T—2毒素及其代谢产物在大鼠胆汁中的排泄和肠肝循环

T-2毒素及其代谢产物的胆汁排泄和肠肝循环分别在胆管插管大鼠和大鼠肠-肝灌流标本上进行了研究。T-2毒素经门静脉注射后4hr内,染毒剂量的68±12％从插管大鼠的胆汁中排出,胆汁中主要产物是HT-2毒素(HT-2)、3′-羟基HT-2毒素(3′-OHHT-2)、3′-羟基T-2毒素(3′-OHT-2)的葡萄糖醛酸结合物。供体大鼠胆汁经固相树脂(XAD-2)小柱提取和薄层层析(TLC)分离,得到纯化的毒素代谢物的葡萄糖醛酸苷,定量后注射进入灌流大鼠肠标本和肠—肝标本的十二指肠内,在2hr的灌流过程中,注射的结合物有22％被肠道分解并重吸收,7.5％经肝脏作用后再由胆汁排出,其中主要是结合产物,由此证明了T-2毒素代谢产物肠肝循环的存在。     

Metabolism of T-2 toxin by rat liver carboxylesterase

The trichothecene T-2 toxin was rapidly hydrolyzed by rat liver microsomal fraction into HT-2 toxin which was the main metabolite. The metabolism was completely blocked by paraoxon, a serine esterase inhibitor, but not affected by EDTA or 4-hydroxy mercury benzoate, inhibitors of arylesterase and esterases containing SH-group in active site, respectively. Among the serine esterases carboxylesterase (EC 3.1.1.1), but not cholinesterase (EC 3.1.1.8) hydrolysed T-2 toxin to HT-2 toxin. Carboxylesterase activity from liver microsomes was separated into at least five different isoenzymes by isoelectric focusing, and only the isoenzyme of pI 5.4 was able to hydrolyse T-2 toxin to HT-2 toxin. The toxicity of T-2 toxin in mice was enhanced by pre-treatment with tri-o-cresyl phosphate (TOCP), a specific carboxylesterase inhibitor. This confirms the importance of carboxylesterase in detoxification of trichothecenes.     

Metabolism of T-2 toxin by rat brain homogenate

HT-2 toxin was the sole metabolite formed when T-2 toxin was treated with homogenate from brain without its blood content. Homogenate from brain with its full blood content produced--besides HT-2 toxin--T-2 triol, neosolaniol, 4-deacetylneosolaniol and T-2 tetraol, i.e. the same metabolites formed by incubation of T-2 toxin with whole rat blood.     

Acute neurobehavioural toxicity of trichothecene T-2 toxin in the rat.

The acute effects of single oral doses, 0.4 and 2.0 mg/kg, of trichothecene T-2 mycotoxin on behaviour, motor performance and nociception were studied in male Wistar rats. Both doses are sublethal and did not cause overt acute signs of intoxication. In the open field test, 2.0 mg/kg of T-2 toxin increased motionlessness and decreased sniffing (P less than 0.05) 4 hr after the administration. The higher dose shortened step-through latencies in the test trial of the 24-hr passive avoidance test (two-way shuttle box). The exponential data analysis showed that, in those rats that did not learn to avoid the dark (unsafe) compartment of the box, the retention after 2.0 mg/kg of T-2 toxin was only 25% of that in controls (P less than 0.001). T-2 toxin had no effect on motor coordination in the rotarod test and in the bridge walking test 7-8 hr after administration. T-2 toxin had no effect on nociception in the hot place test 8.5 hr after administration. The results suggest that T-2 toxin has some inactivating effects on behaviour of rats, and it seems to cause an impairment in the passive avoidance test at dose 2.0 mg/kg.     

Metabolic pathways of T-2 toxin

Among the naturally-occurring trichothecenes found in food and feed, T-2 toxin is the most potent and toxic mycotoxin. After ingestion of T-2 toxin into the organism, it is processed and eliminated. Some metabolites of this trichothecene are equally toxic or slightly more toxic than T-2 itself, and therefore, the metabolic fate of T-2 toxin has been of great concern. The main reactions in trichothecene metabolism are hydrolysis, hydroxylation and deep oxidation. Typical metabolites of T-2 toxin in an organism are HT-2 toxin, T-2-triol, T-2-tetraol, 3'-hydroxy-T-2, and 3'-hydroxy-HT-2 toxin. There are significant differences in the metabolic pathways of T-2 toxin between ruminants and non-ruminants. Ruminants have been more resistant to the adverse effects of T-2 toxin due to microbial degradation within rumen microorganisms. Some plant species are resistant to T-2 toxin, while others are capable of its intake and metabolisation.     

Immunoperoxidase localization of T-2 toxin

Antibody against T-2 toxin was used for monitoring the fate of T-2 toxin in mice given a single po dose of 11 mg/kg by the peroxidase-antiperoxidase (PAP) method. T-2 toxin was demonstrable in the esophagus from 5 min to about 24 hr postdosing. In the stomach, T-2 toxin was detected within the cytoplasm of intact and injured epithelial cells. In the duodenum, T-2 toxin was primarily localized within the surface epithelium and phagocytic elements (macrophages and neutrophils) of the duodenal lamina propria, especially toward the tips of the villi. Following sloughing of duodenal villous tips, the recovering villous tip epithelial cells frequently showed both cytoplasmic and nuclear T-2 toxin. The jejunum showed weak T-2 toxin within the cytoplasm of villous tip epithelial cells only. The ileum never demonstrated T-2 toxin. Tissue response in the gastrointestinal (GI) tract was characterized by transient edema, marked cytolysis and sloughing, and a subsequent leukocytic invasion of the stomach and proximal small intestine. Evidence of severe gastric and less severe duodenal bleeding was apparent and associated with a marked loss of gastric epithelium and intestinal villous tips. The kidney medulla contained the majority of T-2 toxin stain. T-2 toxin was noted within the distal tubular cells, the cells of the collecting tubules, and the epithelium covering the papilla. T-2 toxin was never demonstrated in any of the hepatic tissue examined.     

Enterohepatic circulation of tetracycline in rats.

The absorption of tetracycline hydrochloride excreted in the bile of rats was evaluated using the insitu intestinal preparation. For comparative purposes, the absorption of the drug from an aqueous solution having the same pH as that of the bile was also determined. After 4 hr, the amounts of tetracycline absorbed from the bile and aqueous solutions were 72.92 and 77.34%, respectively. There was no significant difference in the amount of drug accumulated in the gut tissue. The disappearance of the drug from the intestinal lumen was biexponential, and the kinetic parameters appeared to be similar. It was concluded that tetracycline excreted in the bile is readily absorbed from the rat intestine. Accordingly, biliary excretion does not seem to account for a significant elimination of this antibiotic from the body.     

l-carnitine protects rat hepatocytes from oxidative stress induced by T-2 toxin

Context: Oxidative stress and mitochondrial dysfunction are thought to be the main mechanism of T-2 toxin toxicity. T-2 toxin is the most potent trichothecene mycotoxin which is present in agricultural products. L-carnitine, besides its anti-oxidative properties, facilitates the transportation of long-chain fatty acids in to mitochondrial matrix. Objective: In this study we tested whether L-carnitine, an antioxidant and a facilitator for long-chain fatty acid transportation across mitochondrial membranes, could protect rat hepatocytes against toxicity induced by T-2 toxin. Materials and methods: L-carnitine in low and high doses (50 and 500?mg/kg) was administered for five consecutive days to male Wistar rats. Hepatocytes were isolated and freshly exposed to appropriate concentration of T-2 toxin for 2?h followed by oxidative stress and cell death evaluations. Results: Glutathione depletion, ROS overproduction and mitochondrial membrane potential collapse were determined under T-2 toxin exposure. Pretreatment with L-carnitine particularly at high-dose reduced toxicity and prevented the hepatocytes from abnormal caspase-3 activity and apoptosis. Conclusion: Low toxicity of L-carnitine and its mitochondrial protective effects promises an effective way to reduce or prevent the toxicity induced by certain environmental pollutants, including T-2 toxin.     

Effect of T-2 toxin on regional blood flow and vascular resistance in the conscious rat

The acute effect of T-2 toxemia on local blood flow and vascular resistance in hindquarter, mesenteric, and renal vascular beds was continuously measured by the directional pulsed Doppler technique in conscious, male Sprague-Dawley rats. Intravenous injection of T-2 toxin (1 mg/kg) in the conscious rat reduced blood flow and increased vascular resistance in all blood vessels studied but had no significant effect on mean arterial pressure or heart rate. The blood flow in hindquarters gradually decreased to a minimum of -77 +/- 9% (mean +/- SE) 6 hr after the toxin injection. The hindquarter vascular resistance concomitantly increased to a maximum value of +323 +/- 69% above the resistance before toxin administration. Mesenteric and renal blood flow initially increased (slightly) and then gradually decreased. The maximum drop of blood flow, -90 +/- 13% and -76 +/- 13% for the mesenteric and renal vascular beds, respectively, was achieved 4 hr after T-2 toxin injection and the blood flow values remained low for up to 6 hr. Simultaneously with the impairment of blood flow the mesenteric and renal vascular resistance increased to reach the maximal values of +404 +/- 99% and +556 +/- 15%, respectively. In addition, plasma renin activity was markedly elevated (+653 +/- 160%) at the time of reduced renal blood flow. Intravenous injection of the same value of vehicle (10% ethanol in saline) had no significant effect on any of the cardiovascular variables studied. Two of five rats in the T-2 toxin-treated group died within 5 hr after the T-2 toxin injection and only one animal survived 24 hr while all the control animals survived over 24 hr. The results suggest that strong vasoconstriction in skeletal muscle, mesenteric, and renal vascular beds leads to impairment of local blood flow. The ischemia in vital organs together with the earlier reported decrease in cardiac output by T-2 toxin might then be the cause of rapid death in acute T-2 toxemia.     

Cutaneous absorption and decontamination of [3H]T-2 toxin in the rat model

Cutaneous absorption and decontamination of [3H]T-2 mycotoxin using various treatment modalities incorporating water, detergent, sprays, and scrubbing of application sites were examined in the rat model at 5, 30, 60, and 1440 min (24 h) postexposure. Rats were killed immediately after treatment and radiolabeled T-2 remaining in full-thickness skin samples were determined. Absorption and decontamination were followed over time, and decontaminating treatment modalities were evaluated for efficacy. Less than 1% of the applied dose was absorbed in 5 min, and 50% was absorbed in 24 h. At 5 min, 99.5 +/- 0.05% of nonabsorbed (residual) [3H]T-2 was removed, and 58 +/- 5.2% of residual toxin was removed at 24 h with a 2.5% detergent/water spray. When treatment modalities were evaluated at 60 min, a 2.5% detergent/water scrub followed by a detergent/water spray produced optimal decontamination by removing 81 +/- 2.2% of residual toxin. All treatment modalities using detergent and/or water removed significant amounts of toxin (p less than or equal to .0001); a dry scrub was not efficacious. Treatment should be initiated as soon as possible after exposure for best results. However, the stratum corneum acts as a reservoir for the toxin, and decontamination should be carried out even if delayed several hours or days after exposure. Dermal absorption pharmacokinetics found in these studies are similar to those described for other low-molecular-weight compounds, and the decontamination results from T-2 toxin should be applicable to other, similar toxic substances.     

Biotransformation enzyme activities and phase I metabolites analysis in Litopenaeus vannamei following intramuscular administration of T-2 toxin

T-2 toxin (T-2) is a type-A trichothecene produced by Fusarium that causes toxicity to animals. T-2 contamination of grain-based aquatic feed is a concern for the industries related to edible aquatic crustacean species such as the shrimp industry because it can lead to serious food safety issues. T-2, its metabolites, and selected phase I (EROD, CarE) and phase II (GST, UGT, SULT) detoxification enzymes in hemolymph and tissues were monitored at 0, 5, 10 15, 30, 45, and 60?min following T-2 intramuscular administration (3?mg/kg bw) in shrimp (Litopenaeus vannamei). Marked increases of EROD activity in hepatopancreas and CarE activity in hemolymph, gill, hepatopancreas and intestine were observed followed by increases in phase II enzymes (GST, UGT, SULT) in hepatopancreas, hemolymph, intestine and gill, which remained elevated for an extended period. Time-dependent decrease in shrimp tissue T-2 concentration was observed. HT-2 increased up to 15?min. Most other T-2 metabolites were detected but not T-2 tetraol. Enzyme responses on exposure to T-2 were tissue-specific and time-dependent. Detection results indicated that HT-2 may not be the only important metabolite in aquatic crustacean species. Further investigation into T-2 metabolite toxicity is needed to fully understand the food safety issues related to T-2.     

Comparative effects of T-2 toxin and diacetoxyscirpenol on drug metabolizing enzymes in rat tissues

The effects of T-2 toxin and diacetoxyscirpenol on tissue drug-metabolizing enzymes in young male rats were compared. Mycotoxicoses were produced by daily oral administration of toxins at 1.0 mg/kg body weight for 1, 4 or 8 days. Many hepatic, renal and pulmonary oxidative and conjugative enzymes were measured in animals killed 24 hr following the last administration. The effects of the two trichothecene mycotoxins were generally similar. In liver the decrease in microsomal and cytosolic proteins paralleled the decline in total plasma proteins or the increase in plasma GOT activity. Hepatic microsomal cytochrome P-450 decreased in rats receiving trichothecenes for 8 days. This effect was more marked when aminopyrine, benzphetamine, ethylmorphine and ethoxycoumarin dealkylations or aniline and benzopyrene hydroxylations were measured. p-nitrophenol glucuronyltransferase activity was enhanced in animals receiving at least one administration of trichothecenes, whereas there was no change in conjugation to glutathione or acetate. In other tissues, there was no change in any renal enzymes whereas a significant rise in pulmonary monooxygenase was observed in T-2 toxin administered to rats for 4 or 8 days.     

Changes induced by T-2 toxin in the erythrocyte membrane

The interaction of the trichothecene mycotoxin T-2 with guinea-pig erythrocytes was studied. In a time- and dose-dependent manner, T-2 toxin showed a protective antihaemolytic effect in hypotonic solutions. In isotonic environments, T-2 toxin caused membrane changes resulting in an increase in cell volume and a dramatic alteration in red-cell morphology from the biconcave disc to an echinocyte. These results demonstrate that T-2 toxin, in line with other amphipaths, distributes into the outer half of the cell membrane's phospholipid bilayer. This constitutes the first direct demonstration of an initially amphipathic mechanism of action for this toxin. Therefore, it is suggested that the degree of the final toxic effect of T-2 may be influenced by the targeted cell's membrane.     

Enterohepatic circulation of radioactivity following an oral dose of [14C]temazepam in the rat

The enterohepatic circulation of radioactive material after administering [14C]temazepam was evaluated in three sets of male Wistar strain rats connected in pairs by bile duct-duodenum cannulae. After a single oral dose (10 mg kg-1) to the donor rat, the excretion of radioactivity in the urine and faeces of both rats and in the bile of the recipient rat was determined. Mean total recovery of the administered radioactivity was 92.2%. Based on the amount remaining in the donor rat (gastrointestinal tract and faeces), 81.7% of the dose was absorbed by the donor. The total amount recovered from the recipient, 69.4% of original dose (85.1% of donor's absorbed dose), represented the amount excreted in the donor's bile. Similarly, 54.1% of the original dose (77.9% of the transferred biliary excretion from donor) was reabsorbed by the recipient, and the biliary excretion from this animal (45.9% original dose) accounted for 86.% of the amount reabsorbed.     

Role of lipid peroxidation in the toxicity of T-2 toxin

A. , G. , B. and W. . Role of lipid peroxidation in the toxicity of T-2 toxin. Toxicon 25, 1321 – 1328, 1987.—Recent reports suggest that lipid peroxidation may be involved in the toxicity of T-2 toxin. In the present study the influence of T-2 toxin on two parameters of lipid peroxidation was examined: the formation of thiobarbituric acid reactive material in isolated hepatocytes and liver homogenates from rats and ethane exhalation in vivo. In isolated hepatocytes there was no significant increase in thiobarbituric acid reactive material, neither after addition of T-2 toxin in vitro nor when the toxin had been applied to the rats 15 hr before preparation of hepatocytes. In liver homogenates the amount of thiobarbituric acid reactive material was increased up to 50% over the controls, depending on the dose of T-2 toxin. The increased values are difficult to interpret, because the extent of the increase depends on the method used for determination of thiobarbituric acid reactive material. Measuring another parameter of lipid peroxidation, i.e. ethane exhalation, there was no difference between the T-2 toxin treated rats and the controls whereas carbon tetrachloride treated rats exhaled high amounts of ethane. These results suggest that lipid peroxidation does not play a major role in T-2 toxin toxicity.     

Species-specific hemolysis of erythrocytes by T-2 toxin

The tricothecene mycotoxin, T-2 toxin interacts differently with mammalian erythrocytes. Pig, man, rabbit, guinea pig, horse, dog, rat, and mouse erythrocytes are all lysed to a varying degree by T-2 toxin. But cow, sheep, goat, buffalo, and deer erythrocytes are all resistant to hemolysis by T-2 toxin. Since erythrocytes from ruminant animals contain little or no phosphatidylcholine, perhaps the presence of phosphatidylcholine in the membrane is required for the hemolytic action of T-2 toxin. Sheep erythrocytes were used to encapsulate T-2 toxin further confirming the resistance of erythrocytes from animals with ruminant physiology to T-2 toxin lysis.