Background

Aflatoxins (AFs) are common feed contaminants and are one of the common causes of toxin-related pet food poisoning and recalls.

Objective

Currently, there are no validated methods for the detection and quantitation of AFs in biological matrices to diagnose AF exposure in live animals. Following a successful intra-laboratory method development to quantify AFB1 and AFM1 in animal urine by HPLC with fluorescence detection (HPLC-FLD), the present study was conducted to extensively evaluate the method performance in an unbiased manner using blinded samples.

Methods

The evaluation included two stages. First, the performance was verified in the method-originating laboratory in a single-laboratory blinded method test (BMT-S) trial followed by a multi-laboratory blinded method test (BMT-M) trial.

Results

In both trials, accuracy, repeatability, and reproducibility were satisfactory confirming the relatively good ruggedness and robustness of the method and ensuring that it will perform as expected if used by other laboratories in the future.

Conclusions

We extensively evaluated the performance of a quantitative method to detect AFB1 and AFM1 in animal urine by HPLC-FLD by two different laboratories in two separate BMT-S and BMT-M trials. Both BMT results demonstrated the satisfactory accuracy and precision of the method. It is now available to be adopted by other diagnostic laboratories for purposes of diagnosing AF intoxication in animals.

Highlights

A simple urine-based diagnostic test method using HPLC-FLD that originated in a single laboratory now has passed a multi-laboratory evaluation and is now available to be shared with other diagnostic laboratories for purposes of diagnosing AF intoxication in animals so better treatment can be rendered.

Orellanine (OR) toxin is produced by mushrooms of the genus Cortinarius which grow in North America and in Europe. OR poisoning is characterized by severe oliguric acute renal failure, with a mortality rate of 10%-30%. Diagnosis of OR poisoning currently hinges on a history of ingestion of Cortinarius mushrooms and histopathology of renal biopsies. A key step in the diagnostic approach is analysis of tissues for OR. Currently, tissue-based analytical methods for OR are nonspecific and lack sensitivity. The objectives of this study were: (1) to develop definitive HPLC and LC-MS/MS tissue-based analytical methods for OR; and (2) to investigate toxicological effects of OR in mice. The HPLC limit of quantitation was 10 µg/g. For fortification levels of 15 µg/g to 50 µg/g OR in kidney, the relative standard deviation was between 1.3% and 9.8%, and accuracy was within 1.5% to 7.1%. A matrix-matched calibration curve was reproduced in this range with a correlation coefficient (r) of 0.97-0.99. The limit of detection was 20 ng/g for LC-MS/MS. In OR-injected mice, kidney OR concentrations were 97 ± 51 µg/g on Day 0 and 17 ± 1 µg/g on termination Day 3. Splenic and liver injuries were novel findings in this mouse model. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology.

Hydrogen sulfide (H₂ S) is a highly neurotoxic gas. It is the second most common cause of gas-induced deaths. Beyond mortality, surviving victims of acute exposure may suffer long-term neurological sequelae. There is a need to develop countermeasures against H₂ S poisoning. However, no translational animal model of H₂ S-induced neurological sequelae exists. Here, we describe a novel mouse model of H₂ S-induced neurotoxicity for translational research. In paradigm I, C57/BL6 mice were exposed to 765 ppm H₂ S for 40 min on day 1, followed by 15-min daily exposures for periods ranging from 1 to 6 days. In paradigm II, mice were exposed once to 1000 ppm H₂ S for 60 minutes. Mice were assessed for behavioral, neurochemical, biochemical, and histopathological changes. H₂ S intoxication caused seizures, dyspnea, respiratory depression, knockdowns, and death. H₂ S-exposed mice showed significant impairment in locomotor and coordinated motor movement activity compared with controls. Histopathology revealed neurodegenerative lesions in the collicular, thalamic, and cortical brain regions. H₂ S significantly increased dopamine and serotonin concentration in several brain regions and caused time-dependent decreases in GABA and glutamate concentrations. Furthermore, H₂ S significantly suppressed cytochrome c oxidase activity and caused significant loss in body weight. Overall, male mice were more sensitive than females. This novel translational mouse model of H₂ S-induced neurotoxicity is reliable, reproducible, and recapitulates acute H₂ S poisoning in humans.