of the toxin with the concentrations remaining low for periods as long as 48-72 h. MDA content of liver increased significantly after 24-48 h of toxin administration in contrast to the controls. However, MDA levels returned to the control range after 72 h. The concentrations of MDA in liver were inversely related to the vitamin E content of the diet, and were always higher for the toxin-treated animals (significant linear regression between MDA content of liver and the log10 of vitamin E content of the diet). Weights of spleen and thymus decreased after T-2 toxin administration; however, the weight of liver either increased or did not change in the different experiments. In conclusion, T-2 toxin treatment of mice increased lipid peroxidation in the liver as measured by MDA production. This process was maximal after 48 h of T-2 challenge, and decreased thereafter. Plasma alpha-tocopherol levels decreased as soon as 6 h after the toxin challenge, while MDA did not increase until there was a severe depletion of vitamin E. These changes were accompanied by decrease in weight of spleen and thymus.

Identification of ochratoxins and some of their metabolites in bile and urine of rats

The objectives of this study were to develop and evaluate procedures for the confirmation of ochratoxin A (OA), lactone opened OA (OP-OA), ochratoxin B (OB), hydroxy OA (OA-OH) and ochratoxin alpha (Oalpha) and metabolites formed in the rats from these toxins, and to demonstrate that many ochratoxin metabolites can be identified in the bile and urine of rats injected with the different ochratoxins. An esterification procedure in acidified methanol and a lactone hydrolysis procedure in strong base yielded two additional forms of most of the different ochratoxins. The esterification procedure provided a simple, fast and reliable method for the confirmation of the ochratoxins. A total of 20 different metabolites of OA, OP-OA, OB, OA-OH and Oalpha were detected in the urine and the bile of rats of which several were identified. Among these, OA and the recently discovered and toxic form of OA (OP-OA) were readily formed in vivo when either were injected. Procedures developed in this study can be used to confirm and isolate ochratoxins in biological samples and have shown that a new form of OA (OP-OA) along with many other metabolites are formed from OA and related ochratoxins in vivo.

Hydrolysis of ochratoxin A by the microbial activity of digesta in the gastrointestinal tract of rats.

This study established the influence of dietary neomycin sulphate on the rate of hydrolysis of ochratoxin A (OA) by digesta from the intestine, and its effect on the excretion of OA and its hydrolyzed metabolite, alpha ochratoxin (Oα), in the urine and feces of the rat. The first in vitro study demonstrated that digesta from the cecum and the large intestine were able to hydrolyze OA whereas digesta from the small intestine and stomach had very low hydrolytic activity against this substrate. Homogenates of the liver had no hydrolytic activity. The second in vivo study demonstrated that digesta from the large intestine and cecum of the neomycin treated rats was much less effective (P<0.001) in promoting the hydrolysis of OA than digesta from the control rats. Neomycin when added directly to the in vitro system, however, did not affect the rate at which OA was hydrolyzed. In a third study, OA was administered in vivo to control and neomycin-treated rats. Rats fed the neomycin containing diet compared to those fed the control diet had a higher concentration (P<0.005) of blood OA, and a greater cumulative excretion of OA plus Oα over the entire 5 day collection period in the feces (P<0.0001) and a corresponding decrease in the cumulative excretion of OA plus Oα in the urine (P<0.0001). Individually, there was a marked increase in cumulative fecal excretion of OA (P<0.05) and a corresponding decrease in excretion of Oα (P<0.05). Individual OA and Oα values in the urine tended to follow an opposite pattern to that seen in the feces but the differences were not significant (P<0.05). Overall, the results demonstrate that an antibiotic such as neomycin when added to the diet greatly reduces the rate of hydrolysis of OA by digesta from the lower sections of the gastrointestinal tract. Neomycin also alters the pattern of excretion of OA and Oα in the feces and possibly the urine in rats fed OA. These results suggest that intestinal microorganisms affect disposition of OA.

The incidence and distribution of ochratoxin A in western Canadian swine

A survey of swine destined for slaughter in Manitoba was conducted to examine the incidence of ochratoxin A (OA) in swine herds from different regions of Manitoba throughout the year 1989-90. Thirty-six percent of the serum samples which were collected from 1600 pigs contained detectable levels of OA. The identity of this toxin was confirmed using liquid chromatography-mass spectrometry and enzymatic hydrolysis. There was a significant effect of the region from which the herds originated, as well as the season in which the samples were collected on both the incidence (p < 0.001) and concentration of OA (p < 0.001). In July, 65% of the samples contained detectable levels of OA, compared with 38, 21 and 17%, in April, October and January respectively. Furthermore, 24% of the samples collected in July contained greater than 15 ng/ml of OA, while only 2, 9, and 1% of the samples collected in April, October and January respectively, contained greater than 15 ng/ml of OA. Based on the six samples collected from each herd, it appears that the presence and concentration of OA within a herd may be estimated from a limited number of animals per herd.

Dietary cholestyramine reduces ochratoxin A-induced nephrotoxicity in the rat by decreasing plasma levels and enhancing fecal excretion of the toxin

Ochratoxin A (OTA) is a mycotoxin that may contaminate animal feed (oat, barley, and rye) and food (wheat, rice, coffee, beer, pig meat), leading to major health problems (e.g., nephropathy) in several animal species including humans. Several methods have been tested to reduce the toxicity of OTA in animals but with limited success. In rats, the effect of cholestyramine (CHA), a bile acid-binding resin, was investigated on OTA-induced nephrotoxicity and bioavailability. Animals were fed semisynthetic diets containing two levels of OTA: 1 or 3 ppm. At each level of OTA, the diets were enriched with 0.1, 1, and 5% of CHA. The results showed that CHA decreased the concentration of OTA in plasma. At 1 and 3 ppm of OTA in the diet, CHA is effective at a level of 0.1% and 5%, respectively. The excretion of OTA and its metabolites (ochratoxin alpha and hydroxylated ochratoxin A) in bile and urine was also decreased by addition of 5% CHA in the diet. This was associated with an increase of OTA excretion in feces. Enzymuria and renal morphology revealed that dietary CHA can decrease OTA-induced nephrotoxicity, probably by reducing renal exposure to the toxin. In conclusion, CHA can reduce OTA concentrations in plasma as well as reducing nephrotoxicity, which may be attributed to a decrease of bioavailability and/or enterohepatic circulation of the toxin.

Cyclic AMP levels in various tissues of the domestic fowl (Gallus domesticus) as affected by glucagon, epinephrine and insulin.

Abstract 1. 1. The effects of glucagon, epinephrine and insulin on the in vivo time course changes in blood glucose and in blood, muscle and liver cyclic AMP levels of the domestic fowl have been studied. 2. 2. Intravenous injections of glucagon and epinephrine produced a rapid increase in blood cyclic AMP (10- and 6-fold, respectively) followed by a more gradual increase in blood glucose. The patterns of response, however, in blood, muscle and liver, following either glucagon or epinephrine administration were different. Glucagon markedly elevated hepatic tissue levels while it had only a minimal effect on muscle. Epinephrine, in contrast, elevated cyclic AMP levels to a similar extent in both tissues. The decrease in peak cyclic AMP levels following epinephrine treatment was biphasic for both muscle and liver. 3. 3. The time course patterns for the rise and fall of cyclic AMP in liver, muscle and blood were nearly the same suggesting a rapid transfer of tissue cyclic AMP to blood. Maximal cyclic AMP levels occurred 5–10 min and 10–20 min after intravenous injections of epinephrine and glucagon, respectively. 4. 4. Insulin depressed blood glucose levels but elevated blood, muscle and liver cyclic AMP levels. The cyclic AMP pattern following insulin injection is different than that observed in mammals.

Effect of dietary cholestyramine on the elimination pattern of ochratoxin A in rats

Three experiments with rats established the effects of dietary cholestyramine on the disposition of ochratoxin A (OA) and its hydrolysed metabolite, alpha-ochratoxin (O alpha). In the first experiment OA (1 mg/kg) was incorporated into a diet that contained 0, 0.5, 1.0 and 2.0% cholestyramine. Cholestyramine markedly reduced blood concentrations of OA (1.6 to 0.75 micrograms/ml, P less than 0.0001) for all concentrations of the resin. The second experiment demonstrated that 2% cholestyramine added to the diet of rats markedly reduced cumulative urinary OA excretion (26 to 6 micrograms, P less than 0.01) and increased cumulative faecal OA excretion (8 to 38 micrograms, P less than 0.001). The third experiment established the efficacy of cholestyramine (2%) when added to diets containing two concentrations (0 and 6%) of a saturated fat (tallow). The bioavailability of OA as determined by area under the blood concentration curve over 216 hr was 424 micrograms/ml/hr for the control rats and 186 micrograms/ml/hr for the cholestyramine-treated rats (P less than 0.0001). Cholestyramine treatment increased the recovery of OA plus O alpha in the faeces plus urine over a 5-day period from 65.5 to 96.2% (P less than 0.0001). Cholestyramine also greatly increased the amount of OA plus O alpha and particularly of OA excretion in the faeces (105 to 160 micrograms, P less than 0.0001 for OA plus O alpha and 82 to 150 micrograms, P less than 0.0001 for OA) and resulted in a corresponding decrease in the excretion of these compounds in the urine. The concentration of fat in the diet had a much less dramatic effect than cholestyramine, was mainly detected in the urine and was affected by an interaction with cholestyramine (P less than 0.0001). Cholestyramine greatly reduced the concentration of OA plus O alpha (37 v. 8 micrograms) when the content of dietary fat was low but to a much lesser degree when it was high (19 v. 12 micrograms). These results suggest that the concentration of fat in the diet may affect the pattern of OA excretion in the urine. Cholestyramine added to the diet greatly increases the amount of OA eliminated in the faeces and reduces the amount in the urine, and as a result it decreases the amount present in the systemic circulation.

Cholestyramine Protection against Ochratoxin A Toxicity: Role of Ochratoxin A Sorption by the Resin and Bile Acid Enterohepatic Circulation

We have shown that the addition of cholestyramine (CHA, a resin known to bind bile salts in the gastrointestinal tract) to ochratoxin A (OTA)-contaminated rat diets reduced plasma levels of the toxin and prevented OTA-induced nephrotoxicity. To elucidate the mechanism of action of CHA, we carried out in vitro experiments to determine whether the resin may bind the toxin. For comparative purposes, binding of bile salts to the resin was also examined. Results showed that CHA binds both OTA and bile salts (taurodeoxycholate [TDC] and taurocholate [TCA]). Also, CHA showed greater affinity for OTA and TDC than for TCA. At 1 mM concentration, 96% of OTA and 80% of TDC were bound to the resin, while for TCA binding was only 50%. However, saturation of the resin was reached at higher levels with bile acids compared to OTA (3.67 mmol/g resin for TCA and 3.71 mmol/g resin for TDC versus 2.85 mmol/g resin for OTA). To characterize the nature of the binding of the toxin to CHA, NaCl (0 to 200 mM) was added to a fixed amount of OTA or bile acids. As expected, TCA absorption was decreased by the addition of NaCl (<50 mM), indicating electrostatic binding. However, OTA and TDC sorption was decreased only at high concentrations of NaCl (>150 mM), suggesting a stronger binding to the resin than that shown with TCA. Sequential competitive studies demonstrated that CHA binds more OTA than TCA. The results of the in vivo study show the role of bile salts in OTA absorption. The toxins plasma levels at 1 and 3 h after a single oral dose of OTA were significantly decreased in bile salt-depleted rats compared to the control. Thus, the alteration of the bile salt biliary pool and OTA enterohepatic circulation may be an additional mechanism of action of the resin against mycotoxin toxicity.

Organ weights, liver constituents, and serum components in growing chicks fed ochratoxin A

Chicks were fed diets containing 0, 1, 5 and 10 mg/kg ochratoxin A (OA) over a four-week period to study the effects of OA on growth, weight of internal organs, liver RNA, DNA, protein, and glycogen and serum enzymes. Ochratoxin A depressed the rate of growth and relative weight of the bursa of Fabricius, and increased the relative weights of the liver, kidney, pancreas, and various sections of the gastrointestinal tract, but had no effect on the heart and spleen. Concentrations of liver RNA, DNA and protein were decreased while glycogen was increased. Serum alkaline phosphatase and γ-glutamyl transferase activities, and uric acid and creatinine concentrations were elevated while serum proteins, albumin, phosphorus, potassium, and cholesterol were depressed. The effects of OA were time- and dose-dependent.