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Tire tread particles (TTP) are environmentally prevalent microplastics and generate toxic aqueous leachate. We determined the total carbon and nitrogen leachate concentrations and chemical profiles from micron (∼32 μm) and centimeter (∼1 cm) TTP leachate over 12 days. Dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were used to measure the concentration of leached compounds. Nontargeted chemical analysis by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS) was used to compare the chemical profiles of leachates. After leaching for 12 days, DOC was 4.0 times higher in the micron TTP leachate than in the centimeter TTP leachate, and TDN was 2.6 times higher. The total GC×GC/TOF-MS chromatographic feature peak area was 2.9 times greater in the micron TTP leachate than the centimeter TTP leachate, and similarly, the total relative abundance of 54 tentatively identified compounds was 3.3 times greater. We identified frequently measured tire-related chemicals, such as 6PPD, N-cyclohexyl-N'-phenylurea (CPU), and hexa(methoxymethyl)melamine (HMMM), but nearly 50% of detected chemicals were not previously reported in tire literature or lacked toxicity information. Overall, the results demonstrate that smaller TTP have a greater potential to leach chemicals into aquatic systems, but a significant portion of these chemicals are not well-studied and require further risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In recognition of the potential of calreticulin (CRT) protein in enhancing the rate and quality of wound healing in excisional animal wound models, this study was to incorporate CRT via polyblend electrospinning into polycaprolactone (PCL)/type 1 collagen (Col1) nanofibers (NFs; 334 ± 75 nm diameter) as biomimetic extracellular matrices to provide a novel mode of delivery and protection of CRT with enhanced synergistic activities for tissue regeneration. Release kinetic studies using fluoresceinated CRT (CRT-FITC) polyblend NFs showed a burst release within 4 h reaching a plateau at 72 h, with further intervals of release upon incubation with fresh phosphate buffered saline for up to 8 weeks. By measuring fluorescence during the first 4 h of release, CRT-FITC-containing NFs were shown to protect CRT from proteolytic digestion (e.g., by subtilisin) compared to CRT-FITC in solution. CRT incorporated into NFs (CRT-NFs) also showed retention of biological activities and potency for stimulating proliferation and migration of human keratinocytes and fibroblasts. Fibroblasts seeded on CRT-NFs, after 2 days, showed increased amounts of fibronectin, TGF-β1, and integrin β1 in cell lysates by immunoblotting. Compared to NFs without CRT, CRT-NFs supported cell responses consistent with greater cell polarization and increased laminin-5 deposition of keratinocytes and a more motile phenotype of fibroblasts, as suggested by vinculin-capping F-actin fibers nonuniformly located throughout the cell body and the secretion of phosphorylated focal adhesion kinase-enriched migrasomes. Altogether, CRT electrospun into PCL/Col1 NFs retained its structural integrity and biological functions while having additional benefits of customizable loading, protection of CRT from proteolytic degradation, and sustained release of CRT from NFs, coupled with innate physicochemical cues of biomimetic PCL/Col1 NFs. Such synergistic activities have potential for healing recalcitrant wounds such as diabetic foot ulcers.

Coastal reintroduction sites for California condors ( Gymnogyps californianus ) can lead to elevated halogenated organic compound (HOC) exposure and potential health impacts due to the consumption of scavenged marine mammals. Using nontargeted analysis based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC/TOF-MS), we compared HOC profiles of plasma from inland and coastal scavenging California condors from the state of California (CA), and marine mammal blubber from CA and the Gulf of California off Baja California (BC), Mexico. We detected more HOCs in coastal condors (32 ± 5, mean number of HOCs ± SD, n = 7) than in inland condors (8 ± 1, n = 10) and in CA marine mammals (136 ± 87, n = 25) than in BC marine mammals (55 ± 46, n = 8). ∑DDT-related compounds, ∑PCBs, and total tris(chlorophenyl)methane (∑TCPM) were, respectively, ∼7, ∼3.5, and ∼148 times more abundant in CA than in BC marine mammals. The endocrine-disrupting potential of selected polychlorinated biphenyls (PCB) congeners, TCPM, and TCPMOH was determined by in vitro California condor estrogen receptor (ER) activation. The higher levels of HOCs in coastal condors compared to those in inland condors and lower levels of HOC contamination in Baja California marine mammals compared to those from the state of California are factors to consider in condor reintroduction efforts.

Microplastic fibers (MPF) are a ubiquitous marine contaminant, making up to 90% of global microplastic concentrations. Imaging flow cytometry was used to measure uptake and ingestion rates of MPF by blue mussels (Mytilus edulis). Mussels were fed a diet of Rhodomonas salina and MPF concentrations up to 30 MPF mL -1 , or 0.374% of available seston. Filtration rates were greatly reduced in mussels exposed to MPF. Uptake of MPF followed a Holling's Type II functional response with 95% of the maximum rate (5227 MPF h - 1 ) occurring at 13 MPF mL -1 . An average of 39 MPF (SE ± 15, n = 4) was found in feces (maximum of 70 MPF). Most MPF (71%) were quickly rejected as pseudofeces, with approximately 9% ingested and <1% excreted in feces. Mussels may act as microplastic sinks in Gulf of Maine coastal waters, where MPF concentrations are near the order of magnitude as the experimental treatments herein., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the originally published version of this Article, the affiliation details for Kevin P. White inadvertently omitted 'Tempus Labs, Chicago, IL, 60654, USA'. This has now been corrected in both the PDF and HTML versions of the Article.

The oligometastasis hypothesis suggests a spectrum of metastatic virulence where some metastases are limited in extent and curable with focal therapies. A subset of patients with metastatic colorectal cancer achieves prolonged survival after resection of liver metastases consistent with oligometastasis. Here we define three robust subtypes of de novo colorectal liver metastasis through integrative molecular analysis. Patients with metastases exhibiting MSI-independent immune activation experience the most favorable survival. Subtypes with adverse outcomes demonstrate VEGFA amplification in concert with (i) stromal, mesenchymal, and angiogenic signatures, or (ii) exclusive NOTCH1 and PIK3C2B mutations with E2F/MYC activation. Molecular subtypes complement clinical risk stratification to distinguish low-risk, intermediate-risk, and high-risk patients with 10-year overall survivals of 94%, 45%, and 19%, respectively. Our findings provide a framework for integrated classification and treatment of metastasis and support the biological basis of curable oligometastatic colorectal cancer. These concepts may be applicable to many patients with metastatic cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Approximately 85% of all lung cancers are non-small cell histology [non-small cell lung cancer (NSCLC)]. Modern treatment strategies for NSCLC target driver oncogenes and immune checkpoints. However, less than 15% of patients survive beyond 5 years. Here, we investigated the effects of SAR302503 (SAR), a selective JAK2 inhibitor, on NSCLC cell lines and tumors. We show that SAR is cytotoxic to NSCLC cells, which exhibit resistance to genotoxic therapies, such as ionizing radiation, cisplatin, and etoposide. We demonstrate that constitutive IFN-stimulated gene expression, including an IFN-related DNA damage resistance signature, predicts for sensitivity to SAR. Importantly, tumor cell-intrinsic expression of PD-L1 is IFN-inducible and abrogated by SAR. Taken together, these findings suggest potential dual roles for JAK2 inhibitors, both as a novel monotherapy in NSCLCs resistant to genotoxic therapies, and in tandem with immune checkpoint inhibition. Mol Cancer Ther; 17(4); 732-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Multiple therapeutic agents are typically used in concert to effectively control metastatic tumors. Recently, we described microRNAs that are associated with the oligometastatic state, in which a limited number of metastatic tumors progress to more favorable outcomes. Here, we report the effective delivery of an oligometastatic microRNA (miR-655-3p) to colorectal liver metastases using nanoscale coordination polymers (NCPs). The NCPs demonstrated a targeted and prolonged distribution of microRNAs to metastatic liver tumors. Tumor-targeted microRNA miR-655-3p suppressed tumor growth when co-delivered with oxaliplatin, suggesting additive or synergistic interactions between microRNAs and platinum drugs. This is the first known example of systemically administered nanoparticles delivering an oligometastatic microRNA to advanced metastatic liver tumors and demonstrating tumor-suppressive effects. Our results suggest a potential therapeutic strategy for metastatic liver disease by the co-delivery of microRNAs and conventional cytotoxic agents using tumor-specific NCPs., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Various methods exist for the treatment of rectovaginal fistulas, with the choice of repair largely dependent on the fistula location. Options include local repair with mucosal advancement flaps, the use of agents like fibrin glue, and an abdominal approach with resection and colo-anal reconstruction. Traditionally, local repair with mucosal advancement flaps is reserved for simple, low rectovaginal fistulas, while high rectal fistulas require a transabdominal approach.1,2 METHODS: Here, we demonstrate an innovative approach for the treatment of a complex, high rectovaginal fistula in a patient with a hostile pelvis via a transanal approach with robotic assistance. The video demonstrates the basic steps of a repair of a rectovaginal fistula: debridement of the fistula tract, mobilization of the mucosal advancement flap, primary closure of the fistula tract, suturing of the mucosal advancement flap, and flexible sigmoidoscopy to confirm lumen patency and visualization of the closure., Results: By utilizing robotic assistance, we were provided with improved dexterity, precision, and scalability to accomplish the complex task of dissection, suturing, and knot tying in the confines of the rectum., Conclusions: We suggest that this approach can be used on selected patients with high rectovaginal fistula or other rectal pathology who are otherwise not candidates for a transanal approach.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches 1,2 . However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

Background: The selection of patients for oligometastasis-directed ablative therapy remains a challenge. The authors report on clinical and molecular predictors of survival from a stereotactic body radiotherapy (SBRT) dose-escalation trial for oligometastases., Methods: Patients who had from 1 to 5 metastases, a life expectancy of >3 months, and a Karnofsky performance status of >60 received escalating SBRT doses to all known cancer sites. Time to progression, progression-free survival, and overall survival (OS) were calculated at the completion of SBRT, and clinical predictors of OS were modeled. Primary tumor microRNA expression was analyzed to identify molecular predictors of OS., Results: Sixty-one evaluable patients were enrolled from 2004 to 2009. The median follow-up was 2.3 years for all patients (range, 0.2-9.3 years) and 6.8 years for survivors (range, 2.0-9.3 years). The median, 2-year, and 5-year estimated OS were 2.4 years, 57%, and 32%, respectively. The rate of progression after SBRT was associated with an increased risk of death (hazard ratio [HR], 1.44; 95% confidence interval [CI], 1.24-1.82). The time from initial cancer diagnosis to metastasis (HR, 0.98; 95% CI, 0.98-0.99), the time from metastasis to SBRT (HR, 0.98; 95% CI, 0.98-0.99), and breast cancer histology (HR, 0.12; 95% CI, 0.07-0.37) were significant predictors of OS. In an exploratory analysis, a candidate classifier using expression levels of 3 microRNAs (miR-23b, miR-449a, and miR-449b) predicted survival among 17 patients who had primary tumor microRNA expression data available., Conclusions: A subset of oligometastatic patients achieves long-term survival after metastasis-directed SBRT. Clinical features and primary tumor microRNA expression profiling, if validated in an independent dataset, may help select oligometastatic patients most likely to benefit from metastasis-directed therapy. Cancer 2016;122:2242-50. © 2016 American Cancer Society., (© 2016 American Cancer Society.)

Colopericardial fistula after colonic interposition is a rare complication, with few prior reported cases. Management of such cases has usually consisted of resection of the colonic segment with cervical diversion. Here we present a case of successful primary repair of a colopericardial fistula in a 73-year-old woman who had initially undergone a colonic interposition graft 30 years before presentation., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

We present a model of hepatic colorectal metastases which represents monoclonal cell lines double-labeled by luciferase and tdTomato. These cells form liver metastasis in varying numbers and patterns similar to those observed in patients. Using in vivo and ex vivo luminescent and fluorescent imaging we determine the growth kinetics and clonogenic frequency of tumor cells colonizing liver. Molecular profiling detected stable expressional differences between clones consistent with their phenotypes. The data indicate that clinically relevant phenotypes of liver metastases can be modeled in vivo.

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Four different cell models were chosen for comparison of OTA and OTB toxicity: primary porcine (PKC), rat (RPTC) and human renal proximal epithelial cells (HKC) from both sexes and a porcine renal cell line: LLC-PK1. Culture conditions were tested and optimized for each respective cell type (species/sex and origin). All cell types were characterized for epithelial origin and growth patterns and following optimization of dosing strategies and assay procedures, a strict study design was implemented to avoid systemic variations. Due to possible sensitivity differences, three simple endpoints were chosen to provide basic data for interspecies comparison: neutral red uptake, MTT reduction and cell number. Of the endpoints tested neutral red appeared the most sensitive, although all three parameters yielded comparable EC50's. Sex-differences were observed between male and female HKC cells following 96 h exposure to OTA, with HKC(m) being more sensitive than HKC(f). No sex-difference was observed in PKC cells, however, the PKC were approximately 3 and 10 times more sensitive than HKC(m) and HKC(f), respectively, to OTA and OTB. Interestingly, the CI95 of the EC50 values obtained for OTA (15.5-16.5 microM) and OTB (17.0-2 1.0 microM) were comparable in the PKC cells. In contrast, OTB had lower cytotoxicity than OTA in HKC and LLC-PK1 (approx. 2-fold) and no effects in RPTC. Overall, HKC(m) were nearly as sensitive as PKC towards OTA, followed by RPTC, LLC-PK1 and HKC(f), thus suggesting a sex specific sensitivity in humans towards OTA induced cytotoxicity.

Fumonisin B1 (FB1) is a mycotoxin isolated from Fusarium fungi that contaminate crops worldwide. A previous study demonstrated that FB1 promoted preneoplastic foci in initiated rats and induced hepatocellular carcinomas in BD IX rats at 50 parts per million (ppm), but fundamental dose-response data were not available to assist in setting regulatory guidelines for this mycotoxin. To provide this information, female and male F344/N/Nctr BR rats and B6C3F1 Nctr BR mice were fed for two years a powdered NIH-31 diet containing the following concentrations of FB1: female rats, 0, 5, 15, 50, and 100 ppm; male rats, 0, 5, 15, 50, and 150 ppm; female mice, 0, 5, 15, 50, and 80 ppm; male mice, 0, 5, 15, 80, and 150 ppm. FB1 was not tumorigenic in female F344 rats with doses as high as 100 ppm. Including FB1 in the diets of male rats induced renal tubule adenomas and carcinomas in 0/48, 0/40, 9/48, and 15/48 rats at 0, 5, 15, 50, and 150 ppm, respectively. Including up to 150 ppm FB1 in the diet of male mice did not affect tumor incidence. Hepatocellular adenomas and carcinomas were induced by FB1 in the female mice, occurring in 5/47, 3/48, 1/48, 19/47, and 39/45 female mice that consumed diets containing 0, 5, 15, 50, and 80 ppm FB1, respectively. This study demonstrates that FB1 is a rodent carcinogen that induces renal tubule tumors in male F344 rats and hepatic tumors in female B6C3F1 mice.

Two toxigenic strains of the fungus Alternaria alternata (ATCC 56836 and ATCC 66868) were grown on surface-disinfected, fresh, ripe fruits and tested for the production of alternariol (AOH) and alternariol methyl ether (AME). Examined fruits included strawberries; red and green seedless grapes; concord grapes; red delicious, golden delicious, and gala apples; and blueberries. After inoculation, fruits were incubated at 4, 10 degrees C, or room temperature (approximately 21 degrees C) for up to 3 weeks. At weekly intervals, duplicate samples were analyzed for AOH and AME by using liquid chromatography. Results indicated that A. alternata and its metabolites were not a major problem in strawberries due to the presence of fast-growing molds like Rhizopus and Botrytis that outgrew and possibly inhibited Alternaria. Both Alternaria strains showed limited growth on apples, although fast-growing molds were not present after surface disinfection; AOH and AME were produced only by the ATCC 56836 strain on the golden delicious and gala varieties, (ranging from <0.1 to 5 microg/g and <0.1 to 14 microg/g for AOH and AME, respectively). Restricted growth of both strains without toxin production occurred in blueberries, whereas moderate growth and AOH (<0.1 to 3,336 microg/g) and AME (<0.1 to 1,716 microg/g) production took place in grapes.

Fumonisins are toxic metabolites of Fusarium moniliforme, a fungus that occurs widely in corn. Fumonisins causes leukoencephalomalacia in horses and pulmonary edema in swine and have been suggested as a possible cause of an increased incidence of neural tube defects among people living along the Texas-Mexico border. As part of an effort to determine levels of fumonisins in human food, a liquid chromatographic (LC) method was devised for determining fumonisin B1 (FB1) and the total hydrolysis product of FB1 (HB1) in tortillas. The method uses acetonitrile-0.1M phosphate buffer (pH 3; 1 + 1) extraction, solid-phase C18 cleanup, o-phthalaldehyde and 2-mercaptoethanol derivatization, and reversed-phase LC. Average recoveries from tortillas spiked with FB1 and HB1 at 250, 500, and 1000 ng/g were 86.5% for FB1 and 82.6% for HB1. Tortillas (54) and masa (8) from the Texas-Mexico border were analyzed for FB1 and HB1. Average amounts of FB1 and HB1 in tortillas were 187 and 82 ng/g, respectively. Average amounts of FB1 and HB1 in masas were 262 and 64 ng/g, respectively. The results show that fumonisin B1 and its hydrolysis product are present in tortillas consumed by a population experiencing an increased incidence of neural tube defects.

Aminopentol (AP1) is the total hydrolysis product of fumonisin B1 (FB1), the major and best characterized of the fumonisins, which are mycotoxins that are common contaminants of corn and corn meal. Some human populations expected to have significant exposure to AP1 have a high incidence of babies born with neural tube defects (NTD). The embryotoxicity of AP1 was evaluated in cultured rat embryos. Gestation day 9.5 embryos were exposed to 0, 3, 10, 30, 100 or 300 microM AP1 throughout the entire 45-hr culture period. At 100 microM AP1, growth and overall development were reduced significantly. There was also a significant increase in the incidence of abnormal embryos. 29% of the embryos had NTD, and 36% of the embryos had other abnormalities. At 300 microM AP1, the incidence of NTD was 15%, and 85% of the embryos had other abnormalities. These findings suggest that AP1, at concentrations of 100 microM and above, can induce NTD in organogenesis-stage cultured rat embryos. However, these NTD are in conjunction with significant overall retardation of growth and development as well as significant increases in the incidence of other defects. These studies also showed, when compared with previous findings, that AP1 is over 100-fold less toxic than FB1 to cultured rat embryos.

A survey was conducted to evaluate fumonisins FB1 and FB2 in Uruguayan corn products. Sixty-four samples of different local brands were purchased from retail stores during a 15-month period and analyzed for FB1 and FB2 by methanol-water extraction, cleanup with a 1 mL. strong-anion-exchange solid-phase extraction column, and liquid chromatography with o-pthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. Contamination levels for FB1 varied from 50 ng/g (detection limit) to 6342 ng/g. Values were highest in feed samples (up to 6342 ng/g), unprocessed corn kernel (up to 3688 ng/g), and milled products, which included polenta (up to 427 ng/g). They were lowest in processed corn kernel (up to 155 ng/g) and snacks (up to 314 ng/g). FB2 was determined in one-fourth of the total samples and detected at trace levels in only one feed sample. The data demonstrated the natural occurrence of fumonisins in corn products in Uruguay. Feed and polenta that contain fumonisins could be of concern because they are consumed in large amounts and are often the main nutrient source in Uruguay.

A modified liquid chromatographic (LC) method for determining fumonisin B1 (FB1) in corn was applied to canned and frozen sweet corn. The corn is extracted with methanol-water (8 + 2), and the extract is filtered. The filtrate is diluted with water and passed through an immunoaffinity column. After the column is washed with water, FB1 is eluted with methanol-water (8 + 2). The eluate is evaporated to dryness by using a vacuum concentrator, and the residue is dissolved in acetonitrile-water (1 + 1). FB1 is derivatized with o-phthaldialdehyde. The derivative is separated on a reversed-phase C18 LC column using acetonitrile-water-acetic acid (50 + 50 + 1) and quantitated with a fluorescence detector. Recoveries of FB1 from canned and frozen corn spiked over the range of 50-200 ng/g were 76-88%. The limit of determination was about 25 ng/g, and the limit of detection was about 4 ng/g. The method was applied to 97 commercial canned and frozen sweet corn samples collected from different areas of the United States. Sixty samples contained no FB1. Low levels (trace-82 ng FB1/g corn) were found in 35 samples; 235 ng FB1/g was found in 1 canned corn sample, and 350 ng FB1/g was found in 1 frozen corn sample.

Wheat and barley from the 1993 crop year were analyzed for deoxynivalenol (DON). A total of 630 samples were collected by the Federal Grain Inspection Service in 25 states and analyzed using a commercially available, direct competitive, enzyme-linked immunosorbent assay. The limit of determination was about 0.5 micrograms/g. DON contamination in the 483 wheat samples averaged 2.0 micrograms/g and ranged from < 0.5 to 18 micrograms/g. DON contamination in the 147 barley samples averaged 4.2 micrograms/g and ranged from < 0.5 to 26 micrograms/g. About 40% fo the wheat samples and 57% of the barley samples contained DON levels that were greater than the U.S. Food and Drug Administration 1982 advisory level of 2 micrograms/g for DON in wheat designated for milling (human consumption).

An AOAC/IUPAC collaborative study was conducted to evaluate the effectiveness of a multifunctional column for the determination of aflatoxins. The test portion is extracted with acetonitrile-water (9 + 1), the extract is filtered, and the filtrate is passed through the column. The aflatoxins in the eluate are determined by reversed-phase liquid chromatography after derivatization with trifluoroacetic acid. Naturally contaminated corn, almonds, Brazil nuts, peanuts, and pistachio nuts spiked with total aflatoxins at 5, 10, 20, and 30 ng/g were sent to 12 collaborators in the United States, Denmark, France, Japan, and Switzerland. Eleven collaborators completed the study. Average recoveries of total aflatoxins for each spike level for the various commodities (excluding Brazil nuts at 5 ng/g) were 93, 97, 95, and 95%, respectively; the repeatability relative standard deviation (RSDr) ranged from 6.0 to 23.2% and the reproducibility relative standard deviation (RSDR) ranged from 12.0 to 69.4%. The multifunctional column coupled with a liquid chromatographic method for determination of aflatoxins in corn, almonds, Brazil nuts, peanuts, and pistachio nuts has been adopted first action by AOAC INTERNATIONAL.

Alternariol and alternariol methyl ether were tested in the Ames Salmonella typhimurium assay, and both were shown, with and without metabolic activation, to be nonmutagenic to strains TA98 and TA100. The finding of other investigators that alternariol methyl ether is weakly mutagenic to TA98 without metabolic activation could have resulted from the presence of a small amount of one of the highly mutagenic altertoxins in the alternariol methyl ether originally tested.

A direct competitive enzyme-linked immunosorbent assay screening method for aflatoxins at 20 ng/g in corn was studied by 15 collaborating laboratories. Test samples of corn were extracted by blending with methanol-water (8 + 2). The extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of < 30%. Each diluted filtrate was applied to a test device containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the test device was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. A test sample was judged to contain > or = 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; if a blue or gray color developed, the test sample was judged to contain < 20 ng aflatoxins/g. All laboratories correctly identified naturally contaminated corn test samples. Only one false positive was found for controls containing no aflatoxins. The correct responses for positive test samples spiked at levels of 10, 20, and 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 15:1:3) were 67, 97, and 100%, respectively. This method was adopted first action by AOAC INTERNATIONAL as a change in method for 990.34 for screening for aflatoxins B1, B2, and G1 in corn at total aflatoxin concentrations of > or = 20 ng/g.

A thin-layer chromatographic (TLC) method was modified for determination of deoxynivalenol (DON) in 1991 U.S. winter and spring wheat. After extraction with acetonitrile-water (84 + 16) and cleanup on a charcoal-alumina-Celite (7 + 5 + 3) column, acetonitrile was used instead of ethyl acetate to transfer the concentrated extract containing DON. After the extract was evaporated to dryness, the residue was dissolved in methanol and an aliquot was spotted on a high-performance TLC plate. After development with chloroform-acetone-2-propanol (8 + 1 + 1), the plates were sprayed with aluminum chloride solution and heated; DON was quantitated by fluorodensitometry. Average recoveries of DON added to duplicate test portions of wheat at 200, 400, and 800 ng/g were 83, 82, and 72%, respectively. The detection limit was 40 ng/g. The method was applied to 81 test samples of spring and winter wheat. The wheat contained DON levels that ranged from nondetectable to 9330 ng/g (average 1570 ng/g). The results indicate that DON levels were higher in wheat from Missouri, North Dakota, and Tennessee than in wheat from 7 other states. The identity of DON, which was isolated from 21 of the extracts by preparatory TLC, was confirmed by gas chromatography/mass spectrometry in all 21 test samples.

An AOAC/IUPAC (International Union of Pure and Applied Chemistry) collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column for the determination of aflatoxin. The test portion is extracted with methanol-water (7 + 3), filtered, diluted to less than 30% methanol with water, and applied to the affinity column. The column is washed with water and the concentrated aflatoxins are eluted with methanol. Total aflatoxins are determined by solution fluorometry with bromine (SFB), and individual toxins are determined by reverse-phase liquid chromatography with postcolumn derivatization with iodine (PCD). Corn naturally contaminated with aflatoxins, and peanuts, peanut butter, and corn containing added aflatoxins (B1:B2:G1:G2 = 7:1:3:1) were sent to 24 collaborators in the United States, France, Canada, and the Republic of South Africa. Twelve collaborators used the SFB method, 9 used the PCD method, and 3 used both SFB and PCD methods. Twenty collaborators completed the study (10 used the SFB method, 7 used the PCD method, and 3 used both SFB and PCD methods). Test portions were spiked at 10, 20, and 30 ng/g. For SFB analyses, recoveries of total aflatoxins were 123, 105, and 107%, respectively; the relative standard deviation for repeatability (RSDr) ranged from 11.75 to 16.57%, and the relative standard deviation for reproducibility (RSDR) ranged from 10.97 to 33.09%. For PCD analyses, recoveries were 81, 81, and 83%, respectively; the RSDr ranged from 5.20 to 17.22%, and the RSDR ranged from 4.68 to 50.77%. The RSDr for aflatoxins B1 and G1 for spiked test portions ranged from 5.45 to 23.55%, and the RSDR ranged from 4.21 to 57.28%.(ABSTRACT TRUNCATED AT 250 WORDS)

Some common decay organisms of vegetables and ripened fruits are Alternaria species. Even fruits and vegetables kept under refrigeration can be spoiled by Alternaria species because the mold grows at low temperatures. Alternaria alternata is commonly found in grain in areas with a high incidence of esophageal cancer. Three metabolites, altertoxins I, II, and III, have been isolated from A. alternata and have hydroxyperylenequinone structures. Although other perylenequinone metabolites such as stemphyperylenol and stemphyltoxins I, II, III, and IV, have been isolated from Stemphylium botryosum var. lactucum, a plant pathogen and mold, we isolated and identified stemphyltoxin III from A. alternata. This metabolite was tested for mutagenicity in the Ames Salmonella typhimurium plate incorporation assay with and without Aroclor 1254-induced rat S-9 metabolic activation. A positive response was noted with and without metabolic activation in S. typhimurium TA98 and TA1537, and there was a marginal response in strain TA100.

A collaborative study of a liquid chromatographic method for the determination of aflatoxins B1, B2, G1, and G2 was conducted in laboratories located in the United States, Canada, South Africa, and Switzerland. Twenty-one artificially contaminated raw peanuts, peanut butter, and corn samples containing varying amounts of aflatoxins B1, B2, G1, and G2 were distributed to participating laboratories. The test portion was extracted with methanol-0.1N HCl (4 + 1), filtered, defatted with hexane, and then partitioned with methylene chloride. The concentrated extract was passed through a silica gel column. Aflatoxins B1 and G1 were derivatized with trifluoroacetic acid, and the individual aflatoxins were determined by reverse-phase liquid chromatography with fluorescence detection. Statistical analysis of the data was performed to determine or confirm outliers, and to compute repeatability and reproducibility of the method. For corn, relative standard deviations for repeatability (RSDr) for aflatoxin B1 ranged from 27.2 to 8.3% for contamination levels from 5 through 50 ng/g. For raw peanuts and peanut butter, RSDr values for aflatoxin B1 were 35.0 to 41.2% and 11.2 to 19.1%, respectively, for contamination levels from 5 through 25 ng/g. RSDr values for aflatoxins B2, G1, and G2 were similar. Relative standard deviations for reproducibility (RSDr) for aflatoxin B1 ranged from 15.8 to 38.4%, 24.4 to 33.4%, and 43.9 to 54.0% for corn, peanut butter, and raw peanuts, respectively. The method has been adopted official first action for the determination of aflatoxins B1, B2, G1, and G2 in peanut butter and corn at concentrations greater than or equal to 13 ng total aflatoxins/g.

The Ames Salmonella typhimurium assay was used to demonstrate that an extract of the mold Alternaria alternata was mutagenic. The mutagenic extract was fractionated, and the Ames test was used to determine which fractions were mutagenic. Subsequently, altertoxins I and II and a new compound referred to as altertoxin III were isolated by liquid chromatography and shown to be hydroxyperylenequinone compounds by mass spectrometry and infrared, ultraviolet, and proton magnetic resonance spectroscopy. Altertoxins I, II, and III were mutagenic to S. typhimurium TA98, TA100, and TA1537 with and without metabolic activation.

Versicolorin A (VA) and sterigmatocystin (ST) are biosynthetic precursors of aflatoxin B1 (AFB1). The carcinogenicity of these compounds relative to AFB1 was determined with the use of rainbow trout (Salmo gairdneri) embryo exposure. Exposure of 14-day rainbow trout embryos to a 0.5-ppm aqueous suspension of ST for 1 hour produced a 13% incidence of hepatocellular carcinomas among survivors 1 year later. Similar exposure of trout eggs to a 0.5-ppm solution of AFB1 produced a 53% incidence among survivors. Subsequent exposure of 21-day rainbow trout embryos to 5- and 25-ppm solutions of VA resulted in hepatocellular carcinoma incidences among survivors of 42 and 68%, respectively, at 12 months. A 0.5-ppm AFB1 positive control group had a 68% incidence among survivors of hepatocellular carcinomas at 1 year. These results established the carcinogenicity of VA for the first time and confirmed previous reports of ST carcinogenicity. Both compounds were of sufficient potencies to warrant caution as possible human health hazards.

The mold flora was determined for 146 samples of fresh but visibly moldy tomatoes collected from sorting belts in tomato catsup processing plants in California and in Midwestern and Eastern United States. Mold found in 141 of the samples included at least 22 genera, principally Alternaria , Aspergillus , Cladosporium , Fusarium and Penicillium , and 51 species. The California tomatoes were dominated by Geotrichum candidum and species of Aspergillus and Penicillium ; Midwest and East tomatoes were dominated by Alternaria . This suggested that the predominant molds in tomatoes may differ, depending on geographical source. Tenuazonic acid (TA), a toxic metabolite of Alternaria spp., was found in 73 of the samples at a range of 0.4 to 69.7 (average 4.94) μg/g of moldy tissue; however, Alternaria spp. were not found in 35 of the 73 TA-positive samples. It is possible that other molds may produce TA or that the toxin-producing Alternaria died off before our sampling.

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Stemphyltoxin III, (6aR*, 6bS*, 7R*, 8R*)-3,6a, 10-trihydroxy-4,9-dioxo-4,6a,6b,7,8,9-hexahydro-7,8-epoxyperylene, a known metabolite of Stemphylium botryosum var. lactucum, has been identified as a mutagenic metabolite of Alternaria alternata by spectroscopic studies. The 13C-nmr spectral data, which were not reported previously, are presented.

A method is described for the detection and quantitative analysis of xanthomegnin in corn samples. Initial extraction with CHCl3 in the presence of 0.5M H3PO4 is followed by additional purification using silica gel column chromatography. A high pressure liquid chromatograph equipped with a microparticle silica gel column and a 405 nm absorbance detector is used for detection and quantitation of the xanthomegnin. The identity of xanthomegnin is confirmed by thin layer chromatography on silica gel plates developed with benzene-methanol-acetic acid (90 + 5 + 5). The recovery of xanthomegnin added to corn samples at levels of 0.75--9.6 mg/kg averaged 41% with a coefficient of variation of 25%.

Superoxide production by the three 4,9-dihydroxyperylene-3,10-quinone fungal toxins, altertoxins I, II, and III, was stimulated on illumination with broad-spectrum light. As determined previously for cercosporin, superoxide production by illuminated altertoxins was increased by the addition of the reducing substances ergothioneine or urate; ascorbate also effectively increased superoxide production. Illuminated urate alone engendered some superoxide production.

Isolates from Aspergillus ochraceus obtained from green coffee beans were cultured on rice and water. After 20 d of growth the cultures were extracted with chloroform and the extracts were analyzed by high performance liquid chromatography for ochratoxin A (OA), ochratoxin B (OB), xanthomegnin (X), viomellein (V) and vioxanthin (VX). Forty-three percent of the isolates produced OA at an average level of 397 μg of toxin/g rice, 17% produced OB at an average level of 312 μg/g, and 84% produced X, V, and VX at an average level of 281, 417 and 386 μg/g, respectively. The highest levels of toxin production were OA, 2088 μg/g; OB, 3375 μg/g; X, 1562 μg/g; V, 2514 μg/g; and VX, 2054 μg/g. VX has not previously been reported as an A. ochraceus metabolite.

The chemical method for confirmation of the identity of aflatoxin by derivative formation directly on the TLC plate was studied collaboratively by 8 participants. The results show that aflatoxin B-1 was confirmed in 17 of 17 sample extracts representing 15 mu-g aflatoxin B-1/kg peanut butter, in 13 of 16 extracts representing 5 mu-g/kg, and in none of the 7 aflatoxin-free extracts. Collaborators commented that the method was easily performed and gave good results. The method has been adopted as official first action.

The mold genus Alternaria is a widely distributed plant pathogen. Some of these species, e.g., A. alternata, are common decay organisms of fruits and vegetables. Two novel perylene oxide metabolites, altertoxins II and III, have been identified in extracts of A. alternata isolates that exhibit mutagenic responses in the Ames Salmonella typhimurium assay. These identifications were based on mass, optical rotational, and 1H- and 13C-nmr spectral studies. Previous reports of related perylene dione mycotoxins have been clarified.

A high pressure liquid chromatographic (HPLC) method is described for the detection and quantitative determination of satratoxins G and H in cereal grains. The toxins are extracted from the sample by blending with methanol--water (55 + 45) in the presence of hexane, followed by partitioning into chloroform. The chloroform extract is further purified on a 10 g silica gel column. A high pressure liquid chromatograph, equipped with a microparticle silica gel column and a 254 nm absorbance detector, is used for the determination. Additional confirmation of identity is obtained by mass spectrometry or by a brine shrimp bioassay of the HPLC eluates corresponding to the retention times of satratoxins G and H. The recoveries of added satratoxins G and H from wheat samples averaged 65% for G and 71% for H (200--1000 mg/kg). The coefficients of variation (CV) were 15% for G and 14% for H. The lower limit of detection was 200 microgram/kg for wheat. Analysis of corn, oats, and barley samples (400 microgram/kg of each toxin added) gave comparable recoveries. Only the corn extract exhibited HPLC interferences at the retention time for satratoxin H. The method was also used to analyze samples of corn, oats, wheat, barley, and rice on which Stachybotrys atra had been cultured.

A liquid chromatographic (LC) method for determining tenuazonic acid (TA) and alternariol methyl ether (AME) in tomatoes and tomato products is described. The Alternaria metabolites are extracted from a water slurry of the sample with CHCl3, the mixture is centrifuged, and the extract is fractionated on a silica gel column. Reverse phase LC with an ultraviolet detector (for TA) and a fluorescence detector (for AME) connected in series is used for final separation and determination. The limit of determination for TA and AME is 25 and 3 ng/g, respectively, with average recoveries from catsup of 83 and 68%, respectively. The LC method also detects alternariol, but interfering peaks in some samples prevent accurate quantitation. Chemical ionization mass spectrometry (CIMS) is used to confirm TA. Samples (142) of tomatoes collected from commercial processing lines were analyzed; TA was found in 73 samples (0.4-70 micrograms/g).

Four of the metabolites of Penicillium viridicatum 66-68-2 grown on rice cultures were isolated and identified as xanthomegnin, viomellein, rubrosulphin, and viopurpurin. Melting points, elemental analysis, and infrared, ultraviolet, and field desorption and electron impact mass spectra of the isolated compounds were consistent with values reported in the literature for these compounds. In addition, diacetate and triacetate derivatives were prepared, and the chemical and physical analyses of the derivatives were also consistent with literature data. Proton magnetic resonance spectroscopy and thin-layer chromatography were also used for the additional identification of selected compounds.

Fungal isolates from legumes were cultured on rice and examined for production of the toxic mold metabolites xanthomegnin and viomellein. Six of 14 Aspergillus ochraceus isolates produced from 0.3 to 1.3 mg of xanthomegnin per g and 0.1 to 1.0 mg of viomellein per g. One of nine isolates of Penicillium cyclopium produced 0.1 mg of xanthomegnin per g and 0.06 mg of viomellein per g. Three of nine P. viridicatum isolates produced from 0.4 to 1.6 mg of xanthomegnin per g and 0.2 to 0.4 mg of viomellein per g. This is the first report of xanthomegnin and viomellein production by A. ochraeus and P. cyclopium.

Dihydrocitrinone, 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin-7-carboxylic acid, was isolated and identified as a urinary metabolite after oral administration of citrinin to rats. Male and female Osborne-Mendel rats received 30 mg citrinin/kg body weight by oral intubation. The metabolite dihydrocitrinone was present in urine collected at 0-2, 2-4, 4-6, 6-8, and 8-24 h after treatment. Only unchanged citrinin was found in blood collected 24 h after administration of the compound. The metabolite had a blue fluorescence and the same Rf on thin-layer chromatography, the same retention time on reverse-phase high-pressure liquid chromatography, and the same mass spectrum as an authentic sample of dihydrocitrinone.

Corn and oats samples are extracted with acetonitrile-water, followed by partition of the extract against hexane, transfer to chloroform, and elution from a silica gel column. The extract is purified by gel permeation chromatography on an automatic instrument. Reverse phase high-pressure liquid chromatography, using a 254 nm ultraviolet detector and 0.1 M KH2PO4-acetonitrile (7+5) as the mobile phase, is used for quantitation. The average recovery from 6 samples of corn to which 0, 25, 50, and 100 mug sterigmatocystin/kg had been added was 59%, with a coefficient of variation of 8.4%. The average recovery from oats fortified at the same levels was 74%, with a coefficient of variation of 12%. A confirmation procedure based on hemiacetal derivative formation on a thin layer chromatographic plate is also described.