Angela Mally

Proposal of a comprehensive definition of modified and other forms of mycotoxins including “masked” mycotoxins

As the term “masked mycotoxins” encompasses only conjugated mycotoxins generated by plants and no other possible forms of mycotoxins and their modifications, we hereby propose for all these forms a systematic definition consisting of four hierarchic levels. The highest level differentiates the free and unmodified forms of mycotoxins from those being matrix-associated and from those being modified in their chemical structure. The following lower levels further differentiate, in particular, “modified mycotoxins” into “biologically modified” and “chemically modified” with all variations of metabolites of the former and dividing the latter into “thermally formed” and “non-thermally formed” ones. To harmonize future scientific wording and subsequent legislation, we suggest that the term “modified mycotoxins” should be used in the future and the term “masked mycotoxins” to be kept for the fraction of biologically modified mycotoxins that were conjugated by plants.

Comparative analysis of novel noninvasive renal biomarkers and metabonomic changes in a rat model of gentamicin nephrotoxicity.

Although early detection of toxicant induced kidney injury during drug development and chemical safety testing is still limited by the lack of sensitive and reliable biomarkers of nephrotoxicity, omics technologies have brought enormous opportunities for improved detection of toxicity and biomarker discovery. Thus, transcription profiling has led to the identification of several candidate kidney biomarkers such as kidney injury molecule (Kim-1), clusterin, lipocalin-2, and tissue inhibitor of metalloproteinase 1 (Timp-1), and metabonomic analysis of urine is increasingly used to indicate biochemical perturbations due to renal toxicity. This study was designed to assess the value of a combined (1)H-NMR and gas chromatography-mass spectrometry (GC-MS) metabonomics approach and a set of novel urinary protein markers for early detection of nephrotoxicity following treatment of male Wistar rats with gentamicin (60 and 120 mg/kg bw, s.c.) for 7 days. Time- and dose-dependent separation of gentamicin-treated animals from controls was observed by principal component analysis of (1)H-NMR and GC-MS data. The major metabolic alterations responsible for group separation were linked to the gut microflora, thus related to the pharmacology of the drug, and increased glucose in urine of gentamicin-treated animals, consistent with damage to the S(1) and S(2) proximal tubules, the primary sites for glucose reabsorption. Altered excretion of urinary protein biomarkers Kim-1 and lipocalin-2, but not Timp-1 and clusterin, was detected before marked changes in clinical chemistry parameters were evident. The early increase in urine, which correlated with enhanced gene and protein expression at the site of injury, provides further support for lipocalin-2 and Kim-1 as sensitive, noninvasive biomarkers of nephrotoxicity.

Evaluation of Putative Biomarkers of Nephrotoxicity after Exposure to Ochratoxin A In Vivo and In Vitro

The kidney is one of the main targets of xenobiotic-induced toxicity, but early detection of renal damage is difficult. Recently, several novel biomarkers of nephrotoxicity have been identified by transcription profiling, including kidney injury molecule-1 (Kim-1), lipocalin-2, tissue inhibitor of metalloproteinases-1 (Timp-1), clusterin, osteopontin (OPN), and vimentin, and suggested as sensitive endpoints for acute kidney injury in vivo. However, it is not known if these cellular marker molecules may also be useful to predict chronic nephrotoxicity or to detect nephrotoxic effects in vitro. In this study, a panel of new biomarkers of renal toxicity was assessed via quantitative real-time PCR, immunohistochemistry, and immunoblotting in rats treated with the nephrotoxin ochratoxin A (OTA) for up to 90 days and in rat proximal tubule cells (NRK-52E) treated with OTA in vitro. Repeated administration of OTA to male F344/N rats for 14, 28, or 90 days resulted in a dose- and time-dependent increase in the expression of Kim-1, Timp-1, lipocalin-2, OPN, clusterin, and vimentin. Changes in gene expression were found to correlate with the progressive histopathological alterations and preceded effects on traditional clinical parameters indicative of impaired kidney function. Induction of Kim-1 messenger RNA expression was the earliest and most prominent response observed, supporting the use of this marker as sensitive indicator of chronic kidney injury. In contrast, no significant increase in the expression of putative marker genes and proteins were evident in NRK-52E cells after exposure to OTA for up to 48 h, suggesting that they may not be suitable endpoints for sensitive detection of nephrotoxic effects in vitro.

Performance of Novel Kidney Biomarkers in Preclinical Toxicity Studies

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

Mycotoxins and the kidney: Modes of action for renal tumor formation by ochratoxin A in rodents

The mycotoxin ochratoxin A (OTA) is a potent renal carcinogen in rodents. OTA is only slowly eliminated due to high affinity to plasma proteins and inefficient biotransformation. A number of mechanisms have been proposed to account for renal tumor formation. DNA adduct formation, mainly based on the( 32)P-postlabeling assay, was postulated as a mode of action (MoA). However, studies using radiolabeled OTA or MS failed to demonstrate formation of OTA-derived DNA-adducts. While some studies have demonstrated generation of oxidative stress by OTA, the oxidative stress response appears to be not very pronounced and therefore may not explain the high potency of OTA in rodents. A number of recent investigations support the hypothesis that OTA causes disruption of mitosis resulting in blocked or asymmetric cell division. This may present an increased risk of aneuploidy acquisition and may play a critical role in OTA-induced tumor formation. The absence of DNA-adducts derived from OTA supports a thresholded MoA, and a tolerable weekly intake (TWI) of 120 ng OTA/kg bw has been derived by the European Food Safety Authority. The estimated intake of OTA in Europe is below this TWI for most of the population.

Metabonomic study of ochratoxin A toxicity in rats after repeated administration: phenotypic anchoring enhances the ability for biomarker discovery.

For early detection of toxicity and improved mechanistic understanding, GC/MS-, 1H NMR-, and LC/MS-based metabonomics were applied to urine samples from a rodent toxicity study on the mycotoxin and renal carcinogen ochratoxin A (OTA). OTA was administered at doses of 0, 21, 70, and 210 microg/kg body wt for up to 90 days. Urine samples were collected at 24 h intervals 14, 28, and 90 days after the start of treatment and analyzed with GC/MS, 1H NMR, and LC/MS. Principal component analysis and orthogonal projection to latent structures discriminate analysis (OPLS-DA) based on GC/MS and 1H NMR data discriminated controls from animals dosed with 210 microg/kg body wt OTA as early as 14 days and animals dosed with 70 microg/kg body wt 28 days after the start of treatment, correlating with mild histopathological changes in the kidney. Integration of histopathology scores as discriminators in OPLS-DA models resulted in better multivariate model predictivity and facilitated marker identification. Decreased 2-oxoglutarate and citrate excretion and increased glucose, creatinine, pseudouridine, 5-oxoproline, and myo-inositol excretion were detected with GC/MS. Decreased 2-oxoglutarate and citrate excretion and increased amino acid excretion were found with 1H NMR. Increased urinary glucose is a well-established indicator of kidney damage, and altered excretion of TCA cycle intermediates (citrate and 2-oxoglutarate) is found as a general response to toxic insult in many metabonomics studies. Other markers are associated with cell proliferation (pseudouridine), changes in renal osmolyte handling (myo-inositol), and oxidative stress (5-oxoproline), established mechanisms of OTA toxicity. LC/MS was also able to discriminate controls and treated animals but contained more noise, and marker annotation was only speculative due to lack of reference databases. Use of multiple analytical platforms for metabonomics analysis may result in a more comprehensive metabolite coverage and may be applied to obtain mechanistic information from conventional rodent toxicity studies.

Furan in heat-treated foods: Formation, exposure, toxicity, and aspects of risk assessment

Furan is formed in a variety of heat-treated foods through thermal degradation of natural food constituents. Relatively high levels of furan contamination are found in ground roasted coffee, instant coffee, and processed baby foods. European exposure estimates suggest that mean dietary exposure to furan may be as high as 1.23 and 1.01 μg/kg bw/day for adults and 3- to 12-month-old infants, respectively. Furan is a potent hepatotoxin and hepatocarcinogen in rodents, causing hepatocellular adenomas and carcinomas in rats and mice, and high incidences of cholangiocarcinomas in rats at doses ≥ 2 mg/kg bw. There is therefore a relatively low margin of exposure between estimated human exposure and doses that cause a high tumor incidence in rodents. Since a genotoxic mode of action cannot be excluded for furan-induced tumor formation, the present exposures may indicate a risk to human health and need for mitigation. This review summarizes the current knowledge on mechanisms of furan formation in food, human dietary exposure to furan, and furan toxicity, and highlights the need to establish the risk resulting from the genotoxic and carcinogenic properties of furan at doses lower than 2 mg/kg bw.

Modulation of key regulators of mitosis linked to chromosomal instability is an early event in ochratoxin A carcinogenicity

Ochratoxin A (OTA) is a potent renal carcinogen, but little is known regarding the mechanism of OTA carcinogenicity. Early histopathological alterations induced by OTA in rat kidney include single cell death, stimulation of cell proliferation and prominent karyomegaly indicative of blocked nuclear division during mitosis. Based on these observations, it has been suggested that disruption of mitosis by OTA may be the principal cause of cell death and subsequent trigger for cell proliferation to compensate for cell loss. To gain further insight into the molecular mechanism of OTA toxicity, we used targeted quantitative real-time polymerase chain reaction arrays to investigate the expression of genes involved in cell cycle control and mitosis in kidneys of male F344 rats treated with 0, 21, 70 and 210 microg/kg body wt OTA for up to 90 days. Treatment with OTA resulted in overexpression of key regulators of mitosis, including the mitotic protein kinases Polo-like kinase 1, Aurora B and cyclin-dependent kinase 1 (Cdk1Cdc2), several cyclins and cyclin-dependent kinase inhibitors, topoisomerase II and survivin. Immunohistochemical analysis confirmed upregulation of Cdk1, p21(WAF1/CIP1), topoisomerase II and survivin in S3 proximal tubule cells, from which OTA-induced tumors in rats arise, and demonstrated increased phosphorylation of histone H3, a target of Aurora B. Importantly, many of the genes found to be deregulated in response to OTA have been linked to chromosomal instability and malignant transformation, supporting the hypothesis that aberrant mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition, may play a critical role in OTA carcinogenicity.

DNA adduct formation by ochratoxin A: Review of the available evidence

The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumour formation is unknown and conflicting results regarding the potential of OTA to react with DNA have been obtained. While experiments using radiolabelled (3H or 14C) OTA and liquid scintillation counting or accelerator mass spectrometry indicate lack of formation of covalent DNA-adducts, spots detected by 32P-postlabelling have been attributed to treatment with OTA. However, these putative DNA-adducts have not been shown to contain OTA or part of the OTA molecule and so far no structural information has been provided. Consistent with the absence of DNA-binding of radiolabelled OTA, studies on biotransformation in vivo and in vitro indicate that OTA is poorly metabolized and does not form reactive intermediates capable of interacting with DNA. Recently however, the structures of a carbon- and an oxygen-bonded OTA-deoxyguanosine adduct which is formed by photoirradiation of OTA in the presence of deoxyguanosine have been reported and suggested to be involved in OTA carcinogenicity. The aim of this manuscript is to provide an overview of the available literature regarding DNA adduct formation by OTA.

Low Doses of the Carcinogen Furan Alter Cell Cycle and Apoptosis Gene Expression in Rat Liver Independent of DNA Methylation

Background Evidence of potent rodent carcinogenicity via an unclear mechanism suggests that furan in various foods [leading to an intake of up to 3.5 μg/kg body weight (bw)/day] may present a potential risk to human health. Objectives We tested the hypothesis that altered expression of genes related to cell cycle control, apoptosis, and DNA damage may contribute to the carcinogenicity of furan in rodents. In addition, we investigated the reversibility of such changes and the potential role of epigenetic mechanisms in response to furan doses that approach the maximum estimated dietary intake in humans. Methods The mRNA expression profiles of genes related to cell cycle, apoptosis, and DNA damage in rat liver treated with furan concentrations of 0.1 and 2 mg/kg bw were measured by quantitative polymerase chain reaction (PCR) arrays. We assessed epigenetic changes by analysis of global and gene-specific DNA methylation [methylation-specific PCR, combined bisulfite restriction analysis (COBRA), and methylated DNA immunoprecipitation chip] and microRNA (miRNA) analyses. Results The expression profiles of apoptosis-related and cell-cycle–related genes were unchanged after 5 days of treatment, although we observed a statistically significant change in the expression of genes related to cell cycle control and apoptosis, but not DNA damage, after 4 weeks of treatment. These changes were reversed after an off-dose period of 2 weeks. None of the gene expression changes was associated with a change in DNA methylation, although we detected minor changes in the miRNA expression profile (5 miRNA alterations out of 349 measured) that may have contributed to modification of gene expression in some cases. Conclusion Nongenotoxic changes in gene expression may contribute to the carcinogenicity of furan in rodents. These findings highlight the need for a more comprehensive risk assessment of furan exposure in humans.